Alessandro Arcovito

Alessandra Bonamore

Jean Louis

Hazemann ||

Alberto Boffi

Paola D'

Paola D ' Angelo
email: p.dangelo@caspur.it

Unusual proximal heme pocket geometry in the deoxygenated Thermobifida fusca: a combined spectroscopic investigation

Keywords: XANES, EXAFS, bacterial hemoglobin

The spectroscopic properties of the deoxygenated truncated hemoglobin from the actinobacterium Thermobifida fusca have been investigated by means of extended X-ray absorption fine structure (EXAFS), X-ray absorption near edge structure (XANES), and near infrared spectroscopies both at room and cryogenic temperatures. At room temperature the near infrared charge transfer band III occurs at 772 nm, a value that is unusually high for a canonical deoxygenated hemoglobin species, and can only be found as a transient species after photolysis in vertebrate hemoglobins and myoglobins or under strongly dehydrating conditions. EXAFS and XANES quantitative analyses, carried out in parallel with deoxygenated horse myoglobin, revealed an unusually short iron-histidine distance 1.90 ± 0.03 Å, significantly shorter than the deoxygenated horse myoglobin distance of 2.11 ± 0.02 Å. These findings provide novel structural basis for discussing the fine structural geometry of the proximal site, and eventually mapping the coordinates of the metal with respect to the pyrrole nitrogens and the proximal histidine nitrogen.

Introduction

The covalent link between the heme iron and the proximal histidine in hemoglobins is by far the most thoroughly investigated chemical bond in biochemistry. The atomic coordinates and electronic properties of the iron-histidine bond are considered as crucial parameters to adjust the reactivity of the heme-iron atom with the ligand in trans position. Thus, a strong bond with increased electron donation to the iron atom favors dioxygen cleavage (e.g. electron push effect in peroxidases), whereas a weaker bond is a prerequisite to preserve the integrity of the distal ligand in oxygen transport proteins. The stereochemistry of the iron-histidine bond is not only a major factor in determining the function of a hemeprotein, but is also the main conduit that governs the structural transitions at the basis of cooperativity in hemoglobins [START_REF] Perutz | Stereochemistry of cooperative effects in hemoglobin. Bohr effect and combination with organic phosphates[END_REF]. Exogeneous ligand binding to the sixth coordination position entails the displacement of the iron atom (about 0.5 Å) towards the heme plane with consequent shortening of the iron heme-pyrrole nitrogen bonds, and decreasing of the stretching frequency of the proximal ironhistidine bond. In hemoglobins, the proximal histidine follows the iron atom in its movement thus pulling the F helix in a rigid body displacement, that is subsequently transmitted to the helices that form the subunit interface [START_REF] Perutz | The stereochemical mechanism of the cooperative effects in hemoglobin revisited[END_REF]. Multiple spectroscopic investigations have focused on the fine structural and dynamic properties of the iron-histidine bond in order to assess the stereochemical details at the basis of the ligand binding process in hemoglobins and myoglobins.

In recent years, novel, unusual hemoglobin entities have further expanded the scope of the research [START_REF] Friedman | Transient Raman study of hemoglobin: structural dependence of the iron-histidine linkage[END_REF]. In particular, the identification of novel "hemoglobin-like" proteins, widely distributed within prokaryotes and lower eukaryotes, widened the current understanding of hemoglobin functions and fostered a flourishing research on the properties of these unusual globins aimed at understanding their physiological function, their ligand binding properties, and their evolutionary relationships with respect to hemoglobins from higher eukaryotes. Within the bacterial hemoglobin superfamily, the so-called truncated hemoglobins represent a new frontier for hemoglobin research. These proteins are in fact characterized by a compacted globin fold (6 instead of 8 helices) and by an amazing variability of aminoacid side chains in the distal pocket in front of a proximal site characterized by the presence of a canonical proximal histidine [START_REF] Friedman | Transient Raman study of hemoglobin: structural dependence of the iron-histidine linkage[END_REF][START_REF] Scott | Tertiary-structure relaxation in hemoglobin: a transient Raman study[END_REF]. In spite of extensive biochemical, biophysical and physiological studies, the hypotheses on the functional role of truncated hemoglobins are at present weak and rely on possible involvement in the scavenging of NO or oxygen radicalic species, within the framework of the cell response to oxidative or nitrosative stress [START_REF] Friedman | Transient Raman study of hemoglobin: structural dependence of the iron-histidine linkage[END_REF]. Thus, biological investigations are presently running in parallel with physico-chemical characterizations of these novel globins in order to gain structural and functional insights into this amazing protein family. Spectroscopic studies are based on the identification of structure sensitive signals (spectroscopic marker bands) that are related to the stereochemistry of the iron histidine bond. Near infrared absorption

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M A N U S C R I P T spectra are sensitive probes of the heme iron electronic distribution and are strictly correlated to subtle rearrangement of the atomic frame around the critical Fe-His bond. In particular, the band III at about 760 nm has been assigned to a charge transfer band originating from a transition dipole moment involving electron donation from the porphyrin π* to the Fe-d z 2 orbital [START_REF] Eaton | Polarized absorption and linear dichroism spectroscopy of hemoglobin[END_REF][START_REF] Makinen | Iron Porphyrin Part 1[END_REF]. On the basis of this assignment, possible relationships between band III frequency and iron-heme plane distance have been worked out [START_REF] Chavez | Structural and functional significance of inhomogeneous line broadening of band III in hemoglobin and [iron-manganese] hybrid hemoglobins[END_REF]. Nevertheless, near infrared spectroscopy does not allow a direct, quantitative estimate of the relevant atomic coordinates of the iron atom with respect to the heme plane or to the histidine nitrogen. Thus, direct, high resolution measurements of the iron stereochemistry by X-ray diffraction or absorption measurements are required in order to yield a complete picture of the functionally relevant structural rearrangements of the iron atom. In particular, X-ray absorption spectroscopy (XAS) is a powerful method for looking at the Fe-heme local structure [START_REF] Longa | Probing the coordination and geometry of heme-iron ligands in hemoproteins by XANES (X-ray absorption near edge structure)[END_REF]. Recent theoretical advances have made the extended X-ray absorption fine structure (EXAFS) analysis a primary, well established structural tool to investigate bond lengths at a precision of about 0.02 Å [START_REF] Filipponi | X-ray-absorption spectroscopy and n-body distribution functions in condensed matter. I. Theory[END_REF]. Moreover, a quantitative X-ray absorption near edge structure (XANES) analysis has been developed [START_REF] Benfatto | Geometrical fitting of experimental XANES spectra by a full multiple-scattering procedure[END_REF][START_REF] Della Longa | Redox-induced structural dynamics of Fe-heme ligand in myoglobin by X-ray absorption spectroscopy[END_REF] and applied in combination with EXAFS to different system to extract information on bond distances and angular

parameters. An accurate structural characterization has been obtained on metal ionic solutions [START_REF] D'angelo | Combined XANES and EXAFS analysis of Co 2+ , Ni 2+ , and Zn 2+ aqueous solutions[END_REF],

different heme models and heme containing proteins [START_REF] Angelo | Unusual heme iron-lipid acyl chain coordination in Escherichia coli flavohemoglobin[END_REF][START_REF] Angelo | X-ray Absorption Spectroscopy of Hemes and Hemeproteins in Solution: Multiple Scattering Analysis[END_REF][START_REF] Arcovito | An X-ray diffraction and X-ray absorption spectroscopy joint study of neuroglobin[END_REF], showing that a combined analysis of the EXAFS and XANES regions of the spectrum is able to provide unambiguous details on the local structure in solution around the metal center.

In this framework, an investigation on the spectroscopic properties of the ferrous unliganded truncated hemoglobin from the actynomyces Thermobifida fusca (Tf-trHb) [START_REF] Bonamore | A novel thermostable hemoglobin from the actinobacterium Thermobifida fusca[END_REF] has been undertaken

with the aim of characterizing the fine structural properties of the Fe-histidine moiety as compared to ferrous unliganded horse myoglobin (h-Mb). The results of this study bring out relevant differences in the structure of the active site between Tf-trHb and h-Mb.

EXPERIMENTAL PROCEDURES

The hemoglobin from Thermobifida fusca was prepared and purified as described previously [START_REF] Bonamore | A novel thermostable hemoglobin from the actinobacterium Thermobifida fusca[END_REF].

The protein was concentrated up to 1 mM heme with vivaspin PM10 concentrators (Sartorius Spa, Firenze). Protein concentration was determined by using an ε=182000 M -1 cm -1 at 421 nm for the CO derivative.

Near infrared absorption spectra

Near infrared spectra were carried out on a Jasco V570 spectrophotometer equipped with a PbS detector (Jasco Ltd., Japan). Low temperature spectra were measured in an Oxford liquid Nitrogen

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apparatus by diluting concentrated solutions of Tf-trHb in 50 mM phosphate buffer at pH 7.0 with glycerol (3:1 v/v glycerol/protein). A 100 mM solution of sodium dithionite buffered in 0.1 M phosphate buffer at pH 7.0 was then added to the degassed glycerol/protein solution to a final concentration of about 10 mM.

X-ray absorption measurements

Fe K-edge X-ray absorption spectra of h-Mb and Tf-trHb were collected in fluorescence mode at the BM30B beamline of the European Synchrotron Radiation Facility [START_REF] Proux | FAME: A new beamline for X-ray absorption investigations of very-diluted systems of environmental, material and biological interests[END_REF][START_REF] Proux | Feedback system of a liquid-nitrogen-cooled double-crystal monochromator: design and performances[END_REF]. h-Mb was prepared by dissolving metmyoglobin in 1:5 glycerol-water solution buffered at pH 7 (70 mM phosphate) and adding 50 mM of sodium dithionite in nitrogen atmosphere. The final iron concentration was 6 mM. Tf-trHb was prepared by dissolving the protein in 1:5 glycerol-water solution buffered at pH 7 (80 mM phosphate) and adding 50 mM of sodium dithionite in nitrogen atmosphere. The final iron concentration was 4 mM. All the spectra were collected at 15 K. The storage ring was running in the two-third filling mode with a typical current of 170 mA. The monochromator was equipped with a Si (111) double crystal, in which the second crystal was elastically bent to a cylindrical cross section. The energy resolution at the Fe-K edge is 0.5 eV. The X-ray photon beam was vertically focused by a Ni-Pt mirror, and dynamically sagittally focused in the horizontal size. An array detector made by 30 Ge elements of very high purity was used. For each sample 10 spectra were recorded with a 7-s/point collection statistic and averaged. The spectra were calibrated by assigning the first inflection point of the Fe foil spectrum to 7111.2 eV.

