Jean L Whittingham

Juana Carrero-Lerida

Jim A Brannigan

Luis M Ruiz-Pérez

Ana P G Silva

Mark J Fogg

Anthony J Wilkinson

Ian H Gilbert

Keith S Wilson

Dolores González-Pacanowska

James A Brannigan

Luis M Ruiz-Perez

Structural basis for the efficient phosphorylation of AZTMP

Keywords: malaria, thymidylate kinase, ligand complexes, structure, kinetics, drug target

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INTRODUCTION

An essential step in the biosynthesis of the nucleotide dTTP is the phosphorylation of dTMP to dTDP. This reaction is catalysed by thymidylate kinase (TMPK, EC 2.7.4.9, ATP:dTMP phosphotransferase) in a magnesium-dependent manner with ATP as the preferred phosphate donor. Cellular production of dTTP is a highly regulated process [START_REF] Hu | Mitotic control of dTTP pool: a necessity or coincidence?[END_REF][START_REF] Mathews | DNA precursor metabolism and genomic stability[END_REF] that is coordinated with DNA replication in the cell cycle and TMPK has a central role in the control of the dTTP pool size in eukaryotic cells. The enzyme has been widely studied partly owing to its participation in the pathway that leads to the activation of a number of pro-drugs, including azidothymidine (AZT) and acyclovir for the treatment of AIDS and herpes simplex virus infections, respectively [START_REF] Lavie | Structural requirements for efficient phosphorylation of nucleotide analogs by human thymidylate kinase[END_REF][START_REF] Solaroli | Substrate specificity of feline and canine herpesvirus thymidine kinase[END_REF]. TMPK is also being investigated as a drug target in its own right and specific inhibitors of the orthologous enzymes from Bacillus anthracis and Mycobacterium tuberculosis have been developed [START_REF] Carnrot | Mechanisms of substrate selectivity for Bacillus anthracis thymidylate kinase[END_REF][START_REF] Haouz | Enzymatic and structural analysis of inhibitors designed against Mycobacterium tuberculosis thymidylate kinase: New insights into the phosphoryl transfer mechanism[END_REF].

TMPK is found in all organisms requiring de novo pyrimidine synthesis, and crystal structures of the enzyme from a number of different species have been determined [START_REF] Lavie | Structure of thymidylate kinase reveals the cause behind the limiting step in AZT activation[END_REF][START_REF] Lavie | Structural basis for efficient phosphorylation of 3'-azidothymidine monophosphate by Escherichia coli thymidylate kinase[END_REF][START_REF] Kotaka | Structures of S. aureus thymidylate kinase reveal an atypical active site configuration and an intermediate conformational state upon substrate binding[END_REF][START_REF] De La Sierra | X-ray structure of TMP kinase from Mycobacterium tuberculosis complexed with TMP at 1.95 Angstrom resolution[END_REF][START_REF] Ostermann | Potentiating AZT activation: Structures of wild-type and mutant Human thymidylate kinase suggest reasons for the mutants' improved kinetics with the HIV prodrug metabolite AZTMP[END_REF]. The protein is a homodimer with a subunit fold consisting of a five-stranded parallel β-sheet surrounded by 7-11 α-helices. The nucleotide substrates are bound in a groove on the surface of the subunit which is characterised by a number of conserved sequence motifs containing structural elements required for substrate recognition and catalysis, namely the so-called 'P-loop ' [12], G(X) 4 GKS/T, the critical loop DRX motif (Figure 1a) and a 'lid' that partially encloses the phosphate donor and is implicated in catalysis [START_REF] Lavie | Structural basis for efficient phosphorylation of 3'-azidothymidine monophosphate by Escherichia coli thymidylate kinase[END_REF]. Crystal structures of TMPKs with different combinations of ligands have revealed several conformational states of the protein: an open state in the absence of ligand (where the lid is disordered); a partially closed state in the presence of dTMP (lid disordered in most structures, but ordered in the M. tuberculosis enzyme); and a fully closed state in the presence of both co-substrates (lid ordered). Cocrystallisation with bi-substrate analogues such as P 1 -(5'-adenosyl)-P 5 -(5'-thymidyl) pentaphosphate (AP 5 dT), which mimic the ATP-dTMP or ADP-dTDP states [START_REF] Bone | Inhibition of adenosine and thymidylate kinases by bisubstrate analogs[END_REF] has given additional insight into the reaction coordinate [14,[START_REF] Ostermann | Insights into the phosphoryltransfer mechanism of human thymidylate kinase gained from crystal structures of enzyme complexes along the reaction coordinate[END_REF]. After extensive functional and structural studies on TMPK from several species, a general consensus with regard to the mechanism has been achieved. There is a good overview of the mechanism based primarily on structural and functional studies of the human enzyme [START_REF] Ostermann | Insights into the phosphoryltransfer mechanism of human thymidylate kinase gained from crystal structures of enzyme complexes along the reaction coordinate[END_REF]. Due to the complexity of the reaction, there are small differences between species, such as the movement of the P-loop and the lid during catalysis so as to re-orient specific basic side chains to stabilise the transition state complex. These may well have impacts on the specificity and relative kinetics of the enzyme. Sequence variation, particularly in the lid region, is associated with structural alterations around the nucleotide-binding site and relocation of catalytic side chains, leading to speculation that the subtleties of the mechanism vary between species [START_REF] Lavie | Structural basis for efficient phosphorylation of 3'-azidothymidine monophosphate by Escherichia coli thymidylate kinase[END_REF]. Comparison with related systems has given some general insights into the mechanism. In a similar fashion to adenylate kinase, which has the same overall topology as TMPK, the two nucleotide substrates are believed to bind to the enzyme via a random bi-bi mechanism [START_REF] Xiang Rong | An iso-random Bi Bi mechanism for adenylate kinase[END_REF]. It is agreed that phosphate transfer occurs via an associative, rather than a dissociative, mechanism such that the migrating phosphate is bound to both substrates in the transition state [START_REF] Schlichting | Structures of active conformations of UMP kinase from Dictyostelium discoideum suggest phosphoryl transfer is associative[END_REF]. The accumulating negative charge on this phosphate in the transition state is proposed to be stabilised by arginine side chains and one or more magnesium ions.

