Fabien Lacombat

Agathe Espagne

Nadia Dozova

Pascal Plaza
email: pascal.plaza@ens.fr

Pavel Müller
email: pavel.muller@i2bc.paris-saclay.fr

Klaus Brettel

Sophie Franz-Badur

Lars-Oliver Essen
email: essen@chemie.uni-marburg.de

Ultrafast Oxidation of a Tyrosine by Proton-Coupled Electron Transfer Promotes Light Activation of an Animal-like Cryptochrome

The animal-like cryptochrome of Chlamydomonas reinhardtii (CraCRY) is a recently discovered photoreceptor that controls the transcriptional profile and sexual life cycle of this alga by both blue and red light. CraCRY has the uncommon feature of efficient formation and longevity of the semi-reduced neutral form of its FAD cofactor upon blue light illumination. Tyrosine Y 373 plays hereby a crucial role by elongating as fourth member the electron transfer (ET) chain that comprises the tryptophan triad otherwise found in most other cryptochromes and DNA photolyases. Here, we report the full mechanism of light-induced FADH • formation in CraCRY using transient absorption spectroscopy from hundreds of femtoseconds to seconds. Electron transfer starts from ultrafast reduction of excited FAD to FAD •-by the proximal tryptophan (0.4 ps) and is followed by delocalized migration of the produced WH •+ radical along the tryptophan triad (3.7 and 55 ps). Oxidation of Y 373 by coupled ET to WH •+ and deprotonation then proceeds in ~800 ps, without any significant kinetic isotope effect, nor a pH effect between pH 6.5 and 9.0. The FAD •-/Y 373 • pair is formed with high quantum yield (~60%); its intrinsic decay by recombination is slow (~50 ms), favoring reduction of Y 373 • by extrinsic agents and protonation of FAD •-to form the long-lived, red-light absorbing FADH • species. Possible mechanisms of tyrosine oxidation by ultrafast proton-coupled ET in CraCRY, a process about 40 times faster than the archetypal tyrosine-Z oxidation in photosystem II, are discussed in detail.

INTRODUCTION

Cryptochrome blue-light receptors are responsible for various biological photoinduced responses, such as plant photomorphogenesis, circadian rhythm entrainment in animals or presumably magnetoreception in migrating birds. [START_REF] Sancar | Structure and Function of DNA Photolyase and Cryptochrome Blue-Light Photoreceptors[END_REF][START_REF] Lin | Cryptochrome Structure and Signal Transduction[END_REF][START_REF] Chaves | The Cryptochromes: Blue Light Photoreceptors in Plants and Animals[END_REF][START_REF] Hore | The Radical-Pair Mechanism of Magnetoreception[END_REF] The triggering mechanism of cryptochromes is based on the photoreduction of their flavin cofactor (FAD: flavin adenine dinucleotide), in a very similar way as in the so-called photoactivation reaction of photolyases, a group of closely related enzymes able to photorepair UV-damaged DNA. [START_REF] Sancar | Structure and Function of DNA Photolyase and Cryptochrome Blue-Light Photoreceptors[END_REF][START_REF] Cadet | Ultraviolet Radiation-Mediated Damage to Cellular DNA[END_REF][START_REF] Weber | Light-Driven Enzymatic Catalysis of DNA Repair: A Review of Recent Biophysical Studies on Photolyase[END_REF][START_REF] Brettel | Reaction Mechanisms of DNA Photolyase[END_REF][START_REF] Zhong | Electron Transfer Mechanisms of DNA Repair by Photolyase[END_REF] Some bifunctional cryptochromes even act both as light receptors and DNA-repairing enzymes. [START_REF] Selby | A Cryptochrome/Photolyase Class of Enzymes with Single-Stranded DNA-Specific Photolyase Activity[END_REF][START_REF] Pokorny | Recognition and Repair of UV Lesions in Loop Structures of Duplex DNA by DASH-Type Cryptochrome[END_REF][START_REF] Berrocal-Tito | Trichoderma Atroviride PHR1, a Fungal Photolyase Responsible for DNA Repair, Autoregulates its Own Photoinduction[END_REF][START_REF] Bayram | More Than a Repair Enzyme: Aspergillus nidulans Photolyase-Like CryA Is a Regulator of Sexual Development[END_REF][START_REF] Coesel | Diatom PtCPF1 Is a New Cryptochrome/Photolyase Family Member with DNA Repair and Transcription Regulation Activity[END_REF][START_REF] Heijde | Characterization of Two Members of the Cryptochrome / Photolyase Family from Ostreococcus Tauri Gives Insight in the Origin and Evolution of Cryptochromes[END_REF] The evolutionary links between the different members of the photolyase and cryptochrome superfamily (PCSf) may be found in various reports. [START_REF] Kiontke | Crystal Structures of an Archaeal Class II DNA Photolyase and its Complex with UV-Damaged Duplex DNA[END_REF][START_REF] Geisselbrecht | CryB from Rhodobacter Sphaeroides: A Unique Class of Cryptochromes with New Cofactors[END_REF][START_REF] Juhas | A Novel Cryptochrome in the Diatom Phaeodactylum tricornutum Influences the Regulation of Light-Harvesting Protein Levels[END_REF][START_REF] Oliveri | The Cryptochrome/Photolyase Family in Aquatic Organisms[END_REF][START_REF] Zhang | Photolyase: Dynamics and Mechanisms of Repair of Sun-Induced DNA Damage[END_REF] A new type of cryptochrome was recently discovered in the green alga Chlamydomonas reinhardtii, exhibiting homology to animal but not plant cryptochromes, called animal-like cryptochrome (the C. reinhardtii protein is noted CraCRY). In contrast to other cryptochromes, CraCRY appears to be capable of inducing a genetic response not only to blue, but also to red light. [START_REF] Beel | A Flavin Binding Cryptochrome Photoreceptor Responds to Both Blue and Red Light in Chlamydomonas Reinhardtii[END_REF][START_REF] Kottke | Cryptochrome Photoreceptors in Green Algae: Unexpected Versatility of Mechanisms and Functions[END_REF][START_REF] Zou | An Animal-Like Cryptochrome Controls the Chlamydomonas Sexual Cycle[END_REF] This feature is probably related to another unique property of CraCRY, the very efficient formation and stabilization of its semireduced (red light-absorbing) flavin cofactor (FADH • ) that can be easily accumulated under blue light illumination and lives for seconds to minutes (depending on pH). [START_REF] Beel | A Flavin Binding Cryptochrome Photoreceptor Responds to Both Blue and Red Light in Chlamydomonas Reinhardtii[END_REF][START_REF] Spexard | Response of the Sensory Animal-Like Cryptochrome aCRY to Blue and Red Light as Revealed by Infrared Difference Spectroscopy[END_REF] This process relies on the oxidation of the phenolic moiety of the tyrosine residue Y 373 as its mutation to phenylalanine abolishes this effect. [START_REF] Oldemeyer | Essential Role of an Unusually Long-Lived Tyrosyl Radical in the Response to Red Light of the Animal-Like Cryptochrome aCRY[END_REF] Y 373 is located at the distal end of the "canonical" tryptophan triad (W 399 , W 376 and W 322 in CraCRY; see Figure 1A) that is conserved in most members of the PCSf -except the subfamilies of bacterial (6-4) photolyases and class II photolyases -and is involved in the photoreduction of FAD. [START_REF] Chaves | The Cryptochromes: Blue Light Photoreceptors in Plants and Animals[END_REF][START_REF] Brettel | Reaction Mechanisms of DNA Photolyase[END_REF][START_REF] Essen | Photolyases and Cryptochromes: Common Mechanisms of DNA Repair and Light-Driven Signaling?[END_REF] In the canonical PCSf members, the excited flavin abstracts an electron from a nearby (proximal) tryptophan, which gets in turn reduced by a second (medial) tryptophan, before being reduced by a third (distal) one. The distal tryptophan is usually surface-exposed and can hence be reduced by external electron donors such as thiol compounds [START_REF] Heelis | Excited-State Properties of Escherichia Coli DNA Photolyase in the Picosecond to Millisecond Time Scale[END_REF] or NAD(P)H. [START_REF] Engelhard | Cellular Metabolites Enhance the Light Sensitivity of Arabidopsis Cryptochrome through Alternate Electron Transfer Pathways[END_REF][START_REF] Ignatz | Nicotinamide Adenine Dinucleotides Arrest Photoreduction of Class II DNA Photolyases in FADH • State[END_REF] For animal-like cryptochromes and (6-4) photolyases, a fourth aromatic residue, Y 373 in CraCRY and W 360 in the (6-4) photolyase from Xenopus laevis, has been found to act as final electron donor of an extended electron transfer chain. [START_REF] Oldemeyer | Essential Role of an Unusually Long-Lived Tyrosyl Radical in the Response to Red Light of the Animal-Like Cryptochrome aCRY[END_REF][START_REF] Nohr | Extended Electron-Transfer in Animal Cryptochromes Mediated by a Tetrad of Aromatic Amino Acids[END_REF][START_REF] Müller | Discovery and Functional Analysis of a 4th Electron-Transferring Tryptophan Conserved Exclusively in Animal Cryptochromes and (6-4) Photolyases[END_REF][START_REF] Martin | Ultrafast Flavin Photoreduction in an Oxidized Animal (6-4) Photolyase through an Unconventional Tryptophan Tetrad[END_REF][START_REF] Yamamoto | Loss of Fourth Electron-Transferring Tryptophan in Animal (6-4) Photolyase Impairs DNA Repair Activity in Bacterial Cells[END_REF] Transient absorption spectroscopy (TAS) has been extensively used to study the mechanistic details of the photoreduction of photolyases [START_REF] Martin | Ultrafast Flavin Photoreduction in an Oxidized Animal (6-4) Photolyase through an Unconventional Tryptophan Tetrad[END_REF][START_REF] Aubert | Intraprotein Radical Transfer During Photoactivation of DNA Photolyase[END_REF][START_REF] Wang | Femtosecond Dynamics of Flavin Cofactor in DNA Photolyase: Radical Reduction, Local Solvation, and Charge Recombination[END_REF][START_REF] Kao | Ultrafast Dynamics and Anionic Active States of the Flavin Cofactor in Cryptochrome and Photolyase[END_REF][START_REF] Byrdin | Quantum Yield Measurements of Short-Lived Photoactivation Intermediates in DNA Photolyase: Toward a Detailed Understanding of the Triple Tryptophan Electron Transfer Chain[END_REF][START_REF] Liu | Determining Complete Electron Flow in the Cofactor Photoreduction of Oxidized Photolyase[END_REF][START_REF] Liu | Dynamic Determination of Active-Site Reactivity in Semiquinone Photolyase by the Cofactor Photoreduction[END_REF][START_REF] Brazard | Spectro-Temporal Characterization of the Photoactivation Mechanism of Two New Oxidized Cryptochrome/Photolyase Photoreceptors[END_REF][START_REF] Lacombat | Femtosecond Transient Absorption Study of the Primary Photoreduction Events of a Class II Photolyase[END_REF] and cryptochromes, taking advantage of the rather distinct absorption spectra of the species involved (Figure 1B). [START_REF] Kao | Ultrafast Dynamics and Anionic Active States of the Flavin Cofactor in Cryptochrome and Photolyase[END_REF][START_REF] Brazard | Spectro-Temporal Characterization of the Photoactivation Mechanism of Two New Oxidized Cryptochrome/Photolyase Photoreceptors[END_REF][START_REF] Immeln | Primary Events in the Blue Light Sensor Plant Cryptochrome: Intraprotein Electron and Proton Transfer Revealed by Femtosecond Spectroscopy[END_REF] Ultrafast TAS studies have in particular shown that when the flavin is initially oxidized (FAD ox ), the primary electron transfer (ET) takes place in less than one picosecond, [START_REF] Martin | Ultrafast Flavin Photoreduction in an Oxidized Animal (6-4) Photolyase through an Unconventional Tryptophan Tetrad[END_REF][START_REF] Kao | Ultrafast Dynamics and Anionic Active States of the Flavin Cofactor in Cryptochrome and Photolyase[END_REF][START_REF] Liu | Determining Complete Electron Flow in the Cofactor Photoreduction of Oxidized Photolyase[END_REF][START_REF] Brazard | Spectro-Temporal Characterization of the Photoactivation Mechanism of Two New Oxidized Cryptochrome/Photolyase Photoreceptors[END_REF][START_REF] Immeln | Primary Events in the Blue Light Sensor Plant Cryptochrome: Intraprotein Electron and Proton Transfer Revealed by Femtosecond Spectroscopy[END_REF] producing a radical pair of reduced flavin (FAD •-) and oxidized tryptophan (WH •+ ) † . Slower steps observed in the ps regime were assigned to the migration of the electron hole along the tryptophan triad, which competes with partial charge recombination of the radical pairs. The distal WH •+ typically deprotonates in a few hundred nanoseconds, forming the neutral W • radical. [START_REF] Aubert | Intraprotein Radical Transfer During Photoactivation of DNA Photolyase[END_REF][START_REF] Müller | ATP Binding Turns Plant Cryptochrome into an Efficient Natural Photoswitch[END_REF] Besides the tryptophan triad, Liu et al. [START_REF] Liu | Determining Complete Electron Flow in the Cofactor Photoreduction of Oxidized Photolyase[END_REF] demonstrated for the class I CPD photolyase of E. coli (EcCPDI) the involvement of two tryptophans acting as branch lines: one weakly competes with the proximal tryptophan for the reduction of the excited flavin, the other temporarily competes with the medial tryptophan for the reduction of the proximal WH •+ cation. Counterparts of these branch tryptophans are also present in CraCRY, i.e. W 401 and W 332 (shown in gray in Figure 1A). Ultrafast TAS had not yet been applied to CraCRY and the few studies using "classical" TAS [START_REF] Oldemeyer | Essential Role of an Unusually Long-Lived Tyrosyl Radical in the Response to Red Light of the Animal-Like Cryptochrome aCRY[END_REF][START_REF] Nohr | Extended Electron-Transfer in Animal Cryptochromes Mediated by a Tetrad of Aromatic Amino Acids[END_REF] were essentially limited to timescales above 1 µs.