EXAFS data analysis

The EXAFS data analysis has been performed using the GNXAS method [START_REF] Filipponi | X-ray-absorption spectroscopy and n-body distribution functions in condensed matter. I. Theory[END_REF][START_REF] Filipponi | X-ray-absorption spectroscopy and n-body distribution functions in condensed matter. II. Data analysis and applications[END_REF], which is based on the theoretical calculation of the X-ray absorption fine structure signal and a subsequent refinement of the structural parameters. This theory allows the calculation of the interference signal in the cross section by solving the scattering of the photoelectron wave function in an effective muffin tin potential.

In the GNXAS approach, the interpretation of the experimental data is based on the decomposition of the χ(k) signal into a summation over n-body distribution functions γ (n) calculated by means of the multiple-scattering (MS) theory.

Previous investigations have shown that for a quantitative EXAFS analysis of hemeproteins MS fourbody terms have to be accounted for [START_REF] Angelo | Unusual heme iron-lipid acyl chain coordination in Escherichia coli flavohemoglobin[END_REF][START_REF] Angelo | X-ray Absorption Spectroscopy of Hemes and Hemeproteins in Solution: Multiple Scattering Analysis[END_REF][START_REF] Arcovito | An X-ray diffraction and X-ray absorption spectroscopy joint study of neuroglobin[END_REF][START_REF] Bonamore | A novel thermostable hemoglobin from the actinobacterium Thermobifida fusca[END_REF][START_REF] Zhang | Multiple-Edge XAS Studies of Cyanide-Bridged Iron-Copper Molecular Assemblies Relevant to Cyanide-Inhibited Heme-Copper Oxidases Using Four-Body Multiple-Scattering Analysis[END_REF]. The inclusion of these higher order contributions is essential to obtain a good agreement between theoretical and experimental data. The analysis of the two proteins has been carried out starting from the crystallographic coordinates of deoxy myoglobin at 1.15 Å resolution, up to a distance cutoff of 5 Å (PDB code=1A6N). The two-, three-, and four-body

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configurations in each cluster were grouped with a distance tolerance of 0.05 Å. Phase shifts were calculated using the standard muffin-tin approximation. A model EXAFS spectrum was generated by adding all the relevant single-and multiple-scattering contributions, and it was refined against the experimental data by using a least-square minimization procedure in which structural and nonstructural parameters were allowed to float. The quality of the fits was determined by the goodness-of-the-fit parameter R [START_REF] Filipponi | X-ray-absorption spectroscopy and n-body distribution functions in condensed matter. I. Theory[END_REF], and by careful inspection of the EXAFS residuals and their Fourier transforms (FT). A thorough description of the data analysis procedure and all the relevant parameters used in the minimization is given in Ref. [START_REF] Angelo | Unusual heme iron-lipid acyl chain coordination in Escherichia coli flavohemoglobin[END_REF].

Quantitative XANES analysis

The XANES calculations have been performed using the MXAN software procedure [START_REF] Longa | Probing the coordination and geometry of heme-iron ligands in hemoproteins by XANES (X-ray absorption near edge structure)[END_REF][START_REF] Benfatto | Geometrical fitting of experimental XANES spectra by a full multiple-scattering procedure[END_REF]. The analysis of the XANES spectra has been carried out using a cluster of thirty-one atoms, i.e., the porphyrin macrocycle, the histidine imidazole, and the oxygen of an unbound water molecule. The molecular potential for the nearest atoms, i.e., the FeN 5 cluster, was obtained using the spin-unpolarized, self-consistent field method imposing the formal valence of each atom. The exchange correlation part of the potential was determined by the X-α approximation. Inelastic processes were taken into account by a convolution with a phenomenological, energy-dependent, broadening function. All details of this kind of calculation are described in Ref. [START_REF] Della Longa | Quantitative analysis of x-ray absorption near edge structure data by a full multiple scattering procedure: the Fe-CO geometry in photolyzed carbonmonoxy-myoglobin single crystal[END_REF]. A constant experimental error corresponding to a noise-to-signal ratio of 0.012 normalized units (i.e. units for which the absorption jump is equal to 1) was chosen. The minimization of the χ 2 function was performed in the space of the n selected structural parameters (χ 2 /n values are reported in the Result section to quantify the goodness of the fits). They are the core size, the Fe-heme displacement, and distance of the Fe atom from the proximal histidine nitrogen N h . During the minimization procedure, the outer atoms of the histidine and pyrrolic rings rigidly followed the motion of the N h and nitrogen atoms of the porphyrin ring (N p ).

XANES difference spectra Tf-trHb -h-Mb were calculated starting from the 1A6N PDB structure taken as reference, by modifying the MXAN program as already done in a previous study [START_REF] Arcovito | Light-induced relaxation of photolyzed carbonmonoxy myoglobin: a temperaturedependent X-ray absorption near-edge structure (XANES) study[END_REF].

Fitting difference spectra has the important advantage that some systematic errors of the XANES theory, mostly pertaining the photo-electron scattering on the heme plane, can be reduced. As the majority of the signal in solution is due to heme plane scattering, these errors affect the determination of axial parameters and render them, in solution, less accurate than what is reported for the myoglobin P2 1 crystals [START_REF] Della Longa | Quantitative analysis of x-ray absorption near edge structure data by a full multiple scattering procedure: the Fe-CO geometry in photolyzed carbonmonoxy-myoglobin single crystal[END_REF], where the heme plane and heme normal scattering can be experimentally decoupled. In the present case, it is reasonable that theoretical systematic errors associated to the heme plane scattering signals for h-Mb and Tf-trHb are very similar, so that they are partially eliminated in the difference spectrum. Accordingly, the present analysis of the XANES difference spectrum is less sensitive to the

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heme plane parameters (e.g. the Fe-N p distance), but it is more accurate and more sensitive to the heme normal parameters (e.g. the Fe-N h distanceas) as compared to the analysis of absolute XANES absorption spectra.

RESULTS

Near infrared spectra

In order to unveil the fine electronic properties of Tf-trHb we have carried out a direct comparison of the near infrared absorption spectra of this protein with the canonical h-Mb. The Tf-trHb spectra have been acquired both at room (Figure 1, panel A, at 293 K) and low temperature (Figure 1, panel B, at 77 K) revealing the presence of an unusually strong and red shifted band III with respect to h-Mb. In fact, comparison of the spectra of Tf-trHb (Figure 1, solid line) and h-Mb (dashed line) points out that the Tf-trHb displays a high intensity band III peaked at 772 nm, 12 nm higher than that observed in h-Mb. Low temperature spectra confirm this large difference between the absorption peaks of the two proteins The presence of a unusually red shifted band III can stem either from a different electronic properties of the heme pocket or from a unusual geometry of the metal site. To shed light on the origin of this experimental evidence we acquired EXAFS and XANES spectra of Tf-trHb which were able to provide an accurate and independent determination of the local structure of the heme site.

X-ray Absorption spectra

A quantitative estimate of the iron coordination geometry parameters can be obtained from the comparative analysis of the X-ray absorption spectra of Tf-trHb and h-Mb. In Figure 2, the experimental XANES spectra (upper panel) and Fourier Transformed EXAFS spectra (lower panel) of the two samples are superimposed (dotted line h-Mb and solid line Tf-trHb). Looking at the XANES, the main difference between the two spectra is the blue shift of the rising edge of Tf-trHb as compared to h-Mb.

Moreover, the absorption cross section of Tf-trHb increases at about 7150 eV and decreases at about 7170 eV, as evident from the [Tf-trHb -h-Mb] difference spectrum reported in the same panel of Figure 2 (solid gray line). The blue shift of the rising edge can be due to different mechanisms: 1) an increase of the iron redox state, which enhances the energy necessary to extract an electron from the metal [START_REF] Labhardt | X-ray absorption edge fine structure spectroscopy of the active site heme of cytochrome c[END_REF];

2) the binding of a sixth ligand in the case of a five-coordinate Fe-heme [START_REF] Della Longa | Quantitative analysis of x-ray absorption near edge structure data by a full multiple scattering procedure: the Fe-CO geometry in photolyzed carbonmonoxy-myoglobin single crystal[END_REF][START_REF] Della Longa | Polarized X-ray absorption spectroscopy of the low-temperature photoproduct of carbonmonoxy-myoglobin[END_REF]; and 3) a decrease of the first-shell average distance [START_REF] Pin | X-ray absorption spectroscopy of hemoglobin[END_REF]. Both 2 and 3 mechanisms act by changing the ligand field on the metal, and by increasing the energy of the empty electron states with p-symmetry. The UV/Vis spectrum of Tf-trHb (acquired on the same sample exposed to the X-ray beam, data not shown) closely resembles the h-Mb form, thus indicating that a negligible percentage, if any, of oxidized or bound Fe-heme is present.

Note that both proteins have been measured in the presence of an excess of sodium dithionite (see

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method section), and it is well known that prolonged X-ray exposure determines a reduction of the iron heme disfavoring the contamination of oxidized species [START_REF] Della Longa | Redox-induced structural dynamics of Fe-heme ligand in myoglobin by X-ray absorption spectroscopy[END_REF][START_REF] Arcovito | An X-ray diffraction and X-ray absorption spectroscopy joint study of neuroglobin[END_REF]. Therefore, the observed XANES changes are due to a decrease of the first-shell average distance. The h-Mb and Tf-trHb FT moduli of the EXAFS spectra extracted with a three-segmented cubic spline, have been calculated in the interval k = 3.2-14.0 Å -1 with no phase-shift correction applied. In both cases, the high-intensity peak at about 1.5 four nitrogen atoms from the porphyrin (γ (2) Fe-N p ) and the axial nitrogen atom from the histidine (γ (2) Fe-N h ). The latter contribution was treated as a separate single-scattering signal as the Fe-N h bond length is about 0.07 Å longer than the average Fe-N p bond length, according to the crystallographic values (PDB code=1A6N, see Table 1). In addition to the first-shell contributions, the simulation requires four carbon atoms at about 3.4 Å derived from the connecting methylene groups (γ (2) Fe-C h ). A good fit of the experimental data required the inclusion of the Fe-N p -C 14 three-body (γ (3) ) and the Fe-N p -C 14 -C 23 four-body (η (4) ) total contributions. The dominant MS contribution is the Fe-N p -C 14 three-body pathway from the porphyrin, whereas the amplitude of the MS terms associated with the histidine is negligible owing to the low multiplicity. The theoretical signal closely matches the experimental data and the structural parameters obtained from the minimization are all in excellent agreement with the crystallographic values within the reported errors (Table 1).