The TMPKs have been classified into two groups [START_REF] Lavie | Structural basis for efficient phosphorylation of 3'-azidothymidine monophosphate by Escherichia coli thymidylate kinase[END_REF]. Type I enzymes, which include the human, yeast and Plasmodium proteins, have a basic residue in addition to the lysine in the P-loop (Arg 18 in PfTMPK). Type II enzymes including that from Escherichia coli, lack this basic residue in the P-loop; alternatively a basic residue from the lid appears to exert a similar function. TMPKs from different species have varying 3'-azido-3'-deoxythymidine monophosphate (AZTMP) phosphorylation efficiencies and comparative structural and biochemical studies have shown the structural basis for proficient phosphorylation. TMPKs from E. coli and various viruses are able to phosphorylate AZTMP very much more efficiently than the human enzyme [START_REF] Brundiers | Modifying human thymidylate kinase to potentiate azidothymidine activation[END_REF]. The reason for this appears to be the displacement of a catalytically important arginine side chain on the P-loop of the human enzyme in response to AZTMP binding. In contrast the E. coli enzyme, where the P-loop arginine is absent, can accommodate AZTMP without seriously affecting its catalytic properties [START_REF] Lavie | Structural basis for efficient phosphorylation of 3'-azidothymidine monophosphate by Escherichia coli thymidylate kinase[END_REF].

The focus of the present study is to explore the potential of P. falciparum TMPK (PfTMPK) as an anti-malarial drug target. We present a kinetic analysis of PfTMPK in combination with the crystal structure of four ligand complexes: dTMP-ADP, AZTMP-ADP, AP 5 dT and dGMP-ADP. The latter structure provides a structural rationalisation for the broader specificity of the Plasmodium enzyme. Two recent studies by Kitade and colleagues presenting results on the expression and kinetics [START_REF] Kandeel | Molecular characterization, heterologous expression and kinetic analysis of recombinant Plasmodium falciparum thymidylate kinase[END_REF] and on the ligand binding, modelling and mutagenesis [START_REF] Kandeel | Mutational, inhibitory and microcalorimetric analyses of Plasmodium falciparum TMP kinase. Implications for drug discovery[END_REF] of PfTMPK, together with new kinetic data are interpreted in terms of the crystal structures presented herein. The present results confirm that the enzyme has structural and catalytic properties distinct from other members of the family, confirming that it is a good candidate for selective inhibition and rational drug design.

EXPERIMENTAL Materials

AZTMP, 3'-dGMP and AP 5 dT were purchased from Jena Biosciences GmbH. Other nucleotides including ATP (magnesium salt) were purchased from Sigma-Aldrich Company Ltd. Oligonucleotides were synthesized at the Analytical Services of the López-Neyra Institute of Parasitology and Biomedicine. P. falciparum strain 3D7 was provided by J. M. Bautista of the University Complutense of Madrid (Spain). Human erythrocytes and frozen serum were obtained from Centro Regional de Transfusión Sanguínea-SAS (Granada, Spain).