Transient EPR data by Nohr et al. [START_REF] Nohr | Extended Electron-Transfer in Animal Cryptochromes Mediated by a Tetrad of Aromatic Amino Acids[END_REF] on oxidized CraCRY on the µs timescale have shown that fast, unresolved, joint oxidation and deprotonation of Y 373 occurs upon excitation of the flavin. In the same study, monitoring the decay of FAD •-by transient absorption yielded a lifetime of 26 ms that was attributed to recombination of the radical pair FAD •-/Y • ‡ . [START_REF] Nohr | Extended Electron-Transfer in Animal Cryptochromes Mediated by a Tetrad of Aromatic Amino Acids[END_REF] A transient absorption study by Oldemeyer et al. [START_REF] Oldemeyer | Essential Role of an Unusually Long-Lived Tyrosyl Radical in the Response to Red Light of the Animal-Like Cryptochrome aCRY[END_REF] using semi-reduced CraCRY (containing FADH • ) prepared by pre-illumination with blue light also demonstrates oxidation of a tyrosyl residue, in "less than 1 µs", and reports the decay of the corresponding FADH -/Y • pair in 2.6 s. Much slower and only partial oxidation of tyrosyl residues was reported previously during photoreduction of some members of the PCSf: time constants of ~70 µs for the photolyase from A. nidulans, [START_REF] Aubert | Intraprotein Electron Transfer between Tyrosine and Tryptophan in DNA Photolyase from Anacystis nidulans[END_REF] ~1.5 ms for cryptochrome 1 from A. thaliana [START_REF] Giovani | Light-Induced Electron Transfer in a Cryptochrome Blue-Light Photoreceptor[END_REF] and ~1 ms for the CPD class II photolyase from M. mazei. [START_REF] Müller | Sub-Nanosecond Tryptophan Radical Deprotonation Mediated by a Protein-Bound Water Cluster in Class II DNA Photolyases[END_REF] Oxidation and back-reduction of tyrosyl residues has been discovered to be involved in the functioning of several enzymes, like prostaglandin H synthase, galactose oxidase, ribonucleotide reductase or photosystem II (PSII). [START_REF] Stubbe | Protein Radicals in Enzyme Catalysis[END_REF] The case of PSII is of particular importance because the possibility to trigger the reactions by light allowed for detailed time-resolved studies of the reaction mechanism and is still a matter of discussion. [START_REF] Barry | Tyrosine Radicals Are Involved in the Photosynthetic Oxygen-Evolving System[END_REF][START_REF] Tommos | Proton and Hydrogen Currents in Photosynthetic Water Oxidation[END_REF][START_REF] Sjödin | Proton-Coupled Electron Transfer from Tyrosine in a Tyrosine-Ruthenium-Tris-Bipyridine Complex: Comparison with Tyrosine z Oxidation in Photosystem II[END_REF][START_REF] Rappaport | Probing the Coupling between Proton and Electron Transfer in Photosystem II Core Complexes Containing a 3-Fluorotyrosine[END_REF][START_REF] Hammarström | Proton-Coupled Electron Transfer of Tyrosines in Photosystem II and Model Systems for Artificial Photosynthesis: The Role of a Redox-Active Link between Catalyst and Photosensitizer[END_REF][START_REF] Barry | Proton Coupled Electron Transfer and Redox Active Tyrosines in Photosystem II[END_REF][START_REF] Schlodder | Temperature Dependence of the Oxidation Kinetics of Tyr(Z) and Tyr(D) in Oxygen-Evolving Photosystem II Complexes Throughout the Range from 320 K to 5 K[END_REF] The one-electron reduction potential of tyrosine (YH •+ /YH couple) is as high as 1.35 V (vs NHE) in aqueous solution. [START_REF] Tommos | De Novo Proteins as Models of Radical Enzymes[END_REF] However, the oxidant of tyrosine in PSII, the chlorophyll cation P680 + , provides only 1.25 V, [START_REF] Rappaport | Primary Photochemistry and Energetics Leading to the Oxidation of the (Mn)4ca Cluster and to the Evolution of Molecular Oxygen in Photosystem II[END_REF] so that a simple ET from YH to P680 + would be uphill (endergonic). Nevertheless, tyrosine oxidation by P680 + can be as fast as 30 ns. [START_REF] Van Best | Kinetics of Reduction of the Oxidized Primary Electron Donor of Photosystem II in Spinach Chloroplasts and in Chlorella Cells in the Microsecond and Nanosecond Time Ranges Following Flash Excitation[END_REF][START_REF] Brettel | Nanosecond Reduction Kinetics of Photooxidized Chlorophylla II (P-680) in Single Flashes as a Probe for the Electron Pathway, H + -Release and Charge Accumulation in the O 2 -Evolving Complex[END_REF][START_REF] Gerken | Optical Characterization of the Immediate Electron Donor to Chlorophyll A π + in O 2 -Evolving Photosystem II Complexes: Tyrosine as Possible Electron Carrier between Chlorophyll A π and the Water-Oxidizing Manganese Complex[END_REF][START_REF] Styring | Two Tyrosines That Changed the World: Interfacing the Oxidizing Power of Photochemistry to Water Splitting in Photosystem II[END_REF] In fact, the YH •+ † The notation WH •+ indicates the protonated tryptophanyl radical, while W • refers to the deprotonated radical.

‡ Y • stands for the deprotonated tyrosyl radical; the corresponding protonated species is noted YH •+ .

radical is strongly acidic (pKa  -2), [START_REF] Dixon | Determination of Acidity Constants of Some Phenol Radical Cations by Means of Electron-Spin Resonance[END_REF] so when ET is coupled to deprotonation of YH •+ , the overall reaction is well downhill, i.e. exergonic.

Another case of light-induced tyrosine oxidation was more recently discovered in BLUF (blue-light sensing using FAD) photoreceptor proteins. [START_REF] Gauden | Hydrogen-Bond Switching through a Radical Pair Mechanism in a Flavin-Binding Photoreceptor[END_REF][START_REF] Mathes | Redox Modulation of Flavin and Tyrosine Determines Photoinduced Proton-Coupled Electron Transfer and Photoactivation of BLUF Photoreceptors[END_REF][START_REF] Mathes | Hydrogen Bond Switching among Flavin and Amino Acids Determines the Nature of Proton-Coupled Electron Transfer in BLUF Photoreceptors[END_REF] In the dark-adapted Slr 1694 BLUF, ultrafast (tens of picoseconds) ET from Y 8 to the photoexcited FAD cofactor and similarly fast PT from Y 8 H •+ to FAD •-via a glutamine residue yield an Y 8 • /FADH • radical pair. [START_REF] Gauden | Hydrogen-Bond Switching through a Radical Pair Mechanism in a Flavin-Binding Photoreceptor[END_REF][START_REF] Mathes | Redox Modulation of Flavin and Tyrosine Determines Photoinduced Proton-Coupled Electron Transfer and Photoactivation of BLUF Photoreceptors[END_REF] This pair recombines on a 100-ps timescale, but rearrangements of the hydrogen bond network (and a related red shift of the FAD absorption spectrum) persist for seconds and represent the signaling state of the photoreceptor. In the light-adapted Slr 1694 BLUF, formation the of Y 8 • /FADH • pair is even faster (~1ps), by concerted proton-coupled electron transfer. [START_REF] Mathes | Hydrogen Bond Switching among Flavin and Amino Acids Determines the Nature of Proton-Coupled Electron Transfer in BLUF Photoreceptors[END_REF] Proton-coupled electron transfer (PCET) has been the subject of intense theoretical work and experimental studies in chemistry and biology because of its crucial role in avoiding high energy intermediates in chemical and enzymatic catalysis, notably for solar energy conversion. [START_REF] Hammes-Schiffer | Theory of Coupled Electron and Proton Transfer Reactions[END_REF][START_REF] Weinberg | Proton-Coupled Electron Transfer[END_REF][START_REF] Saveant | Electrochemical Approach to Proton-Coupled Electron Transfers: Recent Advances[END_REF][START_REF] Migliore | Biochemistry and Theory of Proton-Coupled Electron Transfer[END_REF][START_REF] Gentry | Synthetic Applications of Proton-Coupled Electron Transfer[END_REF][START_REF] Odella | Controlling Proton-Coupled Electron Transfer in Bioinspired Artificial Photosynthetic Relays[END_REF] As summarized by Hammes-Schiffer and Stuchebrukhov, [START_REF] Hammes-Schiffer | Theory of Coupled Electron and Proton Transfer Reactions[END_REF] two types of coupled reactions can be distinguished. In concerted electron and proton transfer (PT) reactions, both ET and PT occur in one step. [START_REF] Mathes | Hydrogen Bond Switching among Flavin and Amino Acids Determines the Nature of Proton-Coupled Electron Transfer in BLUF Photoreceptors[END_REF] In sequential reactions, the transitions occur in two steps: ET followed by PT (noted ET/PT) or PT followed by ET (PT/ET). In both cases, the first step is typically uphill in energy and the second step is downhill to the final product state. Tyrosine oxidation in the BLUF domain may constitute an exception, which is commented further at the end of Section 4.2.2. A hallmark of a concerted PCET reaction is the observation of a substantial normal kinetic isotope effect (KIE), i.e. the reaction is slowed down when the transferred proton is replaced by a deuteron. Sequential ET/PT and PT/ET typically show a (normal)

KIE only when PT becomes rate limiting.

The absence of a KIE on the oxidation of tyrosine in PSII in 30 ns may point to a sequential PT/ET, where the hydroxyl proton of the tyrosine moves to a nearby histidine residue prior to rate-limiting ET. [START_REF] Rappaport | Probing the Coupling between Proton and Electron Transfer in Photosystem II Core Complexes Containing a 3-Fluorotyrosine[END_REF][START_REF] Schlodder | Temperature Dependence of the Oxidation Kinetics of Tyr(Z) and Tyr(D) in Oxygen-Evolving Photosystem II Complexes Throughout the Range from 320 K to 5 K[END_REF] A slower (few µs) tyrosine oxidation in PSII depleted of the oxygen-evolving complex, however, showed a normal KIE and was hence assigned to concerted PCET. [START_REF] Sjödin | Proton-Coupled Electron Transfer from Tyrosine in a Tyrosine-Ruthenium-Tris-Bipyridine Complex: Comparison with Tyrosine z Oxidation in Photosystem II[END_REF] The presumed oxidant of tyrosine in CraCRY, namely W 322 H •+ , is likely to have an even lower reduction potential (1.07 V for the WH •+ /WH couple in aqueous solution [START_REF] Tommos | De Novo Proteins as Models of Radical Enzymes[END_REF] ) than P680 + in PSII, suggesting that tyrosine oxidation in CraCRY occurs by some type of PCET as well. The main objective of the present work was to resolve the kinetics of tyrosine oxidation in CraCRY and to contribute to establishing its reaction mechanism. Ultrafast pump-probe and real-time transient absorption spectroscopy on timescales from hundreds of fs to hundreds of ms were used to monitor the complete reaction sequence of flavin photoreduction. Wild-type (WT) CraCRY was compared to its Y373F mutant. § This mutation was expected to ablate the electron donor character of Y 373 and enable us to distinguish reactions involving Y 373 from preceding reactions that should not be significantly affected by the mutation.