Å
The EXAFS data analysis of Tf-trHb has been carried out along the line of that of h-Mb. The minimization procedure has been carried out starting from the 1A6N structure of h-Mb and allowing

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distances and angles to float within a preset range of ± 0.3 Å and ± 5°, respectively. The experimental oscillations of the two samples show marked differences in the range between 7 and 12 Å -1 . Inspection of the theoretical signals shows that these differences originate from the modification in frequency and phase of the two body γ (2) Fe-N h signals. As a consequence different first-shell distances have been obtained from the fitting procedure, with a factor index value R=1.009×10 -5 . In particular, whereas the Fe-N p distance does not change in an appreciable way, a decrease of the Fe-N h of 0.21 Å has been observed in going from the h-Mb to Tf-trHb (see Table 1 and2). Therefore, the contraction of the Fe-N h distance is responsible for both the blue shift of edge and the shift of the FT first peak previously described. The Debye-Waller factors obtained for h-Mb and Tf-trHb from the fitting procedures are quite small and very similar between the two proteins. This suggests that in both cases the iron atom is involved in quite stiff bonds with the pyrrole and histidine nitrogens.

XANES results. To further validate the EXAFS results we have performed a comparative analysis of the XANES difference spectrum [Tf-trHb -h-Mb], up to about 180 eV above the edge via the MXAN package [START_REF] Benfatto | Geometrical fitting of experimental XANES spectra by a full multiple-scattering procedure[END_REF]. The best fit (see Figure 4) is obtained for a Fe-N h distance to 1.93±0.03 Å, while the Fe-N p distance remains essentially equal to the starting value of 2.07±0.02 Å, in perfect agreement with the EXAFS results reported in Table 2. The quality of the fit is satisfactory (χ 2 /n=1.74), thus showing that the structural results obtained from the EXAFS analysis are compatible with the XANES data. It is worthwhile to note that the XANES and EXAFS analyses are independent, thus the convergence of the results strongly supports the reliability of the obtained local structure of the heme site.

DISCUSSION

The near infrared data shown in Figure 1 exhibit an unusually red shifted band III in Tf-trHb with respect to h-Mb both at 293 and 77 K. The 760 nm band, present in five coordinate ferrous heme derivatives, has been assigned to a plane polarized charge transfer transition from the porphyrin π system to the iron d xy orbital [START_REF] Friedman | Transient Raman study of hemoglobin: structural dependence of the iron-histidine linkage[END_REF] or, alternatively, to an iron d x 2 y 2 → e g (π ) orbital promotion [START_REF] Scott | Tertiary-structure relaxation in hemoglobin: a transient Raman study[END_REF]. In fact, in h-Mb the 760 nm band was shown to be sensitive to the local structure of the heme within the heme pocket, and in particular to the position of the iron with respect to the heme plane [START_REF] Eaton | Polarized absorption and linear dichroism spectroscopy of hemoglobin[END_REF][START_REF] Sassaroli | Time dependence of near-infrared spectra of photodissociated hemoglobin and myoglobin[END_REF][START_REF] Cordone | Thermal behavior of the 760-nm absorption band in photodissociated sperm whale carbonmonoxymyoglobin at cryogenic temperature: dependence on external medium[END_REF]. In both cases, the structural rearrangements leading to changes in the energy of the iron d orbitals, or the porphyrin π orbital system will affect the energy of the observed transition. The present data provide a clear picture of the structural properties of the iron-proximal histidine bond in the deoxygenated derivative of a truncated hemoglobin, and unambiguously show that the proximal site is widely different

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from that of typical vertebrate hemoglobins and myoglobins. It is interesting to consider the unusual band III position in Tf-trHb in the light of the dynamic behavior observed upon laser photolysis in vertebrate hemoglobins. The band III in deoxygenated vertebrate hemoglobins and myoglobins occurs at 760 nm and transiently shifts to about 770 nm in the nanosecond photoproducts of the CO bound derivatives [START_REF] Sassaroli | Time dependence of near-infrared spectra of photodissociated hemoglobin and myoglobin[END_REF]. The observed shift has been taken as a probe for the presence of a relaxed state in which the transiently generated penta-coordinate heme-iron resides in a position that is close to that typical of the hexa-coordinated, ligand bound species. In hemoglobins the nanosecond phoproducts then evolve towards the strained "unrelaxed" state typical of the "Tense" conformation that is represented, at equilibrium, by the canonical 760 nm band. The photoproduct relaxation, as monitored by near infrared, is thus considered as the most significant structural rearrangement that is functionally relevant to the mechanism of cooperativity. These considerations can be merged with recent transient resonance Raman spectroscopy data obtained in the highly similar truncated HbO from Mycobacterium tuberculosis [START_REF] Samuni | The Absence of Proximal Strain in the Truncated Hemoglobins from Mycobacterium tuberculosis[END_REF]. Both HbO and HbN do not show microsecond or even nanosecond relaxation of the photoproducts obtained with an 8 ns laser pulse. Hence, the iron histidine stretching frequencies of the deoxygenated species and that of the 10 ns photoproduct are identical pointing to the absence of a proximal strain in truncated hemoglobins [START_REF] Samuni | The Absence of Proximal Strain in the Truncated Hemoglobins from Mycobacterium tuberculosis[END_REF]. The presence of band III at 772 nm in Tf-trHb is in line with the observations reported for HbO and provide further evidence that the structure of the proximal site in the static deoxygenated derivative in truncated hemoglobins may be similar to the structure of the transient, unrelaxed photoproduct species in classical globins. Transient spectroscopy measurements in the near infrared region in Tf-trHb will provide a further proof of this picture.

The EXAFS and XANES data directly provide a high resolution map of the iron first shell distances changes with 0.01-0.03 Å accuracy. The measurements, carried out in parallel on the deoxygenated derivatives of h-Mb and Tf-trHb bring out major differences between the two proteins that are manifest in the FT spectrum shown in Figure 2. Quantitative analysis, reported in Table 1 and 2, points out two major features of the Fe-proximal histidine environment in Tf-trHb. First of all, the calculated Fe-N h distance in Tf-trHb is unusually short (1.90 Å) with respect to that of h-Mb (2.14 Å). The 0.24 Å difference in the bond lenght between the two proteins represents a dramatic difference in terms of bond orbital energy and points to an optimal overlap between the iron and N h orbitals as a consequence of a fully relaxed position of the imidazole ring [START_REF] Ellison | Structure of the Deoxymyoglobin Model [Fe(TPP)(2-MeHIm)] Reveals Unusual Porphyrin Core Distortions[END_REF]. The second important structural feature concerns the average out-of-plane position of the iron atom with respect to the heme plane as inferred from the calculated Fe-pyrrole nitrogen distances. In this case, the positioning of the iron atom relative to the pyrrole nitrogens is identical in h-Mb and Tf-trHb (average distances of 2.06 Å in h-Mb, 2.05 Å in Tf-trHb) pointing out that the heme core size must be superimposable in the two proteins. Thus, the differences in the EXAFS and XANES spectra between h-Mb and Tf-trHb are entirely accounted for a

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shorter Fe-N h bond distance with minimal, if any, readjustments of the heme core size. In turn, in agreement with X-ray crystallographic data on the ferric, acetate bound adduct, the stronger Fe-N h bond in Tf-trHb indicates an unrestrained conformation of the proximal histidine. Nevertheless, it should be mentioned that red shifted band III derivatives can also be generated at equilibrium by acting on solvent properties [START_REF] Huang | Near-infrared spectra of Scapharca homodimeric hemoglobin: characterization of the deoxy and photodissociated derivatives[END_REF]. High glycerol concentrations or sol-gel encapsulation are capable of shifting the peak position of band III towards the blue region by promoting dehydration of the protein. The ultimate effect of the removal of protein coordinated water may thus be ascribed to local collapse of the heme pocket with subsequent fine structural rearrangements of the heme iron position.

Hence, in structural terms, the position of band III reflects the fine structural modifications within the iron atom coordination sphere in response to chemical and physical perturbations. Although the present data offer a unique opportunity to correlate the iron coordinates (as measured in EXAFS experiments)

with the position of band III, another possible explanation can be envisaged. In fact, a possible interpretation would be a direct relationship between the Fe-N h distance and the frequency of the electronic transition, assuming that the energy of the porphyrin π system is superimposable in Tf-trHb and h-Mb. However, inspection of the visible spectra suggests that the overall electronic structure of the porphyrin π system is not superimposable. In fact, in Tf-trHb, the peak position of the deoxygenated derivative is at 432 nm and hence red shifted with respect to h-Mb (435 nm) [START_REF] Bonamore | A novel thermostable hemoglobin from the actinobacterium Thermobifida fusca[END_REF]. The Soret peak energies are affected by the overall electron density of the heme thus suggesting that there is a small but significant electron withdrawing effect (which enhances the energy of the π → π* transitions), in Tf-trHb with respect to h-Mb. It has been proposed that there are at least two different mechanisms that may promote π electron withdrawal/donation from/to the porphyrin ring, namely: i) increased hydrogen bonding to N δ of the proximal ligand that promotes electron donation from Fe-N h via the proximal imidazole ring and shortens the iron histidine bond distance, and ii) increased conjugation of the vinyl double bonds with the porphyrin π system that favours electron donation and lowers the π → π* transition energies [31]. In the case of Tf-trHb, the observed blue shift of the Soret band, due to decreased π electron density, might be ascribed mainly to the short iron histidine bond but in the absence of concomitant hydrogen bonding donation to the N δ of the proximal ligand (see also next paragraph) and hence without the "electron push" effect. In turn, the effect of vinyl conjugation still needs to be investigated in detail by appropriate resonance Raman experiments, although no major "out of plane" shift of the double bonds is evident from inspection of the available X-ray structure [START_REF] Bonamore | A novel thermostable hemoglobin from the actinobacterium Thermobifida fusca[END_REF]. In summary, the inverse relationship between the red shifted band III and blue shifted Soret spectrum in Tf-trHb might find a rationale in terms of electronic configuration only assuming an electron withdrawal effect by the proximal imidazole ring, fully protonated at the N δ .