Parasite culture

P. falciparum strain 3D7 was cultured by the method of Trager and Jensen [START_REF] Trager | Human malaria parasites in continuous culture[END_REF] with minor modifications. Cultures were maintained in fresh group O-positive human erythrocytes suspended at 5% hematocrit in RPMI 1640 (GibcoBRL) containing 10% human serum, 3 g glucose, 45 µg hypoxanthine, and 50 µg of gentamicin per liter. The medium was changed daily, as previously described [START_REF] Trager | Human malaria parasites in continuous culture[END_REF].

Expression of PfTMPK in Escherichia coli

DNA encoding full length PfTMPK (633 bp) was amplified by RT-PCR from total RNA obtained by standard procedures from parasites released from infected erythrocytes by saponin lysis [START_REF] Fang | The effects of glucose concentration on the reciprocal regulation of rRNA promoters in Plasmodium falciparum[END_REF]. The primer TKEcoRI (5'-GGAATTCTTATGACCACAAAAAATTAA-3') was used to synthesize the corresponding tmpk cDNA, in the reverse transcriptase reaction, and TKNdeI (5'-GCATATGACTGATGATAAAAAAAA-3') and TKEcoRI were used in a second PCR step to amplify the full coding sequence with NdeI and EcoRI restriction sites placed at the 5' and 3' ends respectively for cloning into the plasmid vector pET28a (Novagen) so as to append a His 6 -affinity purification tag and a thrombin cleavage site to the N-terminus of the PfTMPK peptide sequence. The resulting pET-PfTMPK plasmid was used to transform the E. coli strain BL21 (DE3) and PfTMPK expression was induced by adding 1 mM isopropyl β-D-thiogalactoside (Roche) and incubating for 4 h at 37ºC or by means of auto-induction [START_REF] Studier | Protein production by auto-induction in high-density shaking cultures[END_REF] for a period of 20 h. The cultures were collected by centrifugation and frozen at -80ºC until use.

Purification of recombinant PfTMPK

For purification of PfTMPK, cell pellets were resuspended in 10 ml of binding buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10 mM imidazole, 1 mM DTT) and protease inhibitor cocktail (Pefabloc SC, Roche Diagnostics GmbH) to a final concentration of 2 mM. PSCprotector solution was also added to prevent non-specific covalent attachment of the protease inhibitor to the protein (a problem encountered during initial purification trials). Cells were disrupted by sonication on ice and centrifuged at high speed for 30 min at 4ºC. The cleared lysate was applied to a HiTrap Chelating HP affinity column (Amersham Biosciences) equilibrated with binding buffer. The column was washed with binding buffer containing 20 mM imidazole, and was developed with a linear gradient of 20 to 500 mM imidazole. Fractions containing the enzyme were pooled, passed through a PD-10 column (GE Healthcare) and eluted in 50 mM Tris-HCl pH 7.5, 20 mM NaCl, 1 mM DTT and 50% glycerol. For structural work, the protein was further purified by gel filtration on a HiLoad 16/60 Superdex 75 prep-grade gel-filtration column (Amersham Biosciences) pre-equilibrated with 50 mM Tris-HCl pH 8.5 and 200 mM NaCl. Protein concentration was determined by the method of Bradford [START_REF] Bradford | Rapid and sensitive method for quantification of microgram quantities of protein utilizing principle of protein-dye binding[END_REF], with bovine serum albumin as a protein standard. The purity and molecular mass of the purified protein were established by polyacrylamide gel electrophoresis and electrospray mass spectrometry. The protein was concentrated to 40 mg ml -1 and stored at -80º C.