MATERIALS AND METHODS

Sample preparation

The Chlamydomonas reinhardtii animal-like cryptochrome (CraCRY) samples were prepared as recently described. [START_REF] Franz | Structure of the Bifunctional Cryptochrome aCRY from Chlamydomonas Reinhardtii[END_REF] For transient absorption experiments, we used a 50 mM phosphate buffer at pH 8. shown in SI, Section S1 (Figure S1).

Femtosecond Transient Absorption Spectroscopy (TAS)

Setup

Polarized transient absorption anisotropy was here used to both access the transient population dynamics, through the isotropic transient absorption, and probe orientation of the transient species with the associated transient absorption anisotropy. Anisotropy is indeed particularly useful to help distinguishing the different WH •+ radicals involved in the photoreduction process, as was previously done for other photolyases and cryptochromes. [START_REF] Martin | Ultrafast Flavin Photoreduction in an Oxidized Animal (6-4) Photolyase through an Unconventional Tryptophan Tetrad[END_REF][START_REF] Lacombat | Femtosecond Transient Absorption Study of the Primary Photoreduction Events of a Class II Photolyase[END_REF][START_REF] Immeln | Primary Events in the Blue Light Sensor Plant Cryptochrome: Intraprotein Electron and Proton Transfer Revealed by Femtosecond Spectroscopy[END_REF][START_REF] Byrdin | Polarized Transient Absorption to Resolve Electron Transfer between Tryptophans in DNA Photolyase[END_REF] The pump-probe setup, with white-light continuum probe, has been previously described. [START_REF] Martin | Ultrafast Flavin Photoreduction in an Oxidized Animal (6-4) Photolyase through an Unconventional Tryptophan Tetrad[END_REF][START_REF] Brazard | Spectro-Temporal Characterization of the Photoactivation Mechanism of Two New Oxidized Cryptochrome/Photolyase Photoreceptors[END_REF][START_REF] Lacombat | Femtosecond Transient Absorption Study of the Primary Photoreduction Events of a Class II Photolyase[END_REF] The pump beam was tuned at 475 nm. By comparison with a reference sample of tris-(2,2'-bipyridyl)-ruthenium(II) complex, 72** it was checked § The abridged notations WT and Y373Y will be used to refer to these variants. ** Systematic measurements on the [Ru(bpy) 3 ] 2+ complex were also used to (i) ensure that no major alteration of the transient spectra occurred upon varying the pump-probe delay over the 3.2-ns range of an optical delay line, that the fraction of excited molecules in the probed volume was about 3% (case of WT in H 2 O buffer), which lies in the linear regime.

In order to avoid probing the same region consecutively, the sample cuvette (1-mm optical path) was continuously moved back and forth in horizontal and vertical directions, each pump shot irradiating an area of the order of 0.01 mm². A relatively large volume (~250 µL in a 10-mm wide cell) of sample was used for the experiment with WT-CraCRY in H 2 O buffer. Alternatively, four smaller cells (2-mm wide, containing ~60 µL) were used for WT in D 2 O buffer, each one being irradiated for a shorter time while the others were recovering in the dark. One 60-µL cell was sufficient for Y373F since FADH • does not accumulate so easily in this CraCRY mutant. [START_REF] Oldemeyer | Essential Role of an Unusually Long-Lived Tyrosyl Radical in the Response to Red Light of the Animal-Like Cryptochrome aCRY[END_REF][START_REF] Nohr | Extended Electron-Transfer in Animal Cryptochromes Mediated by a Tetrad of Aromatic Amino Acids[END_REF] Steady-state absorption spectra of the samples were regularly recorded during the experiments to check that no significant accumulation of FADH • or sample degradation occurred. All experiments were performed at room temperature (20 °C).

Data analysis

The polarized differential absorption spectra ( and ), obtained from the transmitted probe ∆𝐴 ∥ Δ𝐴 ⊥ beam intensities, were first corrected from the chirp of the probe beam, which was independently measured by recording cross-phase modulation [START_REF] Ekvall | Cross Phase Modulation Artifact in Liquid Phase Transient Absorption Spectroscopy[END_REF] (XPM) in the pure solvent. These raw polarized spectra were transformed into isotropic transient absorption spectra ( ) and transient anisotropy spectra (r) ∆𝐴 iso using Equations 1 and 2. Scattering of the pump beam was masked in the spectra, both for data analysis and figure presentation.

{ ∆𝐴 𝑖𝑠𝑜 = 1 3 (∆𝐴 ∥ + 2 ∆𝐴 ⊥ ) (Eq. 1) 𝑟 = ∆𝐴 ∥ -∆𝐴 ⊥ ∆𝐴 ∥ + 2 ∆𝐴 ⊥ (Eq. 2)
Notably, the transient absorption spectra of WT samples displayed a weak signal at negative time delays. This signal is due to a long-lived photoproduct (FAD •-/Y • pair [START_REF] Nohr | Extended Electron-Transfer in Animal Cryptochromes Mediated by a Tetrad of Aromatic Amino Acids[END_REF] ) produced by the preceding pump pulse (3 ms before the probe only shot and 6 ms before pump and probe shot), but still detectable because the 2D movement imposed on the sample cell was not fast enough to regenerate entirely the probed sample volume between two probe pulses. This spurious signal has been averaged, smoothed, and subtracted from the raw transient absorption data. The resulting spectra are presented here.

Multiexponential global analysis was performed simultaneously on the parallel and perpendicular data as previously described. [START_REF] Martin | Ultrafast Flavin Photoreduction in an Oxidized Animal (6-4) Photolyase through an Unconventional Tryptophan Tetrad[END_REF][START_REF] Brazard | Spectro-Temporal Characterization of the Photoactivation Mechanism of Two New Oxidized Cryptochrome/Photolyase Photoreceptors[END_REF][START_REF] Lacombat | Femtosecond Transient Absorption Study of the Primary Photoreduction Events of a Class II Photolyase[END_REF] Effective fits of the isotropic and anisotropy data were derived from and (ii) make experiments recorded on different days spectrally comparable (minor corrections based on the [Ru(bpy) 3 ] 2+ complex spectra were applied).

the root fits with Equations 1 and 2. The time constants of the fits will be presented in Table 1 and the corresponding isotropic decay-associated spectra (DADS) provided for reference in SI, Section 4.4 (Figure S10). The global fits are however here more conveniently summarized by the evolution-associated difference spectra (EADS), which correspond to a virtual cascading model with unit quantum yield between state i and state i + 1 and increasing lifetimes. [START_REF] Van Stokkum | Global and Target Analysis of Time-Resolved Spectra[END_REF] The polarized EADS were in turn converted into isotropic EADS and evolution-associated anisotropy spectra (EAAS) using Equations 1 and 2. Spectral analysis of some isotropic EADS were performed as previously described. [START_REF] Lacombat | Femtosecond Transient Absorption Study of the Primary Photoreduction Events of a Class II Photolyase[END_REF] The aim is to fit the spectra by a Beer-Lambert sum of contributions from different species (FAD ox , FAD •-, WH •+ , Y • ), the spectra of which are taken from literature (see Figure 1B and SI, Section S5.1). To improve the quality of the fits, the reference spectra were modified with two intertwined parameters, a shift s and a scaling factor u, so that spectrum () becomes '() = (u(-s)). To avoid ambiguities coming from the overlap of reference spectra, fits were preferred where the number of species in the sum could be kept low. In particular, we focused on the difference between consecutive spectra, as follows: EADS i+1 was fitted with EADS i as a first fit component, multiplied by an adjustable factor () understood as the yield of the i → i + 1 phase, plus additional components reflecting composition changes taking place during this step.

Real-time TAS

Transient absorption kinetics on nanosecond to millisecond timescales were measured on two different setups described in detail elsewhere. [START_REF] Müller | ATP Binding Turns Plant Cryptochrome into an Efficient Natural Photoswitch[END_REF][START_REF] Byrdin | Use of Ruthenium Dyes for Subnanosecond Detector Fidelity Testing in Real Time Transient Absorption[END_REF][START_REF] Müller | Photochemistry of Wild-Type and N378D Mutant E. coli DNA Photolyase with Oxidized FAD Cofactor Studied by Transient Absorption Spectroscopy[END_REF] In experiments on the sub-ns/ns timescales, the samples were excited at 355 nm by a Nd:YAG laser (Continuum Leopard SS-10, pulse duration of 100 ps, repetition rate 2 Hz, and pulse energy in the order of 2 mJ per cm 2 ). In all other experiments, the samples were excited at 475 nm by a Nd:YAG-pumped optical parametric oscillator (OPO; Brilliant B/Rainbow, Quantel, France, pulse duration of 5 ns, repetition rate 2 Hz or single flashes, and energy in the order of 5 mJ per cm 2 ). Laser energies were estimated from the amplitudes of signals obtained with the [Ru(bpy) 3 ]Cl 2 actinometer 77 under otherwise identical conditions and geometry.

Monitoring light was provided by continuous-wave lasers listed previously. [START_REF] Müller | Photochemistry of Wild-Type and N378D Mutant E. coli DNA Photolyase with Oxidized FAD Cofactor Studied by Transient Absorption Spectroscopy[END_REF] 2210 mm cells were used (excitation pulses entered the sample through the 210 mm window; monitoring light through the 22 mm window). The monitoring light beams were attenuated by neutral density filters and, for the experiments on ns to s timescales, were mechanically chopped to produce rectangular light pulses of 140 s duration and energy in the order of 1 J at the entrance of the cell, thus avoiding significant actinic effects. These pulses were synchronized with the excitation laser flash (see Ref. [START_REF] Byrdin | Use of Ruthenium Dyes for Subnanosecond Detector Fidelity Testing in Real Time Transient Absorption[END_REF] for more details).

Signals on the sub-ns/ns timescales were recorded using the Alphalas UPD-200-UP photodiode (rise time <175 ps; sensitive area 0.1 mm 2 ; bandwidth 2 GHz), connected directly to the Infiniium 81004B digital oscilloscope (from Agilent; bandwidth 10 GHz). Signals on the ns/µs timescales were recorded using the Alphalas UPD-500-UP photodiode (rise time <500 ps; sensitive area 0.5 mm 2 ; bandwidth 600 MHz), connected via the Femto HCA electronic signal amplifier (DC-325 MHz, 28 dB) to a the Tektronix DSA602A digital oscilloscope with bandwidth limit set to 100 MHz. Signals on the ms/s timescales were amplified by the AM502 differential amplifier (Tektronix) with a bandwidth limited to 100 kHz.

All experiments were carried out at room temperature (25 °C) and the concentration of both WT and Y373F proteins in most experiments was 60 M. Only for experiments on the ns timescale, a higher concentrated (110 M) WT sample was used in order to improve the signal-to-noise ratio (at all wavelengths except 457 nm).

RESULTS

Femtosecond TAS

Isotropic spectral dynamics

The transient absorption spectra of WT-CraCRY, both WT and Y373F mutant, recorded between 300 fs and 3.2 ns after excitation at 475 nm, are presented in Figure S4 of SI, Section S3.1, together with a detailed description of them. In brief, at early pump-probe delays of (e.g. 300 fs), one recognizes the typical features of excited FAD ox (FAD ox *), with a bleaching band at 447 nm, stimulated emission (SE) at 538 nm and excited-state absorption (ESA) bands around 366, 507 nm and over 570 nm. The following evolution may approximately be divided into three phases. From 0.3 to ~2 ps (Figure S4A), the SE band decays and is substituted by a broad positive band, peaking around 600 nm. The changes in this phase resemble closely those observed in several oxidized PCSf members and attributed to primary charge separation forming a FAD •-/WH •+ pair. [START_REF] Martin | Ultrafast Flavin Photoreduction in an Oxidized Animal (6-4) Photolyase through an Unconventional Tryptophan Tetrad[END_REF][START_REF] Brazard | Spectro-Temporal Characterization of the Photoactivation Mechanism of Two New Oxidized Cryptochrome/Photolyase Photoreceptors[END_REF][START_REF] Lacombat | Femtosecond Transient Absorption Study of the Primary Photoreduction Events of a Class II Photolyase[END_REF][START_REF] Immeln | Primary Events in the Blue Light Sensor Plant Cryptochrome: Intraprotein Electron and Proton Transfer Revealed by Femtosecond Spectroscopy[END_REF] Between ~2 and ~230 ps (Figure S4B), all bands decay partially without much change of their shape, indicating some loss of FAD •-/WH •+ pairs by charge recombination. Between ~230 and 3200 ps (Figure S4C), all bands go on decaying partially but one observes that the transient absorption band at around 600 nm decays to much greater extent and becomes increasingly flatter, consistent with data obtained by real-time TAS and tentatively assigned to reduction of WH •+ by Y 373 (see Section 3.2). The evolution of the transient absorption spectra of WT-CraCRY measured in deuterated buffer is essentially identical (SI, Section S3.2).