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These considerations pave the way to a broader discussion on the architecture of the proximal site with respect to different heme proteins other than globins. First of all, it is of interest to describe the environment of the proximal histidine in Tf-trHb in structural terms, by taking into account that the only available X-ray structure (2BMM in the PDB databank) concerns the ferric, acetate bound derivative. In Tf-trHb, the imidazole ring of His106(F8) is comprised between residues Tyr109, Met151 and the peptide bond of Leu111 (see Figure 5A). None of these residues appear to interact directly with HisF8 but they simply delimit an apolar cavity in which the imidazole ring is almost rotamerically free with the exception of a small angle in which the sulphur atom of Met151 hinders full rotation. Accordingly, by inspecting the position of the imidazole plane with respect to the porphyrin nitrogen plane (Figure 5B), it is apparent that the imidazole plane itself is in a fully staggered (with respect to N p pyrrole atoms) and hence relaxed position. Thus, it might be suggested that the overall architecture of the proximal site in Tf-trHb is such that the iron-histidine bond is free of constraint and is able to reach maximal bonding overlap. Several studies in past years have revealed consistent differences between the structure of the heme in globins and peroxidases [31]. As shown from XAS and high resolution X-ray structures, globins typically have an Fe-N h distance of about 2.1 Å while in the peroxidases this distance is typically shorter by about 0.2 Å, resulting in a typical Fe-N h distance of about 1.9 Å [31][START_REF] Chance | Xray absorption studies of intermediates in peroxidase activity[END_REF][START_REF] Sinclair | Structure of the active site of lignin isozyme H2: native enzyme, compound III, and reduced form[END_REF][START_REF] Sinclair | Active site structure in cytochrome c peroxidase and myoglobin mutants: effects of altered hydrogen bonding to the proximal histidine[END_REF][START_REF] Chance | Movement of Fe with respect to the heme plane in the R-T transition of carp hemoglobin. An extended x-ray absorption fine structure study[END_REF][START_REF] Yang | Crystal structures of CO-, deoxy-and met-myoglobins at various pH values[END_REF][START_REF] Lionetti | X-ray crystal structure of the ferric sperm whale myoglobin: imidazole complex at 2.0 A resolution[END_REF][START_REF] Peisach | An interim report on electronic control of oxygenation of heme proteins[END_REF] (see also Table 3). This was proposed to be crucial to explain the different reactivity observed between the two groups of proteins [START_REF] Sinclair | Active site structure in cytochrome c peroxidase and myoglobin mutants: effects of altered hydrogen bonding to the proximal histidine[END_REF][START_REF] Chance | Movement of Fe with respect to the heme plane in the R-T transition of carp hemoglobin. An extended x-ray absorption fine structure study[END_REF]. A significant feature of the peroxidase structures (either Cytochrome C peroxidase, Ccp, or Horseradich peroxidase, Hrp) is the presence of strongly hydrogen-bonding proximal histidine [START_REF] Peisach | An interim report on electronic control of oxygenation of heme proteins[END_REF]. In CcP, the side-chain carboxylate of Asp235 is H-bonded to both Trp191 and His175, the proximal ligand of the heme iron. The strong H-bond is believed to result in increased electron density on the porphyrin system, which would aid in stabilizing the heme and radical centers in the transition complex. The globins, typified by h-Mb, lack this strong hydrogen bonding to the proximal histidine and exhibit a longer Fe-N h bond. Moreover, the orientation of the imidazole plane with respect to the pyrrole nitrogen plane is such that the proximal histidine is in a fully eclipsed rotameric conformation that precludes optimal overlap of the iron axial and histidine nitrogen orbitals [START_REF] Yang | Crystal structures of CO-, deoxy-and met-myoglobins at various pH values[END_REF][START_REF] Lionetti | X-ray crystal structure of the ferric sperm whale myoglobin: imidazole complex at 2.0 A resolution[END_REF].

In conclusion, the picture that emerges from the comparison of proximal sites among Tf-trHb, typical globins and heme peroxidases is that a combination of proximal effects may be responsible for the tuning of the iron histidine bond distance which in turn is a key determinant for the heme reactivity. In this framework, the unique architecture of the Tf-trHb proximal site lies in between globin and peroxidases. The short iron-histidine distance implies a peroxidase-like environment that suggests a catalytic role towards hydrogen peroxide in truncated hemoglobins. As a matter of fact, a robust peroxidase-like activity has been recently demonstrated in Tf-trHb and in the highly similar truncated

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globin from Mycobacterium tuberculosis [START_REF] Ouellet | Reaction of Mycobacterium tuberculosis truncated hemoglobin O with hydrogen peroxide: evidence for peroxidatic activity and formation of protein-based radicals[END_REF][START_REF] Torge | Peroxidase-like activity of Thermobifida fusca hemoglobin: The oxidation of dibenzylbutanolide[END_REF]. In turn, in Tf-trHb, the short iron-histidine bond is not the consequence of a strong hydrogen bonding to the N δ of the imidazole ring, as is commonly observed in genuine peroxidases. Thus, from the point of view of the proximal site properties, Tf-trHb can be considered as an hybrid between a globin and a peroxidase whereas its biochemical function most likely corresponds to that of the latter protein.

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A C C E P T E D M A N U S C R I P T

  is associated with the first coordination shell around the iron atom. It comprises the four nitrogen atoms of the porphyrin ring, and one nitrogen atom of the proximal histidine. The second set of peaks, in the distance range between 2 and 3.5 Å contains all of the single and multiple-scattering contributions associated with the carbon atoms in the second coordination shell of both the porphyrin plane and the proximal histidine. The third set of peaks beyond 3.5 Å is mainly due to the single-and multiplescattering contributions associated with the third shell of the tetrapyrrole macrocycle atoms. Simple inspection of the Fourier Transformed (FT) spectra offers interesting insight into the structural changes between h-Mb and Tf-trHb. It can be noticed that the first peak of Tf-trHb shows a lower intensity and shorter distance position as compared to h-Mb. This result is in agreement with the blue-shift of the XANES edges and suggests a contraction of the first-shell average distance in Tf-trHb as compared to h-Mb. EXAFS results. A quantitative estimate of the distances of the iron atom from the pyrrolic and histidine nitrogen atoms can be obtained from the analysis of the EXAFS data by means of the GNXAS program [7,19]. The results of the fitting procedure applied to h-Mb EXAFS data are shown in the left panel of Figure 3. The spectrum is dominated by the first-shell two-body signals associated with the

Figure 1 .

 1 Figure 1. IR spectra. Near infrared absorption spectra of deoxygenated Tf-trHb (solid line) in comparison with h-Mb (dashed line). Spectra were measured at 293 K (panel A) and 77 K (panel in 3:1 glycerol/buffer mixtures. Protein concentration was 1.1 mM heme, buffer was 50 mM phosphate at pH 7.0 in the presence of 20 mM sodium dithionite.

Figure 2 .

 2 Figure 2. XAS spectra. Upper panel: XANES spectra of h-Mb (dotted line) and Tf-trHb (solid line), and [Tf-trHb -h-Mb] difference spectrum (gray solid line). Lower panel: Comparison between their Fourier Transform of the experimental EXAFS spectra for h-Mb (dotted line) and Tf-trHb (solid line).

Figure 3 .

 3 Figure 3. EXAFS analysis. Fit of the h-Mb and Tf-trHb EXAFS spectra (left and right upper panels, respectively). From the top to the bottom of each panel, the following curves are reported: the Fe-N p , Fe-N h and Fe-C h first-shell single-scattering signals, the Fe-N p -C 14 total three-body signals, and the Fe-N p -C 14 -C 23 total four-body signals. The total theoretical signals (solid lines) are compared with the experimental spectra (dotted lines) and the residual curve. The lower panels show the nonphase-shiftcorrected Fourier transforms of the experimental data (dotted line), of the total theoretical signal (solid line), and of the residual curves (dashed-dotted line).

Figure 4 .

 4 Figure 4. XANES analysis. Best fit of the XANES difference spectrum [Tf-trHb -h-Mb]. The experimental spectrum is the dotted line while the theoretical curve is the solid one.

Figure 5 .

 5 Figure 5. Close up wiev of the proximal site in Tf-trHb. The X-ray structure (2BMM) of ferric Tf-trHb is characterized by a triad of aminoacids surrounding the proximal histidine (A). The plane of the imidazole ring lies perpendicular and in a fully staggered position with respect to the four pyrrole nitrogen (B).

Table 1 . h-Mb structural parameters.

 1 Structural parameters derived from the EXAFS analysis and from XRD data for h-Mb. Statistical errors are reported in parenthesis.

		Range of crystallographic	EXAFS	EXAFS
		distances (Å) [a]	distance (Å)	Bond variance (Å 2 )
	Fe -N p	2.04-2.11	2.06 (1)	0.005
	Fe -C h	3.36-3.46	3.44(2)	0.004
	C 14 -C 23	1.42-1.48	1.39 (1)	0.011
	Fe -N h	2.14	2.11(2)	0.007

[a] Vojtechovsky, J.; Berendsen, J.; Chu, K.; Schlichting, I.; Sweet, R. M. Biophys. J. 1999, 77, 2153-2174. PDB code: 1A6N

Table 2 . Tf-trHb structural parameters

 2 Structural parameters derived from the EXAFS and XANES analysis for Tf-trHb. Statistical errors are reported in parenthesis.

		EXAFS	EXAFS	XANES
		distance (Å)	bond variance (Å 2 )	distance (Å)
	Fe -N p	2.05(1)	0.006	2.07(3)
	Fe -C h	3.43(1)	0.004	--
	C 14 -C 23	1.34 (1)	0.012	--
	Fe -N h	1.90 (2)	0.008	1.93(3)

Table 3

 3 Fe -N h distance in different globins and peroxidases as determined by XAS amd XRD.

	Protein	Fe -N h (Å)
	Dehaloperoxidase 43	2.18

FIG.5.

ACKNOWLEDGMENT Financial support by the Italian Ministry of University and Research [Linea D1 "ex-60%" 2007-2008 Università Cattolica Sacro Cuore] is gratefully acknowledged. This work was also supported by CASPUR with the Standard HPC Grant 2009 entitled: "A combined X-ray absorption spectroscopy, Molecular Dynamics simulations and Quantum Mechanics calculation procedure for the structural characterization of ill-defined systems". We acknowledge the European Synchrotron Radiation Facility for provision of synchrotron radiation facilities and the staff of BM30B

for the helpful support. We are grateful to Stefano Della Longa for many helpful discussions. Project from Fondazione Cenci Bolognetti to A.B. is also gratefully acknowledged.


Alessandro Spilotros

Matteo Levantino

Antonio Cupane
email: cupane@fisica.unipa.it

Conformational substates of ferricytochrome c revealed by combined optical absorption and electronic circular dichroism spectroscopy at cryogenic temperature

Conformational substates of ferricytochrome c revealed by combined optical absorption and electronic circular

dichroism spectroscopy at cryogenic temperature

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ACCEPTED MANUSCRIPT 2 ABSTRACT. We have investigated the heterogeneity of the Fe(III)-Met80 linkage of horse heart ferricytochrome c by probing the 695 nm charge transfer band with absorption and electronic circular dichroism (ECD) spectroscopy. In order to verify the connection between conformational substates of the Fe(III)-Met80 linkage and the 695 nm band spectral heterogeneity, we have performed experiments as a function of pH (neutral and acidic) and temperature (room and 20 K).

At room temperature, the ECD spectrum is blue shifted with respect to the absorption one; the shift is more pronounced at acidic pH and is compatible with the presence of sub-bands. ECD measurements at 20 K highlighted the heterogeneous nature of the 695 nm band and provided direct experimental evidence for the presence of sub-bands. Indeed, while the absorption spectra remained deceivingly unstructured, the ECD spectra showed well resolved peaks and shoulders. A consistent fit of the 20 K absorption and ECD spectra showed that five Gaussians (each centered at the same frequency in the absorption and ECD spectrum) are able to reproduce the observed lineshapes. A careful analysis of frequency shifts and intensity ratios of these sub-bands enabled us to identify at least three distinct sub-bands arising from taxonomic conformational substates of the Fe(III)-Met80 linkage. In view of the major influence of the Fe(III)-Met80 linkage on the redox potential of ferricytochrome c, we speculate that these spectrally distinguishable substates may have different functional roles.