Activity assays

Recombinant PfTMPK enzyme activity was assayed using a spectrophotometric method by coupling the formation of ADP to the oxidation of NADH in reactions catalyzed by pyruvate kinase and lactate dehydrogenase [START_REF] Blondin | Improved spectrophotometric assay of nucleoside monophosphate kinase activity using the pyruvate kinase/lactate dehydrogenase coupling system[END_REF]. Assays for inhibition determination were performed in a final volume of 1 ml and a standard assay contained 50 mM Tris-HCl (pH 7.4), 50 mM KCl, 5 mM MgCl 2 , 0.2 mM NADH, 1 mM DTT, 1 mM phosphoenolpyruvate, pyruvate kinase (4 U), lactate dehydrogenase (4 U), 50 µM dTMP, 1 mM ATP and 0.83 µg of recombinant enzyme. When K m and V max values for the different nucleotides were determined, a range of concentrations were tested using the standard assay. TMPK activity (1 unit) was defined as that which produces 1 nmol of NAD + per minute and mg of enzyme. K i values were obtained from the expression I50 = K i (1+[S]/K m ) that, for competitive inhibitors, relates the concentration of inhibitor which inhibits activity by 50% (I50) with the K i value. K m is the Michaelis-Menten constant for dTMP and [S] is the concentration of dTMP used in the reaction (50 µM).

Crystallisation, data collection and data processing

To remove the His 6 -tag from the protein prior to crystallisation, biotinylated thrombin (Novagen) was added to a solution containing 1 mg ml -1 PfTMPK in 50 mM Tris-HCl pH 8.5, 200 mM NaCl and 2.5 mM CaCl 2 (1 unit of thrombin per µg of protein). This solution was gently swirled at room temperature for 24 h. The thrombin was then removed from the reaction mix by immobilisation on streptavidin agarose according to the manufacturer's instructions, and a final nickel affinity chromatography step was used to remove any uncleaved PfTMPK. Electrospray mass spectrometric analysis confirmed the identity and purity of the product and the presence of a Gly-Ser-His extension at the N-terminus of the protein (24,973 kDa). Needle-shaped crystals (up to 50 x 50 x 400 µm 3 in size) of four different nucleotide complexes of PfTMPK were obtained using hanging drop vapour diffusion, with a 1:1 mixture of protein solution and reservoir solution in the drop (see Supplementary Table ). Prior to data collection, the crystals were vitrified at 120 K in an appropriate cryoprotectant solution (see Supplementary Table ). X-ray data sets for the four protein complexes (Table 3) collected at the ESRF in Grenoble, were processed using DENZO and SCALEPACK [START_REF] Otwinowski | Processing of X-ray diffraction data collected in oscillation mode[END_REF]. Initial data on the dGMP complex were collected on the ID24 microfocus beam line at the Diamond Light Source from a crystal conglomerate using a 8x8 µm beam to hunt for a single lattice with the help of Gwyndaf Evans, Robin Owen and Danny Axford. This led to a 70 % complete data set at 2.7 Å resolution and allowed the presence of the ligand to be confirmed. While subsequent optimisation led to larger crystals from which complete data were recorded at the ESRF, the power of beam line I24 for analysing tiny crystals was clearly demonstrated by this result.

Structure solution and refinement

The structure of PfTMPK complexed with dTMP and ADP was solved by molecular replacement with the program PHASER [START_REF] Mccoy | Phaser crystallographic software[END_REF] using human TMPK (pdb code 1e2q) as a search model (the two proteins share 39% sequence identity). Searches, carried out in two possible space groups (P3 1 21 and P3 2 21), yielded solutions in space group P3 1 21 only and established the presence of three molecules per asymmetric unit. Refinement was carried out in the CCP4 suite of programs [START_REF] Ccp4 | The CCP4 Suite -Programs for protein crystallography[END_REF] using maximum likelihood methods implemented in REFMAC [29]. 5% of the data were excluded for R-free calculations. The models were manually rebuilt using the program COOT [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF] in conjunction with a 3-fold averaged 2F o -F c electron density map and a F o -F c electron density map. After several rounds of refinement and model building, the dTMP and ADP molecules were built into electron density in the averaged map. Towards the end of the refinement small differences between the three molecules were modelled into a non-averaged 2F o -F c electron density map and two metal ions were located in each of the three molecules. The other three protein complexes were refined starting from the dTMP-ADP complex without its solvent and ligands and rebuilt in a similar manner. Dictionaries for the nucleotide ligands were made using PRODRG [START_REF] Schuettelkopf | PRODRG -a tool for highthroughput crystallography of protein-ligand complexes[END_REF]. Refinement statistics and structural information are listed in Table 3.

Protein Data Bank accession numbers

The atomic coordinates and structure factors for the four PfTMPK complexes have been deposited in the Protein Data Bank, www.ebi.ac.uk (ID codes: dTMP-ADP: 2wwf, dGMP-ADP: 2wwg, AP 5 dT: 2wwh and AZTMP-ADP: 2wwi).