The Y373F mutant (Figure 2D-F) behaves very similarly to the WT as far as the first phase is concerned but exhibits substantial differences afterwards. Firstly, the amplitude of the decay during the second phase is much smaller than for WT and, secondly, the third phase is almost absent; only a very weak decay of all bands without shape alteration occurs between 80 ps and 3.2 ns.

Kinetic analysis

All polarized transient absorption spectra of CraCRY could be globally fitted with a sum of 4 exponential components and a plateau. Table 1 summarizes the obtained time constants (with errors of fit corresponding to a confidence interval of 95%). An illustration of the quality of such fits may be found in SI, Section S4.1, with selected kinetic traces of both isotropic transient absorption and transient anisotropy of WT in H 2 O. Figure 2 (A,C) shows the isotropic EADS of WT and Y373F in H 2 O buffer (for WT in D 2 O see SI, Section S4.2). The evolution phases qualitatively sketched in Section 3.1.1 are here more precisely defined. The initial excited-state decay occurs in about 0.4 ps for both WT and Y373F. The second phase is divided into two kinetic steps, characterized by time constants of 3.7 and 55 ps for WT (4.9 and 56 ps in D 2 O), and 3.4 and 41 ps for Y373F. These time constants are similar in all cases but the amplitude associated to the 41-ps decay of Y373F is, in relative value, smaller than that of the 55-ps decay of WT (Figure 2A,C). As mentioned above, the fourth decay of WT in 710 ps, with conspicuous flattening of the 600 nm band and decay below 350 nm, is almost absent in Y373F. One observes instead a very small homothetic decrease of all bands, in 630 ps. Figure 3 displays the EADS2-5 of WT and Y373F (H 2 O buffer), normalized at the maximum of the bleaching band (NEADS). This normalization approximately compensates for the transient absorption losses due to the recovery of the initial state by charge recombination. Figure S9 (SI, Section S4.3) presents the same data in one-to-one comparison of the NEADS of WT and Y373F. The effect of the Y373F mutation is clearly seen in those figures to block the prominent spectral changes (decay of the absorption bands around 600 nm and 340 nm, relative absorption increase in the 390-430-nm range), observed in WT during the EADS4→EADS5 kinetic step. Minor changes consisting essentially of a small decay of the broad band around 600 nm observed during the EADS3→EADS4 step in the WT are also blocked by the Y 373 mutation.

Transient anisotropy

The raw transient anisotropy spectra are provided for reference in SI, Section S3. The evolutionassociated anisotropy spectra (EAAS) deduced from the global analysis procedure are shown in Figure 2 (B, D) for WT in H 2 O and in Figure S8B for WT in D 2 O buffer. In brief, the four kinetic phases described above can also be clearly distinguished according to their anisotropy. The initial phase is characterized by very large changes between 500 and 650 nm, due to the decay of SE. Subsequently, and starting with the simpler case of Y373F, one observes marked changes between 330 and 375 nm as well as between ca. 510 and 650 nm, during the 2 nd and 3 rd steps. The last kinetic step is accompanied by minimal alterations of the anisotropy. In the case of WT, the anisotropy changes during the 2 nd step are similar to those of Y373F but differences start to appear during the 3 rd step; The marked decay around 510 nm in Y373F is in particular significantly smaller in WT. During the last kinetic step, an increase is observed for WT at this wavelength while no change occurs in Y373F. The final anisotropy spectrum of WT becomes very noisy above ~700 nm and below 350 nm because the corresponding isotropic spectrum (denominator in Equation 2) approaches here zero value. These regions have been masked in Figure 2 (B,D).

Real-time TAS

Sub-nanosecond to nanosecond timescale

To monitor the photoreactions of CraCRY on the sub-ns to ns timescale, we used a sub-ns TAS setup (see Section 2.3) with excitation pulses at 355 nm (i.e. in the near UV absorption band of FAD ox ). Signals recorded at representative wavelengths for WT CraCRY and its Y373F mutant are shown in Figure 4A and 4B, respectively (for other wavelengths, see Figures S2A andS3A). 405 nm in particular lies close to the expected absorption maximum of the Y • radical (see Figure 1B); note that a strong absorption increase due to FAD reduction from FAD ox to FAD •-is also expected at this wavelength. 457 nm is close to the absorption maximum of FAD ox in the blue in order to monitor the negative absorption changes (bleaching) due to the reduction of FAD ox to FAD •-; note that tyrosine and tryptophan radicals exhibit very weak absorptions at this wavelength so their contributions to the signal are expected to be minor compared to those associated with the redox state changes of FAD. At 488 nm, the neutral W • typically absorbs much stronger ( 2.5 ×) than the protonated WH •+ (see Figure 1B). And finally at 562 nm, where essentially only the tryptophan radicals contribute, the opposite is the case -the molar absorption coefficient of the WH •+ radical is much higher ( 3 ×) than that of the W • radical (see Figure 1B).

For the Y373F mutant (Figure 4B), the high-time-resolution signals show essentially only a very slight decline (that will be resolved on a longer timescale, see Section 3.2.2). For the WT, however, one observes prominent kinetic features already on the ns timescale (Figure 4A). In particular, at 562 nm (red trace in Figure 4A), the signal exhibits a major decay from an initial level comparable to that in the Y373F mutant (when corrected for concentration difference) to 20% of its initial amplitude within a few nanoseconds. Similar signals were observed at 515, 594 and 639 nm (Figure S2A), strongly suggesting that they represent disappearance of a WH •+ radical (see the absorption spectrum of WH •+ in Figure 1B). A global fit of all signals (at all measured wavelengths) with two shared time constants (using Equation S1) yielded τ 1 = 550 ps and τ 2 = 5.6 ns at an amplitude ratio of approx. 3:1 for signals at 515 nm and above (see Figure S2B). The 550 ps phase may contain contributions from even faster processes that could not be resolved at the instrumental time resolution of 300 ps (see above for data obtained by ultrafast spectroscopy).

A priori, disappearance of the WH would form the neutral radical W • and should be accompanied by an absorption increase at 488 nm (as observed in the Y373F mutant; see Figure S3B). Instead, the disappearance of WH •+ within a few nanoseconds in WT CraCRY was accompanied by bleaching at 488 nm (green trace in Figure 4A), excluding deprotonation as major process. Back ET from FAD •-to WH •+ should be accompanied by recovery of the bleaching of the absorption band of FAD ox around 450 nm. A partial recovery in approx.

1 ns was indeed observed at 457 nm (blue trace in Figure 4A), but represents only ~20% of the initial bleaching and can hence only account for ~20% of the disappearance of WH •+ .

ET from Y 373 is hence the most plausible major cause of disappearance of WH 1B) that is superimposed to the strong absorption increase due to the preceding formation of FAD •-(Figure 1B). The WT signal at 405 nm (black trace in Figure 4A) shows a minor (10-15%) absorption increase with kinetics consistent with the slower phase (5.6 ns) of disappearance of WH •+ observed at  > 500 nm. Within the first 1 ns after excitation, there was, however, a slight absorption decrease at 405 nm. This is likely to be the result of an overcompensation of a rise by partial decay of the strong absorption of FAD •-due to its recombination with WH •+ (see Discussion).

ACS Paragon Plus Environment

Journal of the American Chemical Society

Because of the likely involvement of tyrosine deprotonation, we followed the kinetics of WH •+ in WT CraCRY also at other pH values. Figure S2C shows the corresponding 562 nm signals at pH 6.5, 7.5, 8.4

(the "standard" pH for all other experiments shown here) and 9.0. All signals exhibit essentially the same decay kinetics and also the same amplitudes of the decay (see SI, Section S2.2 for further details).

Nanosecond to second timescale

To complete our observations on the ns to s timescale, we used a TAS setup with 5 ns time resolution (see Section 2.3), with excitation pulses tuned at 475 nm. In the case of the Y373F mutant (Figure 5 C and D), all signals exhibit a biphasic and complete decay (τ 1 ~ 260 ns, τ 2 ~ 10 µs). At the beginning of the time window of Figure 5C, the amplitude of the signal at 562 nm amounts to ~60 % of the signal at 457 nm and even after the first 260 ns phase, this number still remains as high as ~45 % (Figure 5 C andD). This indicates that tryptophan radicals are present until the decay is completed (in ~70 µs). In most members of the PCSf that contain the canonical tryptophan triad, the distal (3 rd ) WH •+ radical deprotonates in several hundreds of nanoseconds, [START_REF] Aubert | Intraprotein Radical Transfer During Photoactivation of DNA Photolyase[END_REF][START_REF] Müller | ATP Binding Turns Plant Cryptochrome into an Efficient Natural Photoswitch[END_REF] in competition with recombination with FAD •-. [START_REF] Müller | ATP Binding Turns Plant Cryptochrome into an Efficient Natural Photoswitch[END_REF] This also seems to be the case for the Y373F mutant of CraCRY: While the 260 ns phase represents ~30% of the signal decay at 405 and 457 nm (essentially due to FAD •-/WH •+ recombination), it represents ~50% at 562 nm (and even more at higher wavelengths) where both recombination and deprotonation of WH •+ contribute (Figs. 6A andS3). The intrinsic time constants for WH •+ deprotonation and FAD •-/WH •+ recombination derived from these data are ~400 ns and ~850 ns, respectively. The formed FAD •-/W • radical pair decays completely with a time constant of 10 µs (see Figure 5D and Figure S3C), most likely by back ET coupled to re-protonation of the tryptophan. [START_REF] Müller | Photochemistry of Wild-Type and N378D Mutant E. coli DNA Photolyase with Oxidized FAD Cofactor Studied by Transient Absorption Spectroscopy[END_REF] Similarly, in the WT, the much slower FAD S2D and SI Section S2.2). The visible starting decay at the end of the blue trace in Figure S2D indicates that the lifetime of the FADH/Y 373 • pair is in the order of several seconds at pH 6.5.

DISCUSSION

Our experimental results are discussed below in chronological order of the photoinduced events during the light activation of CraCRY, focusing, however, on kinetics and mechanism of fast and efficient tyrosine oxidation that has no precedent in other members of the PCSf and is highly relevant for the understanding of proton coupled electron transfer (Section 4.2). A tentative reaction scheme is provided to facilitate the discussion (Scheme 1).

Reactions preceding tyrosine oxidation

Nature and kinetics of the photoproducts

Starting with the simpler case of Y373F, we find that the isotropic EADS (Figure 2C) are reminiscent of those previously reported for other members of the PCSf [START_REF] Martin | Ultrafast Flavin Photoreduction in an Oxidized Animal (6-4) Photolyase through an Unconventional Tryptophan Tetrad[END_REF][START_REF] Brazard | Spectro-Temporal Characterization of the Photoactivation Mechanism of Two New Oxidized Cryptochrome/Photolyase Photoreceptors[END_REF][START_REF] Lacombat | Femtosecond Transient Absorption Study of the Primary Photoreduction Events of a Class II Photolyase[END_REF] and, more specifically, highly similar to those of the W370F mutant of the 6-4 photolyase from Xenopus laevis (Xl64). [START_REF] Martin | Ultrafast Flavin Photoreduction in an Oxidized Animal (6-4) Photolyase through an Unconventional Tryptophan Tetrad[END_REF] In the latter mutant, the aromatic tetrad serving as ET chain in the WT is analogously reduced to the classical tryptophan triad, as in the Y373F mutant of CraCRY. Accordingly, EADS1 can be straightforwardly assigned to FAD ox * and EADS2 to the FAD •-/WH •+ radical pair formed by photoinduced ET from the proximal tryptophan (W 399 in CraCRY) to FAD ox * in 0.43 ps. EADS3-5 reflect FAD •-/WH •+ states, in which the oxidation hole has migrated along the tryptophan triad (in competition with charge recombination), or relaxed states. In the present case, the initial photoreduction of the flavin produces a particular spectrum (EADS2), characterized by a very flat profile between ca. 500 and 620 nm. This shape becomes rounder in the EADS2EADS3 transition (3.4 ps), and a clearer shallow maximum builds up around 600 nm, as expected for a WH •+ radical. [START_REF] Solar | Oxidation of Tryptophan and Nmethylindole by N 3 • , Br 2 •-, and (SCN) 2 •-Radicals in Light-Water and Heavy-Water Solutions: A Pulse Radiolysis Study[END_REF] As mentioned in the case of Xl64, [START_REF] Martin | Ultrafast Flavin Photoreduction in an Oxidized Animal (6-4) Photolyase through an Unconventional Tryptophan Tetrad[END_REF] and following a hypothesis proposed by Immeln et al. for a plant cryptochrome, [START_REF] Immeln | Primary Events in the Blue Light Sensor Plant Cryptochrome: Intraprotein Electron and Proton Transfer Revealed by Femtosecond Spectroscopy[END_REF] this evolution might be attributed to the cooling of WH •+ in a few ps, assuming this radical is initially produced in a hot state by the very fast primary photoreduction of FAD ox . Alternatively, one might attribute the unique shape of EADS2 to the proximal tryptophanyl radical (W 399 H •+ ), which would supposedly have a distinct absorption spectrum because of its specific environment, i.e. deeply buried inside the protein and in close Coulombic interaction with the anionic semiquinone radical (FAD •-). According to this hypothesis, the change of shape between EADS2 and EADS3 would have to be assigned to a migration of the oxidation hole (WH •+ ) along the ET chain. The transitions EADS3EADS4 (41 ps) and EADS4EADS5 (630 ps) represent only very weak absorption changes in the Y373F mutant.