INTRODUCTION

The three dimensional native structure of proteins is characterized by a highly complex energy landscape in which the global minimum (corresponding to the native conformation) is made up of a large number of quasi-isoenergetic minima (corresponding to the so-called conformational substates). A hierarchical organization of the landscape has been suggested (1, 2), whereby a small number of substates (the taxonomic substates) is, in turn, structured in a large number of higher tier(s) substates (the statistical substates). From the spectroscopic point of view, the statistical substates contribute to the rather large spectral Gaussian widths of protein absorption bands (3-5), while taxonomic substates are responsible for their sub-bands structure, like e.g. in the case of the near infrared charge transfer bands of deoxyhemoglobin, deoxymyoglobin and superoxide dismutase [START_REF] Cupane | Low-temperature optical spectroscopy of native and azide-reacted bovine Cu,Zn superoxide dismutase. A structural dynamics study[END_REF][START_REF] Gilch | Conformational substates of the Fe2+-His F8 linkage in deoxymyoglobin and hemoglobin probed in parallel by the Raman band of the Fe-His stretching vibration and the near infrared absorption band III[END_REF], and of the infrared stretching band of CO bound to myoglobin [START_REF] Frauenfelder | The role of structure, energy landscape, dynamics, and allostery in the enzymatic function of myoglobin[END_REF][START_REF] Ormos | Inhomogeneous broadening in spectral bands of carbonmonoxymyoglobin. The connection between spectral and functional heterogeneity[END_REF][START_REF] Muller | between the taxonomic substates and protonation of histidines 64 and 97 in carbonmonoxy myoglobin[END_REF]. Protein conformational substates have substantial functional relevance, since they are responsible for the heterogeneity observed in many dynamical processes like the binding of ligands to the active site; a role of conformational substates in protein folding/unfolding processes has also been suggested [START_REF] Frauenfelder | The energy landscapes and motions of proteins[END_REF][START_REF] Sullivan | Conformation spaces of proteins[END_REF][START_REF] Frauenfelder | Protein folding is slaved to solvent motions[END_REF].

Cytochrome c is a small globular protein with a molecular weight of about 12.4 kDalton. It contains a single heme group whose central iron atom is coordinated to a histidine residue (His18, proximal side) and a methionine residue (Met80, distal side), besides the four pyrrole nitrogens of the porphyrin. Its main physiological role is to mediate the electron transfer from cytochrome c reductase to cytochrome c oxidase. The absorption spectrum of cytochrome c in the ferric state is characterized by the well known B (Soret) and Q bands and by a weaker charge transfer (CT) band in the near infrared at about 695 nm [START_REF] Schejter | The 695-millimicron Band of Ferricytochrome C and Its Relationship to Protein Conformation[END_REF]. This CT band had initially been assigned to an A 2u (porphyrin) → d z 2 (Fe 3+ ) transition involving a molecular orbital of the porphyrin ring and an atomic orbital of the iron [START_REF] Eaton | Electronic spectrum of single crystals of ferricytochrome-c[END_REF]. However, this band is absent in low-spin ferric forms of cytochrome c whenever the Met80 residue is replaced by a different ligand [START_REF] Rux | Magnetic circular dichroism spectroscopy as a probe of axial heme ligand replacement in semisynthetic mutants of cytochrome c[END_REF][START_REF] Mcknight | Identification of Charge-Transfer Transitions in the Optical-Spectrum of Low-Spin Ferric Cytochrome-P-450 Bacillus-Megaterium[END_REF]. This observation indicated

A C C E P T E D M A N U S C R I P T

that the 695 nm band should be assigned to a S(Met80) → Fe 3+ transition involving atomic orbitals of the methionine sulfur and the iron ion. Indeed, the dependence of this band on the axial ironsulfur linkage has made the band a frequently used tool to monitor the presence of intermediates in the folding/unfolding transition of ferricytochrome c [START_REF] Greenwood | Studies on ferricytochrome c. I. Effect of pH, ionic strength and protein denaturants on the spectra of ferricytochrome c[END_REF][START_REF] Moore | A method for investigating the effect of temperature on the 695 nm band of insoluble cytochrome c[END_REF][START_REF] Myer | Ferricytochrome c. Refolding and the methionine 80-sulfur-iron linkage[END_REF][START_REF] Pearce | Mutation-induced perturbation of the cytochrome c alkaline transition[END_REF][START_REF] Pryse | Kinetics and mechanism of the folding of cytochrome c[END_REF][START_REF] Schweitzer-Stenner | Conformational substates of horse heart cytochrome c exhibit different thermal unfolding of the heme cavity[END_REF]. Structural heterogeneity of the heme pocket is expected to result in a spectral heterogeneity of the 695 nm band. Schweitzer-Stenner and his group have measured the absorption and electronic circular dichroism (ECD) spectra of ferricytochrome c in the 283-333 K temperature interval [START_REF] Dragomir | Optical band splitting and electronic perturbations of the heme chromophore in cytochrome c at room temperature probed by visible electronic circular dichroism spectroscopy[END_REF]. Their data show that the absorption band at 695 nm is shifted with respect to the corresponding ECD band, both bands being highly asymmetric. The authors interpreted this finding as an evidence of the presence of taxonomic conformational substates and proposed the existence of at least three sub-bands (S2, S3, and S4 in their terminology). A frequency shift between absorption and ECD can be attributed to the heterogeneous nature of the spectra; indeed, if the spectra are heterogeneous and if the underlying sub-bands have different rotational and oscillator strengths, the frequency shift can be easily rationalized. A similar argument has been already used by Schweitzer-Stenner and collaborators also in the case of the Soret band of several myoglobin derivatives [START_REF] Schweitzer-Stenner | Asymmetric band profile of the Soret band of deoxymyoglobin is caused by electronic and vibronic perturbations of the heme group rather than by a doming deformation[END_REF], although in that case the origin of spectral heterogeneity is not the presence of conformational substates, but an x-y splitting due to symmetry lowering distortions. Although appealing, such evidences for sub-bands remain indirect since no clear peaks or shoulders could be resolved in the spectra close to room temperature, especially in the ECD ones. This consideration motivated the present study: in order to resolve the spectral heterogeneity of the 695 nm band in a clear-cut way, we have performed absorption and ECD experiments at 290 K and 20 K, exploiting the widely known fact that optical absorption bands of proteins become narrower on lowering the temperature [START_REF] Cupane | Low temperature optical absorption spectroscopy: an approach to the study of stereodynamic properties of hemeproteins[END_REF][START_REF] Cordone | Thermal-Behavior of the 760-Nm Absorption-Band in Photodissociated Sperm Whale Carbonmonoxymyoglobin at Cryogenic Temperature -Dependence on External Medium[END_REF][START_REF] Cupane | Dynamics of various metal-octaethylporphyrins in solution studied by resonance Raman and low-temperature optical absorption spectroscopies. Role of the central metal[END_REF][START_REF] Levantino | The importance of vibronic perturbations in ferrocytochrome c spectra: A reevaluation of spectral properties based on low-temperature optical absorption, resonance Raman, and molecular-dynamics simulations[END_REF].

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MATERIALS AND METHODS

Horse heart cytochrome c was obtained from Sigma-Aldrich. To ensure full oxidation of the iron, a 4-fold molar excess of potassium ferricyanide was added and subsequently removed by prolonged dialysis. The protein solution was mixed with phosphate buffer and glycerol to obtain a sample of 1 mM ferricytochrome c in 65% (vol/vol) glycerol/water and 0.1 M phosphate buffer. The sample, contained in a 1 cm pathlength PMMA cuvette, remained homogeneous and transparent on lowering the temperature down to 20 K. Measurements have been performed at two different pH values, 4 and 7, in order to modulate the spectral heterogeneity while preserving intact functional and spectral properties of the protein [START_REF] Greenwood | Studies on ferricytochrome c. I. Effect of pH, ionic strength and protein denaturants on the spectra of ferricytochrome c[END_REF][START_REF] Verbaro | The pHdependence of the 695 nm charge transfer band reveals the population of an intermediate state of the alkaline transition of ferricytochrome c at low ion concentrations[END_REF].

Absorption spectra were obtained with a Jasco V-570 spectrophotometer operated with a scan speed of 40 nm/min, 1 nm bandwidth, and 1 sec response time. The experimental setup for the cryogenic temperature optical absorption measurements has been described in previous publications [START_REF] Levantino | The importance of vibronic perturbations in ferrocytochrome c spectra: A reevaluation of spectral properties based on low-temperature optical absorption, resonance Raman, and molecular-dynamics simulations[END_REF][START_REF] Santangelo | Ferricytochrome c encapsulated in silica hydrogels: correlation between active site dynamics and solvent structure[END_REF]. For ECD measurements at cryogenic temperatures a Jasco J-715 spectropolarimeter was equipped with a home-built apparatus comprising an Oxford Optistat cryostat and an Oxford ITC 503 temperature controller; spectral parameters were as follows: scan speed = 50 nm/min, bandwidth = 2 nm, response time = 1 sec; 20 accumulations resulted in spectra having good signalto-noise ratio. The frequency calibration of the spectrophotometer and spectropolarimeter were checked by measuring the absorption spectrum of a neodymium glass and the ECD spectrum of a camphor standard, respectively. We estimate that both instruments are calibrated within 20 cm -1 .

At each temperature, the absorption and ECD spectra of a 65% (vol/vol) glycerol/water mixture containing 0.1 M phosphate buffer were measured and subtracted from the corresponding spectra of the protein containing sample. Control experiments made at room temperature showed that, at both pH values, the presence of glycerol does not introduce any relevant spectral alteration in both the absorption and ECD spectral profiles.

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RESULTS AND DISCUSSIONS

Figure 1 reports the absorption and ECD bands at around 695 nm (~14400 cm -1 ) measured at room temperature and normalized with respect to their peaks. Since both bands lie on the tail of a much more intense band, a suitable baseline has been subtracted from each spectrum: while a cubic baseline has been subtracted from the absorption spectra, a linear baseline was enough in the case of the ECD ones (see panels c and f of figure 2 being, as expected, more intense and narrower, remains deceivingly unstructured and gives no direct evidence for the presence of sub-bands. We note that the clear asymmetry of the ~14250 cm -1 absorption peak is not a proof of the presence of sub-bands since this could be related to the coupling of the underlying electronic transition with low frequency vibrational modes [START_REF] Cupane | Dynamics of various metal-octaethylporphyrins in solution studied by resonance Raman and low-temperature optical absorption spectroscopies. Role of the central metal[END_REF][START_REF] Santangelo | Ferricytochrome c encapsulated in silica hydrogels: correlation between active site dynamics and solvent structure[END_REF].