RESULTS AND DISCUSSION

Kinetic parameters and inhibition of PfTMPK

Standard procedures were used to determine the kinetic parameters of recombinant PfTMPK. Plots of specific activity versus substrate concentration gave typical Michaelis-Menten hyperbolic saturation curves. Kinetic constants were determined for a number of different substrates used as phosphoacceptors (Table 1). ATP was used as a phosphate donor in the assays, because it was the preferred donor among those tested (results not shown). The phosphorylating efficiency (k cat /K m ) of PfTMPK was highest for dTMP followed by dGMP, 5FdUMP, dUMP, dIMP and GMP. In addition the K m for ATP was 78.6 µM. This value was obtained at 200 µM dTMP and constant free Mg 2+ concentrations (5 mM). These results are in general agreement with those previously reported [START_REF] Kandeel | Molecular characterization, heterologous expression and kinetic analysis of recombinant Plasmodium falciparum thymidylate kinase[END_REF], with the relative activities having the same rank order although the two assays differ in the absolute measured values. While the assay methodology used in both cases is similar, subtle differences may be due to the different expression systems employed or minor differences in the reaction conditions. It is of particular note that the activity is almost as high for the purine dGMP as it is for dTMP. Surprisingly, phosphorylation of the nucleotide analogue AZTMP was also highly efficient with k cat /K m close to half that for dTMP. This latter value for AZTMP is 200-fold higher than the corresponding value for human TMPK (henceforth hTMPK) and similar to that of the E. coli enzyme (Table 2).

There was no measurable phosphorylation of the nucleosides uridine, thymidine, deoxyuridine, and AZT by PfTMPK. Likewise, purine derivatives such as ganciclovir (9-[(1,3-dihydroxy-2-propoxy) methyl] guanine) and 3'-deoxyguanosine-5'-monophosphate were not substrates. Different nucleotides and nucleosides were tested as inhibitors of dTMP phosphorylation. 5-fluoro-2'-deoxyuridine and 3'-deoxyguanosine-5'-monophosphate showed K i values of 1.6 mM and 0.25 mM respectively while no inhibition was observed with deoxyuridine, 5-fluoro-5'-deoxyuridine or ganciclovir. The K i for AP 5 dT, a bi-substrate inhibitor of several thymidylate kinases [START_REF] Bone | Inhibition of adenosine and thymidylate kinases by bisubstrate analogs[END_REF] was measured by determining the percentage of inhibition at different inhibitor concentrations and assuming competitive inhibition, giving a value of 0.31 µM. The key findings of the kinetic studies are the high activity of PfTMPK on AZTMP, and confirmation of its activity on dGMP. These activities will be discussed in terms of the structural investigations below.

The three dimensional structure

PfTMPK is a homodimeric protein containing 210 amino acid residues per subunit. All four nucleotide complexes crystallised in space group P3 1 21 with three protomers per asymmetric unit, equating to one crystallographically independent dimer, plus a second dimer lying on a crystallographic two-fold axis. We first give a generic description of those features which are common to all four complexes, before describing the structures in detail in the subsequent sections.

The three protomers are essentially identical to one another, with root mean square positional deviations (rms∆) following superposition of all C α atoms of ~0.25 Å. Each subunit has a globular structure consisting of a five-stranded parallel β-sheet (β2-β3-β1-β4-β5) surrounded by eight α-helices (Figure 1b). Interaction with the partner subunit in the dimer occurs predominantly through hydrophobic interactions between α-helices 2, 3 and 5 from each subunit (not shown). The molecules are well-ordered, with only some small loop and chain termini disorder. Superposition of protomer A from all four complexes shows the backbone conformation to be very similar (Supplementary Figure S1) and corresponds to the closed form of the enzyme, as expected for complexes with both nucleotides.

In the dTMP-ADP, AP 5 dT and dGMP complexes, there are two peaks at essentially identical positions in the electron density maps adjacent to the terminal phosphate groups of the nucleotides, whose shape and coordination indicated the presence of two metal ions. These were initially introduced as Mg 2+ ions since magnesium had been included in the crystallisation solutions. However, following refinement, it became clear from the metalligand bond distances (2.3 -2.6 Å) that the ions were in fact sodium. The metal sites are shown for the dTMP-ADP complex in Figures 1b &2c. The two ions, Na (I) and Na (II), are situated 7.0 Å apart with well-defined and independent coordination spheres. In retrospect, this is not surprising as although 25 mM magnesium chloride was present in the crystallisation drops, sodium is also present at large excess, ~1-2 M. These sodium ions in PfTMPK, primarily due to their larger ionic radius, occupy somewhat different positions to those observed for the "biological" Mg 2+ ions seen in the human enzyme (Figure 2c), and some other known structures, which correspond to the two metal ions essential for phosphoryl group transfer. Equivalent sodium ions are likely to be present, but were not modelled, in the lower resolution AZTMP complex. The sodium ions have rather different coordination from the magnesium ions, as described in the next section, with bonds formed to the nucleotides and protein residues.