In the case of WT, the main difference with respect to the Y373F mutant is the final step (710 ps), which leads to a strongly decreased absorption in the red part of the spectrum (see normalized EADS5 in Figure 3). We have assigned this step to proton-coupled ET from Y 373 to the terminal tryptophanyl cation radical W 322 H •+ , yielding a long-lived FAD •-/Y • radical pair (see Section 4.2). We recall that spectral changes not seen in Y373F already arise during the 55-ps phase of WT (see Figure 3 and Figure S9). This observation will be discussed below.

In order to establish more firmly the nature of EADS2 and EADS3, we fitted those spectra with a linear combination of reference spectra of FAD •-and WH •+ , suitably scaled and shifted (see Section 2.2.2 and Equation S4 in SI, Section S5.2), and complemented by the negative FAD ox bleaching contribution. The modification imposed on the FAD •-reference spectrum (Figure S13, SI, Section S5.2) is relatively small, while the WH •+ spectrum needs to be markedly shifted to the red (~20 nm). Such a shift, likely due to the local environment of the tryptophanyl radical inside the protein, has been reported before also for other PCSf proteins. [START_REF] Martin | Ultrafast Flavin Photoreduction in an Oxidized Animal (6-4) Photolyase through an Unconventional Tryptophan Tetrad[END_REF][START_REF] Lacombat | Femtosecond Transient Absorption Study of the Primary Photoreduction Events of a Class II Photolyase[END_REF] The fits shown in Figure S12 are not perfect because the simple alterations imposed on the reference spectra cannot completely reproduce the actual FAD •-and WH •+ spectra inside CraCRY. They nevertheless sufficiently support the hypothesis that EADS2 and EADS3 result from the photoinduced reduction of FAD ox by a tryptophan.

One may finally note for both WT and Y373F that the blue absorption and bleaching bands of EADS2 and EADS3 can be nearly superimposed (Figure 2). This suggests that almost no charge recombination of the FAD •-/WH •+ pair, expected to induce a loss of transient absorption intensity, takes place during the second kinetic phase. The main variation occurs in the red part of the spectrum, which was above suggested to derive either from a cooling of the initial WH •+ radical or from the migration of the oxidation hole to a different tryptophan.

As previously mentioned, the spectral evolution from EADS3 to EADS4 in WT is nearly absent in Y373F (see Figure 3). Since a significant decay of the relative amplitude is observed around 600 nm, the hypothesis of tyrosine oxidation during the 55-ps step has been considered but finally set aside for lack of convincing evidence (see Section 4.2.1 and SI, Section S5.4). Alternatively, the EADS3→EADS4 spectral evolution of WT could be interpreted in terms of migration of the oxidation hole to W 322 and the building of a W 322 H •+ population characterized by a specific (weaker) absorption in the presence of Y 373 (see SI, Section S5.4). As mentioned above, the 55-ps step is accompanied by partial charge recombination. According to the analysis in SI, Section 5.4, the corresponding yield is 23% (for this step only). However, given that the population losses during the preceding steps are probably small (extremely fast step one and almost no evolution of the bleaching band during step two), this number is likely close to the net yield of charge recombination counted from the initially excited flavin.

Migration of the oxidation hole along the Trp triad

In this section, we intend to use the anisotropy data to assign the WH •+ spectroscopic signatures detected in the transient absorption spectra to specific residues. Following an approach previously described in Refs. [START_REF] Martin | Ultrafast Flavin Photoreduction in an Oxidized Animal (6-4) Photolyase through an Unconventional Tryptophan Tetrad[END_REF][START_REF] Lacombat | Femtosecond Transient Absorption Study of the Primary Photoreduction Events of a Class II Photolyase[END_REF] , we chose to probe WH •+ around 610 nm, expecting under these conditions that essentially a single transition of WH •+ is observed. Table 2 gathers the corresponding values of the EAAS (noted r i ), after initial decay of the excited state (averaged over 5 nm). Within experimental errors (of the order of ±0.01), the r i 's of WT and Y373F are rather similar, with a slightly larger difference for r 4 .

At this point, it is interesting to compare the experimental anisotropies to predictions based on the crystal structure of WT-CraCRY (PDB entry 5ZM0) [START_REF] Franz | Structure of the Bifunctional Cryptochrome aCRY from Chlamydomonas Reinhardtii[END_REF] and on known directions of the transition dipole moments of FAD ox and WH •+ , as previously described. [START_REF] Martin | Ultrafast Flavin Photoreduction in an Oxidized Animal (6-4) Photolyase through an Unconventional Tryptophan Tetrad[END_REF][START_REF] Lacombat | Femtosecond Transient Absorption Study of the Primary Photoreduction Events of a Class II Photolyase[END_REF] These predictions may however not be fully comparable to the experimental anisotropies because a small contribution of FAD •-could be present at 610 nm, as suggested by the fact that EADS5 keeps a positive value in this region in spite of the decay of WH •+ . Such a red tail of the FAD •-spectrum has been reported before. [START_REF] Massey | On Existence of Spectrally Distinct Classes of Flavoprotein Semiquinones . A New Method for Quantitative Production of Flavoprotein Semiquinones[END_REF][START_REF] Berndt | A Novel Photoreaction Mechanism for the Circadian Blue Light Photoreceptor Drosophila Cryptochrome[END_REF] From EAAS5 of WT in D 2 O (less noisy than in H 2 O), and neglecting the small amount of W 322 H •+ reduced in 5 ns, the anisotropy of FAD •-at 610 nm is estimated to be of the order of -0.1. If both WH •+ and FAD •-contribute to the experimental anisotropy, the latter should be decomposed as follows: [START_REF] Byrdin | Polarized Transient Absorption to Resolve Electron Transfer between Tryptophans in DNA Photolyase[END_REF] (Eq. 3)

𝑟 exp = 𝑥 𝑟 W + (1 -𝑥) 𝑟 F
where r W and r F are the pure anisotropies respectively attached to WH •+ and FAD •-and x is the relative contribution of WH •+ to the total isotropic signal. Taking x = 0.8 as a crude lower limit (a value of around 0.83 may be obtained from the modified WH •+ and FAD •-of Figure S9), one gets new values listed in Table 3 (line correction), which provide a qualitative idea of the effect of the FAD •-contribution on the predictions.

It first appears that r 2 is relatively close (within errors) to the predicted anisotropy of W 399 H •+ that is expected to be produced after primary reduction of the flavin by the proximal W 399 residue. The possible involvement of W 401 in the first kinetic step is very unlikely since it is placed at a much larger distance of FAD (7.9 Å; Figure 1A) than W 399 (4.0 Å). In EcCPDI where the equivalent tryptophan (W 384 ) is only at 6.3 Å from FAD, Liu et al. found that the ET from W 384 to FAD ox * takes place at a rate of (80 ps) -1 in a mutant lacking the proximal tryptophan. [START_REF] Liu | Determining Complete Electron Flow in the Cofactor Photoreduction of Oxidized Photolyase[END_REF] An even slower rate is to be expected in CraCRY.

During the next step, the anisotropy changes sign and r 3 becomes positive as expected for W 376 H •+ , the medial aromatic residue of the canonical tryptophan triad, however, without reaching its predicted value. Since W 332 is situated at a very close distance from W 399 (3.8 Å; Figure 1A), it is possible that W 332 effectively competes with W 376 for the reduction of W 399 H •+ , as previously reported for EcCPDI by Li et al. [START_REF] Liu | Determining Complete Electron Flow in the Cofactor Photoreduction of Oxidized Photolyase[END_REF] The resulting W 332 H •+ , expected to have a slightly less positive anisotropy than W 376 H •+ (Table 3), is then thought to contribute to the observed r 3 . In addition, the driving force of ET from W 376 to W 399 H •+ may be small, as claimed by Li et al., [START_REF] Liu | Determining Complete Electron Flow in the Cofactor Photoreduction of Oxidized Photolyase[END_REF] for the equivalent residues in EcCPDI, so that r 3 might be diminished by a negative contribution from W 399 H •+ still present at the end of the second ET step.

Finally, one may also hypothesize that r 3 takes a negative contribution from the distal W 322 H •+ , meaning that delocalization of the oxidation hole has already reached the last tryptophan of the triad at this step of the reaction. In any case, it appears that the oxidation hole is never wholly localized on W 376 .

The values of r 4 and r 5 are more readily understood in the case of Y373F. We see that r 4 is quite close to the expectation for W 322 H •+ , and that the match becomes even better for r 5 . This suggests that the third kinetic step (41 ps in Y373F) results in a dominant localization of the oxidation hole on the W 322 site. The last step (630 ps) could either sign the final localization of the oxidation hole on W 322 , if it were not already completed and/or a rearrangement of the protein that stabilizes the FAD •-/W 322 H •+ pair (possibly required for a complete localization of the oxidation hole on W 322 ) and eventually blocks the weak charge recombination channel still observed during this step. In the case of WT, the fourth and possibly third kinetic steps are marked by the oxidation of the Y 373 residue. Since Y • does not contribute at 610 nm, Equation 3 would still apply but the x ratio would be smaller than in the absence of Y • . In the limiting case of r 5 , one may assume that the amount of WH •+ is relatively small and take x = 0 as a rough approximation. This leads to the estimation of r F mentioned above (-0.1; from WT in D 2 O). The case of r 4 is interesting because Equation 3 would predict a value situated somewhere between ca. -0.06 and -0.1 (depending on x) provided the oxidized tryptophan is W 322 (see Table 3, line raw). The fact that r 4 has a less negative value than that (Table 2; WT) suggests that the localization of the oxidation hole on W 322 is not finished at that step, and that other oxidized tryptophans contribute to this value, in particular W 376 H •+ because of its positive expected anisotropy. This consequently means that oxidation of Y 373 begins while there is still a certain degree of delocalization over different tryptophans. It might reciprocally be speculated that the replacement of tyrosine by phenylalanine somehow enhances protein and water reorganization leading to full localization of the oxidation hole on W 322 during the third kinetic step, as observed for Y373F.

Oxidation of tyrosine Y 373

Kinetics and spectral changes

In our attempts to establish the kinetics and the mechanism of oxidation of tyrosine Y 373 in CraCRY, we focused on kinetic and spectral features that depended on the presence of this tyrosine residue, i.e.

were absent or strongly modified in the Y373F mutant.

Our results obtained by both pump-probe and real-time TAS agree that reduction of a tryptophan cation radical WH reduction phase with a time constant of ~5 ns and approx. one third of the amplitude of the ~600-700 ps phase. Neither variation of the pH (between 6.5 and 9.0; Figure S2C) nor use of a deuterated buffer solution (Figure S8) had a significant effect on the reduction kinetics of WH •+ .

Looking for a direct evidence of ultrafast tyrosine oxidation, we used the spectrally-resolved ultrafast S2) and the modified reference spectra are presented in Figure 6C. The WH •+ and Y • spectra providing the best fit of our experimental data are found to be both red-shifted relative to solution spectra, by ~25 and 8 nm, respectively (Figure 6C). It is worth noting that a 9-nm red shift of the Y • spectrum, as compared to a reference spectrum in water, was also reported by Oldemeyer et al. in their time-resolved spectroscopic study. [START_REF] Oldemeyer | Essential Role of an Unusually Long-Lived Tyrosyl Radical in the Response to Red Light of the Animal-Like Cryptochrome aCRY[END_REF] The fit also indicates that FAD •-/WH •+ recombination (competing with the oxidation of Y 373 ) occurs with a yield of 22%. We similarly attempted to fit EADS4 according to Equation S5 to check the hypothesis of early oxidation of Y 373 (see Section 4.1.1). This approach however led to a conflicting result, a large blue shift of the modified Y • spectrum, and was not considered conclusive (see more details in SI, Section S5.4).

The kinetics of tyrosine oxidation should show up in our sub-ns/ns real time TAS data at 405 nm (within the absorption band of Y • ). The absorption rise due to the formation of Y • may, however, be compensated of even overcompensated by the partial recombination of FAD •-/WH •+ that is accompanied by an absorption decrease at 405 nm (see spectra in Figure 1B). Such a compensation is apparent in the ultrafast TAS data: while the uncorrected amplitudes around 400 nm slightly decrease from 10 ps to 3.2 ns, (Figure S4 (B,C) and Figure S7), there is an increase after correction for recombination of FAD •-/WH •+ (compare NEADS3, 4 and 5 in Figure 3A). Similarly, no rise with τ ≈ 600 ps was observed by real-time TAS at 405 nm (black trace in Figure 4A). A minor rise with τ ≈ 5 ns, in line with the slower phase of WH •+ reduction, is however visible.