Following the idea of Schweitzer-Stenner and coworkers [START_REF] Dragomir | Optical band splitting and electronic perturbations of the heme chromophore in cytochrome c at room temperature probed by visible electronic circular dichroism spectroscopy[END_REF][START_REF] Verbaro | The pHdependence of the 695 nm charge transfer band reveals the population of an intermediate state of the alkaline transition of ferricytochrome c at low ion concentrations[END_REF][START_REF] Shah | Structural changes of horse heart ferricytochrome C induced by changes of ionic strength and anion binding[END_REF], we complemented the absorption data with ECD data on the very same samples. The ECD spectrum of ferricytochrome c at 20 K and pH 7 (Figure 2d) shows an asymmetric peak at ~14500 cm -1 and a clearly resolved peak at ~15000 cm -1 ; the high frequency wing of the spectrum is characterized by a positive peak a ~15400 cm -1 and negative one at ~15800 cm -1 . The residual marked asymmetry of the peak at 14500 cm -1 seems to suggest the presence of further sub-bands. Indeed, the ECD spectrum of the sample at pH 4 (Figure 2e) gives direct evidence of this hypothesis. This spectrum is very well resolved: it shows a shoulder at ~14250 cm -1 , a peak at ~14500 cm -1 , and a clearly resolved peak at ~15000 cm -1 ; as for the pH 7 sample, the high frequency wing is characterized by peaks at approximately 15400 and 15800 cm -1 . Note that the small band at about 13650 cm -1 reported by Dragomir et al. [START_REF] Dragomir | Optical band splitting and electronic perturbations of the heme chromophore in cytochrome c at room temperature probed by visible electronic circular dichroism spectroscopy[END_REF] and named S1 by the authors, is never observed in our experiments (both in absorption and ECD) even at 20 K.

Based on direct evidence given by the 20 K ECD spectrum at pH 4, we conclude that the 695 nm absorption and ECD bands of ferricytochrome c are composed of several spectrally distinguishable sub-bands having different oscillator and rotational strengths. These spectroscopically distinct subbands likely correspond to different structures, i.e. to different conformational substates. Note that this spectral heterogeneity is already evident by inspection of the raw data, and it does not depend on the details of the baseline subtraction procedure.

To obtain quantitative information on the composite structure of the 695 nm band, and to check the consistency of our hypotheses, we analyzed simultaneously the absorption and ECD spectra measured at 20 K between 13500 and 16500 cm -1 ; five different Gaussian components sharing the same peak frequencies in the absorption and ECD spectra have been used to reproduce the observed spectral profiles. Figure 3 shows the result of the fitting procedure and demonstrates that the absorption and ECD spectra can be well fitted with a single set of five Gaussian sub-bands that are named A1 to A5 in the figure. Best fit parameters for the areas (I k , k=1...5), peak frequencies (ν k , k=1...5), and widths (σ k , k=1...5) of the five Gaussian components are reported in Table 1. The fractional areas of the sub-bands are reported in Figure 4.

The data in Figures 3 and4 and in Table 1 show that:

-The area of the sub-bands depends sizably on pH, particularly in the case of the ECD spectrum: indeed, at pH 4, the ratio I 2 /I 1 is ~1.37 (i.e. the rotational strength of A1 is smaller than that of A2), while at pH 7 it is ~0.83 (i.e. the rotational strength of A1 is larger than that of A2). This makes the ECD spectrum at pH 4 more resolved than the corresponding absorption spectrum.

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(Figure 4): e.g., in the case of the sample at pH 7, I 2 /I 1 is ~0.45 and ~0.83 for the absorption and ECD spectrum, respectively, while, at pH 4, I 2 /I 1 is ~0.50 for absorption and ~1.37 for ECD.

-Frequency separations between sub-bands depend weakly on pH and are ∼300 cm -1 (ν 2 -ν 1 ), ∼700 cm -1 (ν 3 -ν 1 ), ∼1100 cm -1 (ν 4 -ν 1 ), and ∼1530 cm -1 (ν 5 -ν 1 ).

-Although they were left as free parameters in the fitting procedure, the widths of the sub-bands in the ECD spectrum are very similar to those of the absorption one (Table 1). A significant discrepancy is observed only in the case of the A4 sub-band (at ~15400 cm -1 ) whose width is, however, very sensitive to the details of the baseline subtraction procedure, especially for the ECD spectrum.

On the basis of our data we conclude that the spectrally distinguishable A1, A2, and A3 components are different sub-bands of the same p (S) → d π (Fe 3+ ) transition ( 16) arising from different cytochrome c taxonomic conformational substates. Supporting evidence for this conclusion comes from the following arguments:

1) Though it would seem possible to assign the different sub-bands to different p(S) → d π (Fe 3+ ) transitions, the observed sub-bands frequency differences are at variance with such conclusion. In fact, the magnetic circular dichroism (MCD) data reported by McKnight et al. [START_REF] Mcknight | Identification of Charge-Transfer Transitions in the Optical-Spectrum of Low-Spin Ferric Cytochrome-P-450 Bacillus-Megaterium[END_REF] suggest a wavenumber difference of nearly 1000 cm -1 between, e.g., p y → d yz and p z → d yz , while the ν 2 -ν 1 and ν 3 -ν 1 frequency differences are sizably smaller than 1000 cm -1 .

2) Our spectra rule out the hypothesis of a vibronic origin of these sub-bands since the I 2 /I 1 and I 3 /I 1 ratios scale differently in the absorption and ECD spectra. As a matter of fact, it is possible to demonstrate that vibronic replicas in the ECD spectra are simply proportional to those in the absorption ones [START_REF] Moffit | Optical Activity in Absorbing Media[END_REF][START_REF] Caldwell | Vibronic theory of circular dichroism[END_REF].

Coming to the origin of the high-frequency wing centered at ~15400 cm -1 (~650 nm) and corresponding to the A4 and A5 Gaussian components, our data do not allow us to make an

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unambiguous assignment since the arguments used above cannot be applied. Indeed, the ν 4 -ν 1 and ν 5 -ν 1 frequency separations are greater than 1000 cm -1 , and indeterminations in the baseline subtraction procedure (that has a stronger influence in this frequency region) do not allow us to draw definitive conclusions on the intensity scaling. The high frequency wing could, thus, well originate also from a different CT transition, or from porphyrin skeletal vibrations coupled to the main CT transition [START_REF] Eaton | Electronic spectrum of single crystals of ferricytochrome-c[END_REF][START_REF] Mcknight | Identification of Charge-Transfer Transitions in the Optical-Spectrum of Low-Spin Ferric Cytochrome-P-450 Bacillus-Megaterium[END_REF][START_REF] Hu | Complete Assignment of Cytochrome-C Resonance Raman-Spectra Via Enzymatic Reconstitution with Isotopically Labeled Hemes[END_REF]. As far as the first hypothesis is concerned, the most reasonable assignment, following the arguments of McKnight et al. [START_REF] Mcknight | Identification of Charge-Transfer Transitions in the Optical-Spectrum of Low-Spin Ferric Cytochrome-P-450 Bacillus-Megaterium[END_REF], is that of a p z (S) → d yz (Fe 3+ ) transition as opposed to the p y (S) → d yz (Fe 3+ ) transition at 695 nm. According to such assignment the low frequency transition is expected to be z-polarized, while the high frequency one should be x,y-polarized. Accurate low temperature polarized absorption, resonance Raman with excitation in the near-infrared, and spectral hole burning experiments on the 695 nm band would be needed in order to make an unambiguous assignment of the high frequency side-bands.

In the absence of direct structural evidence, we can only speculate about the structural origin of the A1, A2, and A3 sub-bands. In view of the charge-transfer character of the associated transition, the iron-methionine linkage must obviously be involved. Different orientations of the S-Fe bond might be a plausible origin. However, the pH dependence of sub-bands intensity ratios suggests that they may be related to substates having different protonation states of residues pointing inside the heme pocket (close to the iron-methionine linkage). Finally, since the spectral heterogeneity is more pronounced for ECD than for absorption, a difference in the overall conformation and chirality of the heme pocket is also expected. In view of the major influence of the Fe 3+ -Met80 linkage on the redox potential of the protein (34), we speculate that the above spectrally distinguishable substates may as well have different functional roles. Such hypothesis should be tested and taken into account also in future studies employing the 695 nm band as a conformational marker sensitive to the folding/unfolding of cytochrome c. shows that the ferricytochrome c spectrum in the 13500-16500 cm -1 (610-740 nm) region can be decomposed in five sub-bands that we label A1-A5. Detailed inspection of frequency shifts and intensity ratios enabled us to identify components A1, A2 and A3 as sub-bands of the same p y (S) → d yz (Fe 3+ ) electronic transition, arising from different cytochrome c taxonomic substates.

For A4 and A5 the assignment is more ambiguous and needs further experimental investigations with different spectroscopic techniques.

The present results highlight the potentialities of combining low temperature absorption and ECD for the resolution of highly heterogeneous protein spectra. 1.

FIGURES AND TABLE CAPTIONS

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  below). The peak of the ECD band is blue shifted with respect to the absorption one in the ferricytochrome c sample at pH 7 (Figure1a,b); this effect is even larger in an analogous sample at pH 4 (Figure1c,d). A frequency shift between an absorption band and its corresponding ECD one can be interpreted as an evidence of spectral heterogeneity as already mentioned in the Introduction. To further investigate such heterogeneity we have performed measurements at cryogenic temperatures. Figure2shows the absorption spectra at 290 and 20 K of ferricytochrome c at pH 7 (panel a) and pH 4 (panel b); the baseline subtracted from the spectrum measured at 20 K (pH 4) is depicted in panel c. The low temperature absorption spectrum, although

  of low temperature optical absorption and ECD spectroscopies unambiguously

Figure 1 .

 1 Figure 1. Room temperature absorption and ECD spectra of horse heart ferricytochrome c at pH 7

Figure 2 .Figure 3 .

 23 Figure 2. Absorption (panel a-c) and ECD (panel d-f) spectra of ferricytochrome c at 20 K (black

Figure 4 .

 4 Figure 4. Fractional areas of the Gaussian sub-bands for the absorption (panel a) and ECD (panel b)

Table 1 .

 1 Best fit parameters of the five Gaussian sub-bands (A1-A5) used to fit simultaneously the absorption and ECD spectra at 20 K. The typical uncertainty on the best fit values for the areas (I k , k=1...5) is ≤0.005, while that for frequencies (ν k , k=1...5) and widths (σ k , k=1...5) is ≤20 cm -1 .