PfTMPK has the same general fold and mode of substrate binding as other TMPKs with known structures (www.ebi.ac.uk), suggesting a common mode of action. Thus, PfTMPK would be expected to bind the two nucleotide co-substrates independently of one another, with binding accompanied by stepwise closure of the P-loop and lid so that phosphate transfer can be facilitated by magnesium ions, arginine side chains, and other essential conserved residues.

A superposition of the dTMP-ADP complex of PfTMPK on the equivalent complex of the human enzyme shows that the cores of the molecules are very similar (see below), with some minor differences evident in the loops remote from the active site, and some significant deviations in the loops close to the active site (Supplementary Figure S2). A set of sub-states of the closed form of the human enzyme, with the lid and P-loop being either in the open, half-closed or closed form depending on the ligand bound have been described [START_REF] Ostermann | Insights into the phosphoryltransfer mechanism of human thymidylate kinase gained from crystal structures of enzyme complexes along the reaction coordinate[END_REF]. Comparison of the backbone conformation of PfTMPK with that of the human enzyme complexes shows that it corresponds most closely to the closed sub-conformation of the lid and P-loop (Supplementary Figure S3). Indeed in terms of the "degree of closure", PfTMPK appears to be even more tightly closed around the nucleotides in the four complexes than in the equivalent human structures. In all, the comparison of PfTMPK and hTMPK reveals significant and consistent conformational changes in the catalytically important P-loop and lid regions. These conformational differences together with natural side chain variations may account for differences in specificity and rate in the two enzymes.

The PfTMPK-dTMP-ADP complex

The structure of the PfTMPK-dTMP-ADP complex was refined at a resolution of 1.9 Å. A prominent groove on the surface of each subunit forms the binding site for dTMP and the preferred phosphate donor, ATP, which bind co-linearly to facilitate phosphate group transfer. Residues of the lid region flank the bound nucleotides, partially enclosing the binding site.

The presence of both dTMP (substrate) and ADP (product) results, as stated above, in the closed form of the enzyme with the lid fully ordered. The dTMP is buried, the thymine base being maintained in position by hydrogen bonds (both directly and indirectly via ordered water molecules) to the side chains of Arg 78 and Tyr 153 , and a ring stacking interaction with Phe 74 (Figure 2a). The ribose ring is loosely packed between the side chains of Tyr 153 and Arg 99 , and forms one direct hydrogen bond to Asp 17 in the P-loop. There is clear evidence for the binding of two sodium ions (Figure 2c). The phosphate group makes direct interactions with the side chain of Arg 99 and Na (I) which is penta-coordinate, its ligands consisting of a phosphate oxygen (from dTMP), the side chain oxygen atoms of Asp 17 and Glu 154 and two water molecules (Figure 2c). The existence of a third highly mobile water molecule to complete the octahedral coordination sphere cannot be excluded. Arg 99 , which forms part of the highly conserved critical loop, is the only residue in the structure whose main chain bond angles lie in a disallowed region of the Ramachandran plot. This phenomenon has been observed in other thymidylate kinases, and can be presumed to be associated with catalysis since steric strain in well refined crystal structures correlates with functional significance [START_REF] Herzberg | Analysis of the steric strain in the polypeptide backbone of protein molecules[END_REF]. The residues involved in dTMP binding are highly conserved and superposition of the dTMP-bound TMPK structures available from the PDB shows that the mode of binding is very similar in each.