In an attempt to reconstruct the kinetics of tyrosine oxidation from the real-time TAS trace at 405 nm by correcting for the FAD •-contribution, we assumed that essentially only FAD ox , FAD •-and Y • contribute at 405 nm (neglecting the weak contribution from WH •+ ; see Figure 1B) and that the kinetics of FAD •-population is represented by the kinetics of the bleaching at 457 nm (blue trace in Figure 4A;

here we also neglect a weak contribution from WH •+ ). Subtraction of the 457 nm trace (inverted and rescaled to the same initial amplitude) from the 405 nm trace yielded a "corrected" 405 nm signal (Figure 6A) that we consider to be a coarse approximation of the kinetics of tyrosine oxidation (see SI, Section S2.1 for more details on how the corrected 405 nm signal was constructed). A biexponential fit of the corrected signal (using Equation S2) yielded time constants of 820 ps (with an amplitude of ~6 mOD) and 6.0 ns (with an amplitude of ~3 mOD). The tyrosine oxidation hence follows essentially the same biphasic kinetics as WH •+ reduction (~600-700 ps and ~5 ns) observed above 500 nm, confirming that Y 373 is oxidized by a tryptophanyl cation radical, most likely the distal W 322 H •+ .

Let us finally emphasize that, according to our spectral analysis (Figure 6B), the oxidized tyrosine is observed in its deprotonated Y • form. The protonated YH •+ radical cation has recently been reported in a flavoenzyme to have a strongly displaced spectrum around 490 nm [START_REF] Nag | Identification of the TyrOH •+ Radical Cation in the Flavoenzyme TrmFO[END_REF] , which would not match our observations (see also the lack of induced absorption at 488 nm in the real-time TAS data; green trace in Figure 4A). Tyrosyl deprotonation is thus intimately involved in the process that must be considered a case of proton-coupled electron transfer (PCET). [START_REF] Hammes-Schiffer | Theory of Coupled Electron and Proton Transfer Reactions[END_REF] It should be noted that, according to the above analyses, the observed 600-700 ps time constant results from a competition between reduction of WH •+ by PCET from Y 373 and approximately four times slower recombination with FAD •-. The intrinsic rate constant of the major phase of PCET would thus be of the order of (800-900 ps) -1 . We will adopt an approximate value of (800 ps) -1 for the discussion of the PCET mechanism presented in the following section.

Reaction mechanism of tyrosine oxidation

As discussed above, the reduction of WH •+ by PCET from Y 373 in CraCRY mostly proceeds with an intrinsic rate constant of (800 ps) -1 . This is substantially faster than the fastest phase of oxidation of the so-called tyrosine-Z in oxygen-evolving PSII (τ  30 ns). Remarkably, in both cases the reaction kinetics was found to be independent of pH (between 6.5 and 9.0 in CraCRY (Figure S2C) and between 4.5 and 7.5 in PSII [START_REF] Meyer | O 2 Evolution and Chl a + II (P-680 + ) Nanosecond Reduction Kinetics in Single Flashes as a Function of pH[END_REF] )

and was not significantly affected by deuteration of the buffer solution (Table 1). [START_REF] Karge | Effects of Hydrogen Deuterium Exchange on Photosynthetic Water Cleavage in Ps II Core Complexes from Spinach[END_REF][START_REF] Haumann | Photosynthetic Oxygen Evolution: H/D Isotope Effects and the Coupling between Electron and Proton Transfer During the Redox Reactions at the Oxidizing Side of Photosystem II[END_REF][START_REF] Schilstra | Proton/Hydrogen Transfer Affects the S-state-Dependent Microsecond Phases of P680 + Reduction During Water Splitting[END_REF] The structure of CraCRY (Figure 1A) strongly suggests that the electron acceptor is W 322 H + , the distal member of the canonical Trp triad, because it is located at only 3.8 Å edge-to-edge distance from Y 373 .

The proton acceptor is likely to be aspartate D 321 that forms a hydrogen bond with the Y 373 hydroxyl group (3.0 Å; Figure 1A). [START_REF] Franz | Structure of the Bifunctional Cryptochrome aCRY from Chlamydomonas Reinhardtii[END_REF] In the following, we discuss possible reaction mechanisms in the framework of limiting cases of PCET following the classification by Hammes-Schiffer and Stuchebrukhov [START_REF] Hammes-Schiffer | Theory of Coupled Electron and Proton Transfer Reactions[END_REF] and Liu et al. [START_REF] Liu | Accelerating Proton-Coupled Electron Transfer of Metal Hydrides in Catalyst Model Reactions[END_REF] The reaction pathways considered for CraCRY and our notations are shown in Scheme 1. We note that based on published reduction potentials and pK a values in aqueous solution of the amino acid side chains involved, namely E°(WH [START_REF] Dixon | Determination of Acidity Constants of Some Phenol Radical Cations by Means of Electron-Spin Resonance[END_REF] pK a (YH/Y -) ≈ 9.9, [START_REF] Tommos | De Novo Proteins as Models of Radical Enzymes[END_REF] and pK a (DH/D -) = 3.9 [START_REF] Grimsley | A Summary of the Measured pK Values of the Ionizable Groups in Folded Proteins[END_REF] , the overall PCET reaction would be downhill by 0.07 eV. ET as the first step would, however, be uphill by 0.28 eV and PT as the first step even by 0.35 eV. As discussed below, these values may be modified considerably by the protein environment and by interactions between the species.

•+ /WH) ≈ 1.07 V vs. NHE, 54 E°(YH •+ /YH) ≈ 1.35 V, 54 E°(Y • /Y -) ≈ 0.65 V, 54 pK a (YH + /Y  ) ≈ -2,
(I) Sequential ET/PT (blue pathway in Scheme 1)

(Ia) PT is "fast" (k PT >> k BET )
PT takes immediately place when the "activated" state

[W 322 H Y 373 H •+ •••D 321 -] is reached by uphill ET.
The uphill ET is hence rate limiting.

k PCET = k FET (Ib) PT is "slow" (k PT << k BET )
Several ET "attempts" are needed to complete the reaction by PT. In other words, PT occurs from an ET pre-equilibrium in which the "activated" state [W 322 H Y 373 H •+ •••D 321 -] has low population.

k PCET = k PT K ET with K ET = k FET /k BET = exp(-G°E T /k B T) (II) Sequential PT/ET (red pathway in Scheme 1) (IIa) ET is "fast" (k ET >>k BPT )
The ET takes place immediately when the "activated" state [W 322 H •+ Y 373 -•••HD 321 ] is reached by the uphill PT. The uphill PT is hence rate limiting.

k PCET = k FPT (IIb) ET is "slow" (k ET <<k BPT )
Several PT "attempts" are needed to complete the reaction by the ET. In other words, the ET occurs from a PT pre-equilibrium in which the "activated" state

[W 322 H •+ Y 373 -•••HD 321 ] has low population. k PCET = k ET K PT with K PT = k FPT /k BPT = exp(-G°P T /k B T) (III) Concerted PCET (green pathway in Scheme 1)
ET and PT occur together in one step, avoiding formation of a high energy intermediate.

No simple rate equation can be given.

Among these cases, Ib and IIa are expected to show a significant normal H/D KIE because the rate of forward PT enters directly into the rate equation. For concerted PCET (case III), a substantial normal KIE is considered to be a hallmark. [START_REF] Hammes-Schiffer | Theory of Coupled Electron and Proton Transfer Reactions[END_REF] † † Because of the absence of a significant KIE for W 322 H •+ reduction by Y 373 in CraCRY, we focus on the two remaining cases.

For sequential ET/PT with "fast" PT (case Ia), the overall rate is given by the (uphill) forward ET. The expected rate of this ET step at room temperature may be estimated using the following semi-empirical expression for the rate (in s -1 ) of endergonic ET in proteins at room temperature: 89 𝑘 𝑒𝑛 ET (Eq. 4)

log 𝑘 𝑒𝑛 ET = 13.0 -(1.2 -0.8𝜌)(𝑅 -3.6) -3.1( - 𝐺°+  ) 2 /  - 𝐺°/0.06
where R is the edge-to-edge distance between electron donor and acceptor (in Å), G° the (positive) standard Gibbs free energy (in eV) of the ET and  the reorganization energy (in eV).  represents the packing density of protein atoms in the volume between electron donor and acceptor. † † A recent theoretical work [START_REF] Hazra | Isotope Effects on the Nonequilibrium Dynamics of Ultrafast Photoinduced Proton-Coupled Electron Trasfer Reactions in Solution[END_REF] presented special cases with negligible KIE, in which highly excited (several eV) electron-proton vibronic states dictated the dynamics. Such highly excited states are not expected to play a role in PCET between tyrosine and tryptophan in CraCRY.

Using R = 3.8 Å (Figure 1A), G° = 0.28 eV (see above),  = 1 eV (considered as "unremarkable" in Ref. 89 and  = 0.76, an average value for ET reactions in proteins, [START_REF] Page | Natural Engineering Principles of Electron Tunnelling in Biological Oxidation-Reduction[END_REF] Equation 4 yields = (250 ns) -1 , i.e. 𝑘 𝑒𝑛 ET more than two orders of magnitude slower than the rate of ~(800 ps) -1 as deduced from the observed kinetics (see above). The somewhat arbitrarily chosen value of  is not critical in this evaluation because the term (R -3.6) is small. The also not well known  has more impact, but even when choosing the optimal value  = G°, the calculated rate of (6.1 ns) -1 is still slower by an order of magnitude than the observed rate. Most critical in this evaluation is the endergonic degree of the reaction. Keeping  = 1 eV, a value of G° = 0.0642 eV would be required to get a rate of (800 ps) -1 , i.e. far below the value of 0.28 eV estimated above. We however do not exclude that the protein environment and the interactions between the species may have such a strong effect on the energetics, for the following reasons:

(1) The reduction potential of WH •+ /WH embedded in the protein matrix is expected to be higher than in aqueous solution (1.07 V) because the positive charge is less stabilized in a low dielectric environment. E.g., the reduction potentials of members of the canonical Trp triad in oxidized EcCPDI were estimated to ≥ 1.48, 1.41 and 1.24 V vs NHE 37 in the order of increasing distance from FAD, similar to estimations that can be deduced from the energetics in semi-reduced EcCPDI. [START_REF] Byrdin | Quantum Yield Measurements of Short-Lived Photoactivation Intermediates in DNA Photolyase: Toward a Detailed Understanding of the Triple Tryptophan Electron Transfer Chain[END_REF] The large drop from the medial to the distal Trp in EcCPDI can be understood as a stabilization of the positive charge by the proximity of the aqueous phase [START_REF] Byrdin | Quantum Yield Measurements of Short-Lived Photoactivation Intermediates in DNA Photolyase: Toward a Detailed Understanding of the Triple Tryptophan Electron Transfer Chain[END_REF][START_REF] Liu | Determining Complete Electron Flow in the Cofactor Photoreduction of Oxidized Photolyase[END_REF] and may be less Case Ia further requires that the subsequent PT is much faster than back ET (k PT >>k BET ). Back ET would be exergonic and its rate may be estimated by Equation 5. [START_REF] Page | Natural Engineering Principles of Electron Tunnelling in Biological Oxidation-Reduction[END_REF] (Eq. 5) log 𝑘 𝑒𝑥 ET = 13.0 -(1.2 -0.8𝜌)(𝑅 -3.6) -3.1(  𝐺°+  ) 2 /  Using G° = -0.0642 eV,  = 1 eV and the other parameters as above, one obtains

(68 ps) - 𝑘 BET = 𝑘 𝑒𝑥 ET = 1 .
Based on the solution pK a values given above, PT from Y 373 H •+ to D 321 -would be downhill by 0.35 eV.

The PT distance would be at least 1.0 Å based on the 3.0 Å hydrogen bond length in the crystal structure (Figure 1A) and assuming an O-H distance of 1.0 Å both in the reactant Y 373 H •+ and in the product D 321 H.