Table 1 .

 1 Best fit parameters of the five Gaussian sub-bands (A1-A5) used to fit simultaneously the absorption and ECD spectra at 20 K. The typical uncertainty on the best fit values for the areas (I k , k=1...5) is ≤0.005, while that for frequencies (ν k , k=1...5) and widths (σ k , k=1...5) is ≤20 cm -1 .

	1.	2. Abs pH7	3. ECD pH 7	4. Abs pH 4	5. ECD pH 4
	6. I 1 (a.u.)	7. 0.410	8. -0.323	9. 0.425	10. -0.244
	11. I 2 (a.u.)	12. 0.185	13. -0.268	14. 0.211	15. -0.335
	16. I 3 (a.u.)	17. 0.159	18. -0.170	19. 0.178	20. -0.200
	21. I 4 (a.u.)	22. 0.196	23. 0.107	24. 0.210	25. 0.081
	26. I 5 (a.u.)	27. 0.129	28. -0.088	29. 0.129	30. -0.081
	31. ν ν ν ν 1 (cm -1 )	32. 14310		33. 14280
	34. ν ν ν ν 2 (cm -1 )	35. 14600		36. 14590
	37. ν ν ν ν 3 (cm -1 )	38. 15000		39. 15000
	40. ν ν ν ν 4 (cm -1 )	41. 15390		42. 15420
	43. ν ν ν ν 5 (cm -1 )	44. 15830		45. 15810
	46. σ σ σ σ 1 (cm -1 )	47. 160	48. 150	49. 160	50. 150
	51. σ σ σ σ 2 (cm -1 )	52. 160	53. 150	54. 160	55. 150
	56. σ σ σ σ 3 (cm -1 )	57. 160	58. 150	59. 160	60. 150
	61. σ σ σ σ 4 (cm -1 )	62. 320	63. 130	64. 320	65. 140
	66. σ σ σ σ 5 (cm -1 )	67. 150	68. 150	69. 150	70. 150

ACKNOWLEDGMENT. We thank Prof. M. Leone for providing very helpful discussions, Dr. E.

Vitrano for generous help in developing the system for ECD measurements at cryogenic temperatures, and Mr. G. Napoli of the cryogenic laboratory for technical help.


Yoran Margaron

Luciana Bostan

Jean-Yves Exposito

Maryline Malbouyres

Ana- Maria Trunfio-Sfarghiu

Yves Berthier

Claire Lethias
email: c.lethias@ibcp.fr

Tenascin-X increases the stiffness of collagen gels without affecting fibrillogenesis

Keywords: extracellular matrix, Ehlers-Danlos Syndrome, tenascin-X, collagen, elastic modulus

Tenascin-X is an extracellular matrix protein whose absence leads to an Ehlers-Danlos Syndrome in humans, mainly characterised by connective tissue defects including the disorganisation of fibrillar networks, a reduced collagen deposition, and modifications in the mechanical properties of dense tissues. Here we tested the effect of tenascin-X on in vitro collagen fibril formation. We observed that the main parameters of fibrillogenesis were unchanged, and that the diameter of fibrils was not significantly different when they were formed in the presence of tenascin-X. Interestingly, mechanical analysis of collagen gels showed an increased compressive resistance of the gels containing tenascin-X, indicating that this protein might be directly involved in determinating the mechanical properties of collagen-rich tissues in vivo.

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INTRODUCTION

Tenascin-X (TNX) is a huge extracellular matrix glycoprotein that has a multidomain structure consisting of an N-terminal region involved in oligomerisation, a series of epidermal growth factor (EGF)-like repeats, a variable number of fibronectin-type III (FNIII) modules and a C-terminal domain homologous to fibrinogen (Fbg) [START_REF] Jones | The tenascin family of ECM glycoproteins : structure, function, and regulation during embryonic development and tissue remodeling[END_REF]. Several observations suggest that TNX is involved in the deposition and/or stabilisation of collagen fibrils, and in influencing the mechanical properties of connective tissues. Indeed, TNX deficiency leads to the appearance of an Ehlers-Danlos Syndrome in humans [START_REF] Burch | Tenascin-X deficiency is associated with Ehlers-Danlos syndrome[END_REF]. Major clinical symptoms consist of skin hyperextensibility and joint laxity, while ultrastructural analyses reveal abnormalities in collagen fibril networks and elastic fibre morphology. Mice deficient in TNX partly reproduced this phenotype [START_REF] Mao | Tenascin-X deficiency mimics Ehlers-Danlos syndrome in mice through alteration of collagen deposition[END_REF], and fibroblasts isolated from these mice failed to deposit collagen in cell culture [START_REF] Schalkwijk | A recessive form of the Ehlers-Danlos syndrome caused by tenascin-X deficiency[END_REF]. Moreover, we have previously demonstrated that TNX is localised at the surface of collagen fibrils within tissues [START_REF] Lethias | Flexilin : a new extracellular matrix glycoprotein localized on collagen fibrils[END_REF]. This association might be explained by the interaction of TNX with several molecular components of these fibrils i.e. decorin, fibrillar and FACIT collagens [START_REF] Elefteriou | Binding of tenascin-X to decorin[END_REF][START_REF] Lethias | A model of tenascin-X integration within the collagenous network[END_REF][START_REF] Egging | Interactions of human tenascin-X domains with dermal extracellular matrix molecules[END_REF]. More recently, it was shown that TNX has elastic properties, due to the extension/refolding of its constitutive FNIII modules [START_REF] Jollymore | Nanomechanical properties of tenascin-X revealed by single-molecule force spectroscopy[END_REF]. Taken together, these data suggest that TNX might affect collagen fibrillogenesis and/or act as a bridge between collagen fibrils and consequently modulate the mechanical properties of connective tissues. In order to test these hypotheses, we analysed the consequences of TNX-collagen interaction in vitro, by studying the influence of TNX on collagen fibrillogenesis, and by measuring the mechanical properties of collagen gels polymerised in the presence of TNX.

MATERIALS AND METHODS

Production and purification of recombinant TNX

Recombinant full length bovine TNX was produced in mammalian HEK293 cells as previously described [START_REF] Lethias | A model of tenascin-X integration within the collagenous network[END_REF][START_REF] Lethias | Identification and characterization of a conformational heparin-binding site involving two fibronectin type III modules of bovine tenascin-X[END_REF]. TNX was further purified from the culture medium by two chromatographic steps: firstly on heparin-Sepharose (GE Healthcare), and secondly on Q-Sepharose (GE Healthcare) [START_REF] Lethias | A model of tenascin-X integration within the collagenous network[END_REF]. TNX fractions were dialyzed against phosphate-buffered saline (PBS) and stored at -80°C. Protein concentration was determined using the QuantiPro BCA assay kit (Sigma) according to the manufacturer's instructions.

Preparation of acid-soluble collagen

Acid-soluble collagen was extracted from tail tendons of young rats using standard procedures. All steps were performed at 4°C. Tendons were dissected in 0.2 M NaCl, then dilacerated and transferred to 0.5 M acetic acid/0.2 M NaCl for 48 h. The extract was centrifuged at 20,000 g for 20 min, and the resulting supernatant was dialysed against 0.5 M acetic acid/0.9 M NaCl. After centrifugation (20,000 g, 20 min), the pellet was treated with 70% ethanol, dried in a sterile atmosphere, and dissolved in 0.001 M HCl. Collagen concentration was determined by both QuantiPro BCA assay kit and amino-acid analysis.

Collagen fibrillogenesis assays

Collagen solutions (3 to 5.6 mg/mL) were neutralised on ice using 0.5 M NaOH and diluted to a final concentration of 1 mg/mL by the addition of PBS containing recombinant TNX or control protein. Fibrillogenesis was initiated by warming the solutions to 37°C in a thermostated cuvette, and gelation was detected by monitoring turbidity at 400 nm in a DU 640 spectrophotometer (Beckman). Assays were performed in triplicate and a minimum of three independent experiments were carried out. Pepsin-extracted bovine collagen was purchased from BD Biosciences (Le Pont de Claix, France).

Scanning electron microscopy

Collagen gels, prepared as described for the fibrillogenesis assay, were briefly rinsed in 0.15 M sodium cacodylate (Sigma) pH 7.4 then fixed for 1 hour in 2% glutaraldehyde (Electron Microscopy Sciences) diluted in the same buffer. After further rinsing, gels were treated for 45 min with 1% osmium tetroxide diluted in 0.1 M sodium cacodylate. Samples were then dehydrated by a series of graded ethanol solutions, impregnated in hexamethyldisilazane (Sigma), and air-dried. After coating with gold-palladium by sputtering, the gels were observed with a Hitachi S800 microscope (operating at 15 kV) at the "Centre Technologique des Microstructures" (Université de Lyon). Collagen fibril diameters were measured using NIH Image J software.

Mechanical tests

Neutralised collagen solutions were prepared as described in the fibrillogenesis section, and 10.5 mL aliquots were transferred to 60 mm diameter cell culture Petri dishes (Falcon). Gelation was carried out for 30 min at 37°C, then collagen gels were stored at 4°C in the presence of PBS until mechanical testing. Collagen gel samples had a cylindrical form, with diameter ~50 mm and height ~3 mm, and their mechanical resistance was measured by compression testing in an unconfined configuration.

Step-wise compression tests were performed with an Advanced Rheometric Expansion System (ARES) equipped with a maximum cell load of 20 N and controlled gap (hi) (TA Instruments). The load assembly consists of two stainless steel 50 mm diameter parallel plates above and below the sample. In order to control the boundary conditions, the stainless steel platens were sonicated twice for 10 min at 25°C in ethanol before use. For each test, a cycle of 4 strains was performed at an imposed speed deformation of 0.02 mm/s, the compression level representing 5% of the sample thickness. Every compression strain ramp was followed by a period of stress relaxation of 600 s. This period of stress relaxation was necessary to reach the balance of stress equilibrium characteristic to cartilage biphasic materials [START_REF] Ateshian | Finite deformation biphasic material properties of bovine articular cartilage[END_REF].

The normal force and the gap thickness were directly measured by the rheometer sensors. To reproduce the same initial load condition, the test began after having submitted each sample to a compressive force of 0.02 N followed by a 600 s relaxation period. Stressrelaxation curves were generated for each sample, including compressive and relaxation stresses, calculated as normal force divided by gel area, and were drawn as a function of time.

Stress-strain curves were generated for each sample by taking only into consideration the compression peak stress, where the compressive strain was calculated as (h0-hi)/h0 (where h0 is the initial gap). The linear regression slope of the stress-strain curve was used to estimate the apparent elastic modulus of each gel.

For each type of gel (collagen only, collagen and BSA, collagen and TNX), four samples were tested, and each test was repeated three times to verify the reproducibility of measurements.