The ADP, bound close to the surface of the protein, is protected by the lid and is secured in position by a number of interactions (Figure 2b). These include stacking between the adenine base and the guanidine moiety of Arg 145 , a hydrogen bond between the base and the main chain oxygen of Arg 182 , three hydrogen bonds between the phosphate groups and the atoms of residues Lys 21 , Ser 22 and Thr 23 (P-loop), and a direct interaction with the second sodium ion, Na (II). Na (II) has an octahedral coordination sphere comprising a phosphate oxygen from ADP, the side chain carboxylate of Asp 98 , the side chain oxygen atom of Ser 19 and three water molecules. The catalytically important Asp 17 of the P-loop has a full complement of polar interactions, including hydrogen bonds to Tyr 107 and Gln 159 . The side chain of the adjacent Arg 18 points away from the substrate, hydrogen bonding to the side chain of Glu 154 . Superposition of the structure of PfTMPK-dTMP-ADP with the equivalent complex of hTMPK (pdb code 1e2d, sequence identity 40%, sequence homology 60%) shows a highly conserved topology (rms∆ 1.35 Å over 195 C α atoms) and mode of nucleotide binding (Supplementary Figure S2); the dTMP binding sites are essentially identical. However, there are small but significant differences in the lid regions, which differ in conformation, and in the P-loops, which have a relative displacement of 2.1 -2.5 Å on main chain atoms and differences in side chain conformations. For the human enzyme, the P-loop displacement results in a shift in position of the phosphate groups of ADP and a different mode of metal binding reflecting the presence of sodium rather than magnesium (Figure 2c). The different conformation for Asp 15 results in Mg (I) in hTMPK having a quite different coordination sphere, consisting of six water molecules and an indirect rather than direct interaction with dTMP, compared to that of Na (I) in PfTMPK (Figure 2c). A likely reason for the difference in the position of the P-loop is a natural variation in the substrate binding site, Phe 105 (human) → Tyr 107 (Plasmodium). The tyrosine side chain of PfTMPK forms a hydrogen bond (2.6 Å) with the side chain of Asp 17 , which helps to tether this catalytically important residue in close proximity to the terminal phosphate groups of the nucleotide co-substrates (Supplementary Figure S4). The phenylalanine in the human enzyme lacks the capacity to do this. There is a tyrosine at this position in yeast TMPK and this residue has been cited as the reason for its enhanced catalytic activity relative to hTMPK [START_REF] Wöhrl | Expressing engineered thymidylate kinase variants in human cells to improve AZT phosphorylation and human immunodeficiency virus inhibition[END_REF]. The other P-loop side chain believed to be important for catalysis, Arg 18 (Arg 16 in hTMPK), is directed away from the nucleotide binding site in PfTMPK and hTMPK, in both structures making hydrogen bonding interactions with lid residues. Residue Lys 37 in yeast TPMK corresponds to Arg 41 and Tyr 43 in the human and P. falciparum enzymes, respectively (Figure 1a). These natural variations in the substrate binding site may provide scope for the design of selective inhibitors against PfTMPK.

The structural basis for dGMP phosphorylation

In view of the kinetic observation that PfTMPK phosphorylates dGMP with a k cat /K m value close to that for dTMP, we determined the structure of the dGMP-ADP complex, which was refined to a resolution of 2.4 Å. Two sodium ions were modelled at equivalent positions to those in the dTMP complex, but with reduced electron densities reflecting the lower resolution of the analysis. The purine nucleotide is accommodated in the pyrimidine binding pocket (Figure 3a) with negligible changes in the protein structure compared to the dTMP-ADP structure (rms∆ 0.25 Å over 209 C α atoms). The guanine base is coplanar with the dTMP thymine and just requires the displacement of a single highly ordered water molecule, which in the dTMP-ADP complex hydrogen bonds to atom O2 of thymine and the hydroxyl groups of residues Tyr 153 and Ser 108 . The ribose and the phosphate groups of dGMP have closely similar conformations and positions to those of dTMP and the ADP binding sites are essentially identical.