Proton transfer in hydrogen bonds can be as fast as tens of femtoseconds over a distance of approx. ‡ ‡ Our data by ultra-fast spectroscopy indicate that W 322 H + is less stabilized in the WT than in the Y373F mutant; see Section 4.1.2. 0.6 Å in the excited state of 1-hydroxy-2-acetonaphtone [START_REF] Kim | Excited State Intramolecular Proton Transfer Dynamics of 1-Hydroxy-2-Acetonaphthone[END_REF] or at most a few picoseconds over approx. 0.7 Å (O-H•••O hydrogen bond length of 2.7 Å ) in green fluorescent protein [START_REF] Chattoraj | Ultra-Fast Excited State Dynamics in Green Fluorescent Protein: Multiple States and Proton Transfer[END_REF][START_REF] Shinobu | Proton Wire Dynamics in the Green Fluorescent Protein[END_REF] but depends very strongly on the transfer distance, decreasing typically by one order of magnitude per 0.07 Å. [START_REF] Krishtalik | The Mechanism of the Proton Transfer: An Outline[END_REF] It is hence questionable that proton transfer from Y 373 H •+ to D 321 -can be faster than back ET from W 322 to Y 373 H •+ . We note, however, that formation of Y 373 H •+ may induce structural changes (e.g. due to electrostatic interaction with D 321 -) that decrease the proton transfer distance relative to the (dark state) crystal structure.

For sequential PT/ET with "slow" ET (case IIb), the overall rate is determined by the product of the PT (pre-)equilibrium constant K PT and the rate constant k ET of the subsequent ET. There should be no significant KIE, [START_REF] Hammes-Schiffer | Theory of Coupled Electron and Proton Transfer Reactions[END_REF] because the effects on the forward and backward PT are usually similar and cancel each other out in the rate expression. § § Based on the solution values given above, PT from Y 373 H to D 321 -would be uphill by G°P T(1) = 0.35 eV and K PT = 10 -6 . Subsequent ET from Y 373 -to W 322 H •+ would be downhill by 0.42 eV, yielding an ET rate of (1.5 ps) -1 according to Equation 5 with the other parameters as above, and an overall rate of (1.5 µs) -1 .

Assuming the optimal reorganization energy  = -G° = 0.42 eV, the ET rate would be (130 fs) -1 and the overall rate (130 ns) -1 , still very far off the experimental rate of (800 ps) -1 . To obtain the rate of (800 ps) -1 , the PT (pre-)equilibrium constant would have to be > 1.610 -4 , corresponding to G°P T < 0.22 eV and a pK a difference < 3.8 (as compared to 6.0 according to solution values; see above). We would not exclude such a strong modification of the PT equilibrium, because:

(1) pK a values of amino acid side chains in proteins can deviate substantially from solution values. [START_REF] Grimsley | A Summary of the Measured pK Values of the Ionizable Groups in Folded Proteins[END_REF] (2) The presence of W 322 H + close to Y 373 is likely to stabilize Y 373 -by electrostatic interaction and hence to decrease the endergonicity of PT from Y 373 to D 321 -.

Case IIb further requires that (downhill) back PT is much faster than ET (k ET <<k BPT ), i.e. well in the subpicosecond range. As discussed above for case Ia, it is questionable that such a fast proton transfer is possible at the distance in the (dark-state) crystal structure. One should, however, not exclude the possibility that formation of W 322 H •+ induces a shortening of the hydrogen bond between Y 373 and D 321 .

Our experimental data indicated that a minor fraction of Y 373 was oxidized at a slower rate of (5 ns) -1 . This may reflect some conformational heterogeneity of CraCRY that affects ET or PT distances or energetics, but may also be explained by an energetic relaxation of W 322 H + due to reorganization of § § A remarkable exception was recently reported for PCET involving a metal hydride with an appended pyridine base: a substantial inverse KIE was observed and attributed to an isotope effect on the PT (pre)equilibrium constant due to differences in the zero point energy of the metal-H and H + -pyridine vibrations. [START_REF] Liu | Accelerating Proton-Coupled Electron Transfer of Metal Hydrides in Catalyst Model Reactions[END_REF] the protein environment that occur in competition with the oxidation of Y 373 in 800 ps (large white arrow in Scheme 1). Relaxed W 322 H + would have a lower reduction potential, and hence, for sequential ET/PT (case Ia), ET would be more uphill and the overall reaction would slow down. In the sequential PT/ET scenario (case IIb), ET would be less downhill and the overall reaction would slow down as well (except if ET occurred in the Marcus inverted region (i.e. -G° > ), which is highly unlikely given the moderate driving force). The free energy decrease due to relaxation would have to be in the order of 0.07 and 0.2 eV in cases Ia and IIb, respectively, to account for a slowdown of the overall reaction to ~5 ns.

We finally would like to compare tyrosine oxidation in CraCRY with oxidation of tyrosine-Z by P680 + in oxygen evolving PSII that has been studied experimentally for 40 years and is still the subject of controversial discussions. [START_REF] Tommos | Proton and Hydrogen Currents in Photosynthetic Water Oxidation[END_REF][START_REF] Sjödin | Proton-Coupled Electron Transfer from Tyrosine in a Tyrosine-Ruthenium-Tris-Bipyridine Complex: Comparison with Tyrosine z Oxidation in Photosystem II[END_REF][START_REF] Rappaport | Probing the Coupling between Proton and Electron Transfer in Photosystem II Core Complexes Containing a 3-Fluorotyrosine[END_REF][START_REF] Hammarström | Proton-Coupled Electron Transfer of Tyrosines in Photosystem II and Model Systems for Artificial Photosynthesis: The Role of a Redox-Active Link between Catalyst and Photosensitizer[END_REF][START_REF] Barry | Proton Coupled Electron Transfer and Redox Active Tyrosines in Photosystem II[END_REF][START_REF] Schlodder | Temperature Dependence of the Oxidation Kinetics of Tyr(Z) and Tyr(D) in Oxygen-Evolving Photosystem II Complexes Throughout the Range from 320 K to 5 K[END_REF] The fastest kinetics in PSII has a time constant of 30 ns [START_REF] Van Best | Kinetics of Reduction of the Oxidized Primary Electron Donor of Photosystem II in Spinach Chloroplasts and in Chlorella Cells in the Microsecond and Nanosecond Time Ranges Following Flash Excitation[END_REF][START_REF] Brettel | Nanosecond Reduction Kinetics of Photooxidized Chlorophylla II (P-680) in Single Flashes as a Probe for the Electron Pathway, H + -Release and Charge Accumulation in the O 2 -Evolving Complex[END_REF][START_REF] Gerken | Optical Characterization of the Immediate Electron Donor to Chlorophyll A π + in O 2 -Evolving Photosystem II Complexes: Tyrosine as Possible Electron Carrier between Chlorophyll A π and the Water-Oxidizing Manganese Complex[END_REF] and does not show a significant KIE. [START_REF] Karge | Effects of Hydrogen Deuterium Exchange on Photosynthetic Water Cleavage in Ps II Core Complexes from Spinach[END_REF][START_REF] Haumann | Photosynthetic Oxygen Evolution: H/D Isotope Effects and the Coupling between Electron and Proton Transfer During the Redox Reactions at the Oxidizing Side of Photosystem II[END_REF][START_REF] Schilstra | Proton/Hydrogen Transfer Affects the S-state-Dependent Microsecond Phases of P680 + Reduction During Water Splitting[END_REF] The reduction potential of P680 + /P680 is 1.25 V, [START_REF] Rappaport | Primary Photochemistry and Energetics Leading to the Oxidation of the (Mn)4ca Cluster and to the Evolution of Molecular Oxygen in Photosystem II[END_REF] i.e. 0.1 V below the solution potential of YH + /YH, and probably even more below that of Y Z H + /Y Z H in a protein. [START_REF] Tommos | De Novo Proteins as Models of Radical Enzymes[END_REF] The edgeto-edge distance between P680 and Y Z is 9.0 Å, [START_REF] Schlodder | Temperature Dependence of the Oxidation Kinetics of Tyr(Z) and Tyr(D) in Oxygen-Evolving Photosystem II Complexes Throughout the Range from 320 K to 5 K[END_REF] as compared to 3.8 Å between Y 373 and D 321 in CraCRY.

Y Z forms a strong H-bond with a histidine residue (O-N distance, 2.5 Å, [START_REF] Umena | Crystal Structure of Oxygen-Evolving Photosystem II at a Resolution of 1.9 Å[END_REF] as compared to 3.0 Å for the hydrogen bond between Y 373 and D 321 in CraCRY). As for tyrosine oxidation in CraCRY, the absence of a significant KIE suggests either a sequential ET/PT with PT being "fast" (case Ia) or a sequential PT/ET with ET being "slow" (case IIb) *** . Case Ia seems, however, to be excluded in PSII because the rate limiting uphill ET from Y Z to P680 + is expected to take ≥500 ns according to Equation 4 using  = 0.75, R = 9 Å,  = 0.77 eV (all from Ref. [START_REF] Schlodder | Temperature Dependence of the Oxidation Kinetics of Tyr(Z) and Tyr(D) in Oxygen-Evolving Photosystem II Complexes Throughout the Range from 320 K to 5 K[END_REF] ) and G° ≥ 0.1 eV. A recent analysis suggested sequential PT/ET with a PT (preequilibrium constant of 0.16 and an ET rate of (7.7 ns) -1 . [START_REF] Schlodder | Temperature Dependence of the Oxidation Kinetics of Tyr(Z) and Tyr(D) in Oxygen-Evolving Photosystem II Complexes Throughout the Range from 320 K to 5 K[END_REF] QM/MM calculations on PSII indicate that the two energy minima of the proton location are very shallow and only 0.4 Å apart, 100 presumably allowing ultrafast and nearly activationless PT.

For the even faster tyrosine oxidation in CraCRY, the substantially longer hydrogen bond between Y 373 and D 321 in the dark state crystal structure does not support a sequential PT/ET mechanism unless formation of W 322 H •+ substantially shortens this bond and shifts the PT (pre-)equilibrium towards Y 373 - •••HD 321 (see above). The very short ET distance in CraCRY, however, could enable sequential ET/PT, *** Concerted PCET has been suggested for tyrosine oxidation in PSII with inactivated oxygen evolution, [START_REF] Sjödin | Proton-Coupled Electron Transfer from Tyrosine in a Tyrosine-Ruthenium-Tris-Bipyridine Complex: Comparison with Tyrosine z Oxidation in Photosystem II[END_REF] where the environment of tyrosine-Z is substantially modified. [START_REF] Hammarström | Proton-Coupled Electron Transfer of Tyrosines in Photosystem II and Model Systems for Artificial Photosynthesis: The Role of a Redox-Active Link between Catalyst and Photosensitizer[END_REF][START_REF] Schlodder | Temperature Dependence of the Oxidation Kinetics of Tyr(Z) and Tyr(D) in Oxygen-Evolving Photosystem II Complexes Throughout the Range from 320 K to 5 K[END_REF] In this case tyrosine oxidation by P680 + is slower and pH dependent (time constants from 300 ns at pH 9 to 50 µs at pH 4 [START_REF] Conjeaud | The Effect of pH on the Reduction Kinetics of P-680 in Tris-Treated Chloroplasts[END_REF][START_REF] Schlodder | pH Dependence of Oxygen Evolution and Reduction Kinetics of Photooxidized Chlorophyll aII (P-680) in Photosystem II Particles from Synechococcus Sp[END_REF] and shows a significant normal H/D KIE at pH<8. [START_REF] Ahlbrink | Function of Tyrosine Z in Water Oxidation by Photosystem II: Electrostatical Promotor Instead of Hydrogen Abstractor[END_REF][START_REF] Diner | Hydrogen Bonding, Solvent Exchange, and Coupled Proton and Electron Transfer in the Oxidation and Reduction of Redox-Active Tyrosine Y z in Mn-Depleted Core Complexes of Photosystem II[END_REF][START_REF] Hays | Role of D1-His190 in the Proton-Coupled Oxidation of Tyrosine Y z in Manganese-Depleted Photosystem II[END_REF] provided that the protein environment decreases the endergonic character of ET from Y 373 H to W 322 H •+ relative to the solution value (see above).

Tyrosine oxidation during photoactivation of the Slr 1694 BLUF domain of Synechocystis PCC6803

occurs by sequential ET (from Y 8 to the photoexcited FAD cofactor; edge-to-edge distance, 3.5 Å) and PT (from Y 8 H •+ to FAD •-via Q 50 ) on a time scale of tens of picoseconds, [START_REF] Gauden | Hydrogen-Bond Switching through a Radical Pair Mechanism in a Flavin-Binding Photoreceptor[END_REF] i.e. two orders of magnitude faster than tyrosine oxidation by W 322 H •+ in CraCRY. A main cause of the much faster kinetics is likely the much more favorable energetics of ET in Slr 1694 BLUF, which was estimated to be downhill because of the high energy of FAD ox *, [START_REF] Gauden | Hydrogen-Bond Switching through a Radical Pair Mechanism in a Flavin-Binding Photoreceptor[END_REF]101,102 but uphill in the oxidation of Y 373 in CraCRY. The term PCET has been used for the overall process of tyrosine oxidation during photoactivation of Slr1694 BLUF, [START_REF] Mathes | Hydrogen Bond Switching among Flavin and Amino Acids Determines the Nature of Proton-Coupled Electron Transfer in BLUF Photoreceptors[END_REF][START_REF] Migliore | Biochemistry and Theory of Proton-Coupled Electron Transfer[END_REF] in Fig. 3A). No significant further losses were detected by real time TAS up to 70 µs (Fig. 6). The quantum yield of the initial ET from W 399 to excited FAD ( 1 FAD ox *) in ~0.4 ps is likely to be virtually 100% as this process is several orders of magnitude faster than the lifetime of singlet excited flavins in the absence of quenching by ET (a few ns 103 ). We conclude that the FAD •-/Y 373 • pair is formed with a quantum yield of ~60%.