A C C E P T E D M A N U S C R I P T

RESULTS AND DISCUSSION

Influence of TNX on collagen fibrillogenesis

The effect of TNX on collagen fibril formation was tested in vitro by monitoring turbidity during the gelation process. As shown in Fig. 1, TNX did not seem to affect collagen fibrillogenesis except at high concentrations where a slight delay in the initiation step was apparent. However, the same effect was obtained by the addition of a control protein (BSA), which does not specifically interact with fibrillar collagens. A careful examination of the fibrillogenesis parameters led to the same conclusion (Table I). Neither the lag phase, nor the fibrillogenesis rate or the absorbance plateau were significantly modified by the presence of TNX. These results are somehow contradictory with previous results [START_REF] Minamitani | Modulation of collagen fibrillogenesis by tenascin-X and type VI collagen[END_REF] showing a diminished lag phase and an increased absorbance plateau of collagen solutions when fibrillogenesis assays were done in the presence of TNX.

The first point of divergence that might explain the discrepancy between our results and those of Minamitani et al. [START_REF] Minamitani | Modulation of collagen fibrillogenesis by tenascin-X and type VI collagen[END_REF] concern the source of the collagen. Their collagen solution was from a commercial source, and gave rise to slow fibrillogenesis in control conditions (2h for their lag phase instead of 5 min in our experiments and elsewhere [START_REF] Sullivan | Matricellular hevin regulates decorin production and collagen assembly[END_REF][START_REF] Giudici | Mapping of SPARC/BM40/osteonectinbinding sites on fibrillar collagens[END_REF]). One explanation could be that the commercial collagen was pepsinised and consequently devoid of telopeptides, which have been shown to be important for fibrillogenesis [START_REF] Kuznetsova | Does the triple helical domain of type I collagen encode several molecular recognition and fiber assembly while telopeptides serve as catalytic domains?[END_REF]. To test this hypothesis, we performed fibrillogenesis assays with bovine pepsinised collagen and indeed observed a slower fibrillogenesis. However, even in these conditions, the presence of TNX did not modify the fibrillogenesis parameters compared to the control protein BSA (data not shown).

The differences observed might also result from the concentration and presence of FNIII repeats present in the recombinant TNX used during the fibrillogenesis assays. These discrepancies seem not to be due to the lower TNX-concentrations (0.2 to 0.5 µg/mL) used in Ref. 12, since we did not observe any differences in the fibrillogenesis parameters in this range of concentration (data not shown). Moreover, these authors used a small isoform lacking the central FNIII modules, whereas we produced the full-length protein comprising 30 FNIII domains. Interestingly, another group has shown that the 29 th FNIII module of TNX accelerates collagen fibrillogenesis [START_REF] Egging | Interactions of human tenascin-X domains with dermal extracellular matrix molecules[END_REF], but this modulatory effect seems to be masked in

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larger constructs. Taken together, these data indicate that the modulatory effets of TNX on collagen fibrillogenesis seems to be dependent on its constitutive domains.

With the aim of determining if TNX had an influence on the morphology of the collagen fibrils, we examined the gels by scanning electron microscopy after the gelation process was completed. The resulting micrographs are shown in Fig. 2, and no striking difference was highlighted by comparison of gels formed in the presence of TNX or control proteins (BSA, fibronectin). Consistent with this observation, measurement of collagen fibril diameter distribution did not show an effect of TNX on this parameter (Fig. 3).

Effect of TNX on the mechanical properties of collagen gels

Considering the interaction of TNX with fibrillar collagens [START_REF] Lethias | A model of tenascin-X integration within the collagenous network[END_REF][START_REF] Egging | Interactions of human tenascin-X domains with dermal extracellular matrix molecules[END_REF] and the elastic properties of TNX [START_REF] Jollymore | Nanomechanical properties of tenascin-X revealed by single-molecule force spectroscopy[END_REF], we decided to examine the physical behaviour of the collagen gels formed in the presence of TNX by rheometry, and to compare them to control gels consisting of collagen alone, or collagen and BSA.

The average stress-relaxation response under unconfined compression for the three types of gel is shown in Fig. 4A. The highest stress values were obtained for collagen/TNX gels while there was no obvious difference between collagen/BSA and collagen gels. Voytik-Harbin on collagen gels [START_REF] Kreger | Hyaluronan concentration within a 3D collagen matrix modulates matrix viscoelasticity, but not fibroblast response[END_REF]. This difference can be explained in two ways. The first concerns the boundary condition. In this study, the gel sample was free to slide on the stainless steel platen, which can decrease the apparent compressive elastic modulus compared to that obtained when the gel sample is fixed on the stainless steel platen. Secondly, the collagen concentration that we used is two-fold lower than in the previously published study

(1 mg/mL instead of 2 mg/mL). In agreement with the latter explanation, it has been shown that both linear modulus and failure stress values increase linearly with collagen gel concentrations in the range 0.3 to 3 mg/mL [START_REF] Roeder | Tensile mechanical properties of three-dimensional type I collagen extracellular matrices with varied microstructures[END_REF].

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The effect of TNX on the mechanical properties of collagen gels was further examined in a dose-response experiment shown in Fig. 5. The results clearly demonstrate an increase in elastic modulus correlated with an augmentation of TNX concentration. Finally, since previous experiments were performed with rat collagen combined with bovine TNX, we confirmed these results by the use of bovine collagen gels (data not shown). Firstly, values of elastic modulus in control conditions were similar to those obtained with rat collagen, and secondly, the increase in elastic modulus in the presence of TNX was of the same order of magnitude.

Thus, by these rheological studies, we can conclude that the presence of TNX within the collagen gels significantly increased their mechanical compressive behaviour, in a dosedependent manner.

Conclusion

As demonstrated by Mao et al. [START_REF] Mao | Tenascin-X deficiency mimics Ehlers-Danlos syndrome in mice through alteration of collagen deposition[END_REF], TNX deficiency in mice strongly influences the mechanical properties of skin. Indeed, these authors observed an increased deformability and reduced tensile strength of this tissue in the absence of TNX. The reduced fibrillar collagen content of the tissues and the failure of fibroblasts to deposit collagen in cell cultures led to the conclusion that TNX is critical for the cell-mediated fibrillogenesis process. Our results demonstrate that TNX increases collagen gel stiffness, probably by reinforcing the association between collagen fibrils. The dose-response effect that we observed might be of significant importance in vivo since in humans, different phenotypes (classical Ehlers-Danlos Syndrome versus joint hypermobility only) are observed in patients homozygous or hetereozygous for TNX mutations, indicating a gene-dosage effect [START_REF] Zweers | Haploinsufficiency of TNXB is associated with hypermobility type of Ehlers-Danlos syndrome[END_REF]. These data suggest that TNX should be involved directly in determining the mechanical properties of tissue components and might in part explain how TNX contributes to the mechanical resistance in vivo.

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  The average stress-strain response is plotted in Figs4B and 4C, in terms of the peak compression values (PCV; Fig.4B) or the equilibrium level values (ELV; Fig.4C). The linear regression based on these data showed that the compressive elastic modulus of the collagen/TNX gel was 529.2±94 Pa for PCV and 20.7±7.4 Pa for ELV. These values are approximately twice as high as the apparent compressive elastic modulus of the collagen/BSA gel (PCV=262.1±73 Pa and ELV=7.8±1.6 Pa) or the collagen gel (PCV=280.8±51 Pa and ELV=8.5±2.9 Pa). These values are smaller in comparison to the data obtained byKreger and

Fig. 1 :Fig. 2 :

 12 Fig. 1: Effect of TNX on collagen fibrillogenesis. Fibril formation was measured by monitoring collagen solution turbidity at 400 nm. The different curves represent control experiment with collagen alone ( ), collagen plus TNX at 5 µg/mL (s) or 30 µg/mL (66.6 nM, v), collagen plus BSA at 4.4 µg/mL (66.6 nM, ), or collagen plus fibronectin at 16.7 µg/mL (66.6 nM, ). Each point is the mean of triplicate values and error bars are SEM. The data shown are representative of three independent experiments.

Fig. 3 :

 3 Fig. 3: Fibril diameter distribution within collagen gels. Measurements were performed on scanning electron micrographs obtained from control (black bars) or TNX-containing (grey bars) gels. A minimum of two-hundred fibrils were measured for each condition. Error bars are Standard Deviation Deviation, and a statistical analysis of the fibrils diameters distribution was performed by non-parametric Mann-Whitney U tests. Each sample was compared to the PBS condition.

Fig. 4 :Fig. 5 :

 45 Fig. 4: Stress-relaxation response of collagen gels. (A) Stress versus compression time curves. The purple, blue, and yellow curves correspond to collagen gels prepared in the presence of PBS, TNX (66.6 nM) and BSA (66.6 nM) respectively. The black curve represents the gap between the two platens. For each condition, four different samples were tested by three cycles of three successive compressions. For each type of gel, the average stress-strain response is plotted by taking into account the mean of the peak compression values (B) or of the equilibrium level values (C). The linear regression lines shown allowed the calculation of the apparent compressive elastic modulus for each gel tested. Error bars represent min/max values values and an ANOVA test was performed as statistical analysis. Each sample was compared to PBS. ***p<0.001.

Figure 1 A

 1 Figure 1

Table I :

 I Collagen fibrillogenesis parameters corresponding to Fig.1experiments. Lag time, rate of fibrillogenesis, and absorbance plateau were calculated according to the method published by Sullivan et al.[START_REF] Minamitani | Modulation of collagen fibrillogenesis by tenascin-X and type VI collagen[END_REF].

		0,3
	Abs. 400 nm	0,2 0,1			Lag time (min)	Rate of fibrillogenesis (1x10 TNX 5 µg/mL PBS TNX 30 µg/mL -2 /min) BSA 4.4 µg/mL	Absorbance plateau (400 nm)
						FN 16.7 µg/mL
	Control TNX (5 µg/mL) 0 0	5	10	5.1 ± 0.2 5.5 ± 0.1 15 20	25	6.5 ± 0.1 5.8 ± 0.4 30	0.34 ± 0.08 0.34 ± 0.04
	TNX (30 µg/mL) BSA (4.4 µg/mL)		6.6 ± 0.4 6.6 ± 0.4 Time (min)		6.3 ± 0.3 6.3 ± 0.4	0.35 ± 0.01 0.34 ± 0.07
	Fibronectin (16.7 µg/mL)	6.6 ± 0.4		6.2 ± 0.2	0.33 ± 0.01

Acknowledgements: This work was supported by a grant from the Ligue Nationale contre le Cancer (Comité Départemental de l'Ardèche), and Y. Margaron received a PhD fellowship from the Ligue Nationale contre le Cancer. The authors are grateful to Dr M.C. Ronzière (IBCP, Lyon) for providing acid-soluble collagen extracted from bovine skin and to D.J.S.

Hulmes for revising english.

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