The FAD •-/Y 373 • pair in isolated WT CraCRY at pH 8.4 finally decayed by recombination in tens of milliseconds (Figure 5B, inset), in line with a previous report. [START_REF] Nohr | Extended Electron-Transfer in Animal Cryptochromes Mediated by a Tetrad of Aromatic Amino Acids[END_REF] We found that this recombination is in competition with protonation of FAD •-to form the neutral, red light-absorbing radical FADH • that is the primary target for CraCRY-based red light responses of Chlamydomonas cells. [START_REF] Kottke | Cryptochrome Photoreceptors in Green Algae: Unexpected Versatility of Mechanisms and Functions[END_REF][START_REF] Zou | An Animal-Like Cryptochrome Controls the Chlamydomonas Sexual Cycle[END_REF]104 The protonation was accelerated and hence more efficient at lower pH (see Figure S2D and SI, Section S2.2 for more details), explaining a previous observation [START_REF] Spexard | Response of the Sensory Animal-Like Cryptochrome aCRY to Blue and Red Light as Revealed by Infrared Difference Spectroscopy[END_REF] Remarkably, on a time scale up to a few nanoseconds, there are less losses by recombination in in the Y373F mutant protein (~20%) than in WT (~40%; Figures 3A and3C). This may indicate that the FAD • /W 322 H •+ pair relaxes more rapidly and/or more deeply in the mutant (cf. end of Section 4.1.2), increasing the barrier for recombination via reformation of the primary pair FAD •-/W 399 H •+ (see Scheme 1). The presence of Y373 as fourth member of the ET chain in WT CraCRY appears, however, to be required to obtain a long-lived radical pair as FAD •-/W 322 • in the Y373F mutant recombines in 10 µs as compared to 50 ms for the FAD •-/Y 373 • pair in WT.

CONCLUDING REMARKS

The main result of the present study is our demonstration of a very fast photoinduced proton-coupled electron transfer (PCET) [START_REF] Hammes-Schiffer | Theory of Coupled Electron and Proton Transfer Reactions[END_REF] It is finally worth recalling that CraCRY is uniquely characterized by its ability to function as a red light receptor in vivo and to accumulate FADH • in vitro even in the (alleged) absence of external reductant. [START_REF] Beel | A Flavin Binding Cryptochrome Photoreceptor Responds to Both Blue and Red Light in Chlamydomonas Reinhardtii[END_REF] Our results demonstrate that the FAD •-Y 373 • pair is formed with a high quantum yield (~60%) and has a long intrinsic lifetime (~50 ms due to recombination). The semi-reduced FAD may therefore be easily

Figure 5

 5 Figures S2 and S3 for additional wavelengths). Note that absolute signal amplitudes are not directly comparable to those of the sub-ns setup due to different excitation conditions and different protein concentrations. Upon flash excitation, the signals recorded for the WT CraCRY up to 70 µs (Figure 5A, 5B) exhibit step-like absorption changes, containing a very small and unresolved fast kinetic phase in ≤ 10 ns (minor growth at 405 nm, minor decay at 562 nm and essentially no visible change at 457 nm). All signals remain essentially constant within the time frames of Figure 5A and 5B and decay only on a much longer timescale (in ~50 ms, see inset of Figure 5B). The amplitudes of the 562 nm signals amount to mere ~10% of the amplitudes of the 457 nm signals (in absolute values) in WT CraCRY, indicating that there are virtually no tryptophan radicals (protonated or deprotonated) left when the initial fast kinetic steps are over. The remaining small positive signal at 562 nm can be attributed to the red tail in the absorption spectrum of the FAD •-radical (Figure 1B).

  Figure 6B (in EADS5 - EADS4 form, where 1 - is the yield of charge recombination occurring during the EADS4  EADS5 step) provides a convincing support that WH •+ indeed undergoes reduction by a tyrosine. The optimized parameters are available in SI, Section S5.3 (TableS2) and the modified

  pronounced in CraCRY because of the presence of Y 373 . ‡ ‡ (2) The negative charge of aspartate D 321 -in close proximity of Y 373 may decrease the YH •+ /YH reduction potential due to electrostatic stabilization of the positive charge.

  despite ET as the first step of the sequential ET/PT being downhill (cf. Introduction). A very recent work named it electron-coupled double proton transfer. 102 4.3. Fate of the FAD •-/Y 373 • pair and efficiency of FAD photoreduction The light-induced ET and PT reactions leading to FAD reduction occur in competition with nonproductive recombination (see Scheme 1). The quantum yield of FAD reduction can be estimated from the time course of the amplitude of the bleaching of the main absorption band of oxidized FAD around 450 nm. Our data by ultrafast TAS indicate that in WT CraCRY 40-45% of the initially formed FAD •- (represented by EADS2 in Fig. 3A) is lost during formation of the FAD •-/Y 373 • pair (represented by EADS5

  that the amplitude of blue light-induced very long-lived bleaching of the FAD absorption band in the blue (attributed to formation of FADH • ) increased strongly with decreasing pH. One would expect that the FADH • /Y 373 • pair recombines via activated re-formation of FAD •-/Y 373 • (the blue trace in Figure S2D (pH 6.5) was measured up to 600 ms and indicated an onset of recombination with a time constant in the order of seconds) and that light-generated FADH • in CraCRY can live for minutes only when its recombination partner Y 373 • is quenched by reduction. Given the unusually long lifetime of the FADH • /Y 373 • pair, it seems likely that even trace amounts of reducing agents have enough time to act upon the Y 373 • radical, leading to accumulation of metastable FADH • .

  from tyrosine to tryptophan in WT-CraCRY, forming the neutral (deprotonated) tyrosyl radical of Y 373 • in ~800 ps without significant H/D KIE. This PCET step is about 40 times faster than formation of the tyrosyl radical Y Z • in oxygen evolving PSII. Our results may either be explained by a sequential ET/PT with fast PT or a sequential PT/ET with slow ET (see detailed discussion in Section 4.2.2). Quantum mechanical/molecular mechanical (QM/MM) calculations may provide crucial information to decide which mechanism is actually preferred by CraCRY and how it is implemented. In particular, it would be useful to estimate the rate of PT from Y 373 H •+ to D 321 -in order to evaluate whether sequential ET/PT as fast as 800 ps and not rate-limited by PT is possible. As far as the validity of PT/ET is concerned, it would be interesting to determine if the formation of W 322 H •+ could induce any shortening of the hydrogen bond between Y 373 and D 321 , thereby allowing a very fast back PT from D 321 H to Y 373 -(k ET <<k BPT ; see Section 4.2.2), which in turn could ensure the lack of a KIE. Further experimental developments should involve checking the identity of the proton acceptor by point mutation of D 321 to a non-H-acceptor residue and studies of the temperature dependence of the tyrosine oxidation kinetics for estimating the barrier of the putative uphill step in sequential PCET.

  stabilized by protonation of FAD •-(to form the red-absorbing FADH • ) and by reduction of its partner Y 373 • by even trace amounts of residual reducing agents. All these factors are congruent with the unique properties of CraCRY in vivo.

FIGURES Scheme 1 .

 1 FIGURES

Figure 1 .

 1 Figure 1. (A) Local structure of CraCRY in the flavin photoreduction site (PDB entry 5ZM0 70 ; resolution 1.6 Å). FAD is represented in yellow, the conserved tryptophan triad in green, Y 373 in cyan, D321 in blue and the side tryptophans as reported by Li et al. 37 in grey. Closest edge-to-edge distances are indicated in Å. (B) Reference species spectra used for spectral analysis (see SI, Section S5.1).

Figure 4 .

 4 Figure 4. Flash-induced absorption changes on a ns timescale for (A) ~110 µM WT and (B) ~60 µM Y373F mutant CraCRY at four characteristic wavelengths (see Figures S2A and S3A for signals at additional wavelengths). The samples were excited at 355 nm by 100 ps pulses of an energy of ~2 mJ per cm 2 . Individual traces in both panels are averages of 64 flashes recorded with a repetition rate of 2 Hz.

Figure 5 .

 5 Figure 5. Flash-induced absorption changes on sub-µs to µs timescales for ~60 µM WT (A & B) and Y373F mutant (C & D) CraCRY at three characteristic wavelengths (see Figure S3B and C for additional wavelengths). The samples were excited at 475 nm by 5 ns pulses of an energy of ~5 mJ per cm 2 (~4 mJ for the Y373F mutant). Individual traces in panels A and B (WT) are averages of four single flash signals spaced by ~1 minute, those in panels C and D (Y373F) are averages of 16 flashes recorded with a repetition rate of 2 Hz. The trace in the inset of panel B is a single-flash signal recorded at 405 nm.

Figure 6 .

 6 Figure 6. (A) Black: signal constructed by subtraction of the inverted and normalized 457 nm signal in Figure 4A from the signal at 405 nm (ibid.). The constructed signal should reflect the crude kinetics of Y • formation by "correcting" the 405 nm ΔA signal for contributions from FAD •-; red: bi-exponential fit of

  •-/Y 373 • recombination presumably proceeds via back ET and re-protonation of the tyrosine. It is of note that, at lower pH, a non-negligible fraction of FAD •-gets protonated (in competition with back ET), creating a much longer-lived FADH • /Y 373 • pair (see Figure

Table 3

 3

(line raw) gathers the anisotropies expected for each WH •+ radical of the ET chain if one could probe them independently.

Table 1 .

 1 TABLES Time constants of the global multiexponential fit of the polarized transient absorption spectra of CraCRY, WT in H 2 O and D 2 O buffer, and Y373F in H 2 O buffer. Fit errors (95% confidence interval) are indicated after the ± sign. The coefficient of determination (R²) is given in the last column.

	protein	buffer	 1 (ps)	 2 (ps)	 3 (ps)	 4 (ps)	R²
	WT	H 2 O	0.39 ± 0.02	3.7 ± 0.8	55 ± 6	710 ± 80	0.999468
		D 2 O	0.37 ± 0.02	4.9 ± 1.0	56 ± 9	600 ± 70	0.999278
	Y373F	H 2 O	0.43 ± 0.01	3.4 ± 0.4	41 ± 4	630 ± 110	0.999904

Table 2 .

 2 Evolution-associated anisotropies taken at 610 nm (r i = EAAS i (610 nm), averaged over 5 nm), after initial decay of the excited state.

	protein	buffer	r 2	r 3	r 4	r 5
	WT	H 2 O	-0.062	0.041	-0.017	-0.053
		D 2 O	-0.062	0.071	-0.027	-0.101
	Y373F	H 2 O	-0.047	0.063	-0.042	-0.059

Table 3 .

 3 Expected intrinsic anisotropies attached to the different WH •+ radicals of the photoreduction site, according to the crystal structure of WT-CraCRY (PDB entry 5ZM0).[START_REF] Franz | Structure of the Bifunctional Cryptochrome aCRY from Chlamydomonas Reinhardtii[END_REF] Errors indicated after the ± sign correspond to variations of ± 5° of the directions of both FAD ox and WH •+ transition moments within the molecular plane. The bottom line proposes at tentative correction of these values, including a contribution from FAD •-(see text).

		W 399	W 376	W 322	W 401	W 332
	raw	0.004 ± 0.07	0.27 ± 0.05	-0.06 ± 0.07	-0.20 ± 0.02	0.20 ± 0.07
	correction	-0.02 ± 0.07	0.20 ± 0.05	-0.07 ± 0.07	-0.18 ± 0.02	0.14 ± 0.07

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ACS Paragon Plus Environment Journal of the American Chemical Society ACKNOWLEDGEMENTS This work obtained financial support by the International Max Planck research school for Environmental, Cellular and Molecular Biology (SF), the Deutsche Forschungsgemeinschaft (LOE: FOR 1261-2, ES 152/12-1), the Air Force Office of Scientific Research (AFOSR; grant No FA9550-14-1-0409) and by the French Infrastructure for Integrated Structural Biology (FRISBI: ANR-10-INBS-05). ACS Paragon Plus Environment Journal of the American Chemical Society

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*Lars-Oliver Essen: Email: essen@chemie.uni-marburg.de. Phone: +49 64212822032.

Conflicts of interest

There are no conflicts to declare.