The structure of the human dTMP-ADP complex is superimposed on the PfTMPK complex in Figure 3a. From the close similarity of the two structures, it might be expected that the human enzyme would also bind dGMP. However, in spite of the fact that the dTMP binding site of hTMPK is very similar to that of PfTMPK, particularly around the pyrimidine binding pocket, several studies have demonstrated that dGMP is not a substrate for the human enzyme [START_REF] Topalis | Substrate specificity of vaccinia virus thymidylate kinase[END_REF][START_REF] Su | Molecular cloning and expression of the human deoxythymidylate kinase gene in yeast[END_REF][START_REF] Auvynet | Phosphorylation of dGMP analogs by vaccinia virus TMP kinase and human GMP kinase[END_REF]. In human cells it appears that a guanylate kinase very efficiently phosphorylates dGMP and GMP [START_REF] Auvynet | Phosphorylation of dGMP analogs by vaccinia virus TMP kinase and human GMP kinase[END_REF]. A survey of the P. falciparum genome (www.PlasmoDB.org) shows the existence of a guanylate kinase and a UMP-CMP kinase which have potential functions in dGMP phosphorylation, as guanylate kinases are capable of dGMP phosphorylation [START_REF] Van Rompay | Phosphorylation of nucleosides and nucleoside analogs by mammalian nucleoside monophosphate kinases[END_REF], likewise certain UMP kinases such as that from yeast have been shown to phosphorylate dGMP [START_REF] Jong | Characteristics, substrate analysis, and intracellular location of Saccharomyces cerevisiae UMP Kinase[END_REF]. A recent study has revealed that the P. falciparum guanylate kinase utilizes dGMP with very low specificity, which is of interest in respect of the potential contribution of PfTMPK to cellular dGDP formation and purine nucleotide metabolism in this parasite [START_REF] Kandeel | The exceptional properties of Plasmodium deoxyguanylate pathways as a potential area for metabolic and drug discovery studies[END_REF]. Site-directed mutagenesis has been previously used to evaluate the contributions of selected residues to substrate binding and catalysis [START_REF] Kandeel | Mutational, inhibitory and microcalorimetric analyses of Plasmodium falciparum TMP kinase. Implications for drug discovery[END_REF]. The mutation of the conserved Phe 74 , whose side chain stacks with the thymine base, to an alanine eliminated activity almost entirely. Introduction of individual point mutations between the human and parasite enzymes (Figure 1a) into PfTMPK, S108T, Y43R and Y43L had negligible effects on activity. The structure of PfTMPK reveals that although these residues are close to the active site, they are not directly involved in nucleotide binding. Substitution of Ala 111 by Lys, resulted in a dramatic reduction in activity of PfTMPK, especially in dGMP phosphorylation. Ala 111 resides on a sevenresidue loop on the outside of the molecule which has quite different conformations in the two enzymes. As a result, Ala 111 and K 109 , rather than coinciding, are displaced by 3.7 Å from one another (C α ), and located 10 -12 Å away from the dTMP ligand exerting no direct influence on binding. Indeed the A111K mutation in PfTMPK would be expected to cause a steric clash with the side chain of Tyr 153 which is in van der Waals' contact with both the base and the sugar of dTMP. Displacement of the tyrosine side chain would be predicted to affect substrate binding, and this would be exacerbated for the larger dGMP, in keeping with the kinetic data.

Vaccinia virus TMPK is also capable of phosphorylating purine nucleotides [START_REF] Topalis | Substrate specificity of vaccinia virus thymidylate kinase[END_REF] and recent structural analysis has revealed a dimer arrangement which is orthogonal and not anti-parallel 11 as in other TMPKs. This unusual subunit tilting is closely correlated with enzyme stability and with the presence of a cavity allowing broader substrate specificity [40]. Apart from Plasmodium and vaccinia TMPKs, the only other kinase that is known to handle both dGMP and dTMP is the bacteriophage T4 deoxynucleotide kinase. The structure of the phage enzyme is similar to the fold of the NMP kinases, but with a different dimerisation mode and no apparent conservation of the active site residues [START_REF] Teplyakov | Crystal structure of bacteriophage T4 deoxynucleotide kinase with its substrates dGMP and ATP[END_REF].

The PfTMPK-AP 5 dT complex

The bisubstrate inhibitor AP 5 dT [START_REF] Bone | Inhibition of adenosine and thymidylate kinases by bisubstrate analogs[END_REF] has been co-crystallised with TMPK from a number of species [START_REF] Haouz | Enzymatic and structural analysis of inhibitors designed against Mycobacterium tuberculosis thymidylate kinase: New insights into the phosphoryl transfer mechanism[END_REF]14,[START_REF] Ostermann | Insights into the phosphoryltransfer mechanism of human thymidylate kinase gained from crystal structures of enzyme complexes along the reaction coordinate[END_REF]. In the PfTMPK complex, AP 5 dT is bound at full occupancy with the same mode of binding in all three protomers (Figure 3b). The five phosphate groups of the inhibitor are labelled PA-PE, phosphate PA being closest to the thymidine group. Superposition with the PfTMPK-TMP-ADP complex shows that the effect of bisubstrate binding on the protein conformation is minimal, with rms∆ for all main chain atoms of 0.3 Å. The adenosine and thymidine moieties of AP 5 dT superpose closely with the nucleoside species of ADP and dTMP, as do the phosphates PA, PD and PE with the phosphate of dTMP and the β and α phosphates of ADP respectively. The two bridging phosphate groups of AP 5 dT, PB and PC, are bound next to the P-loop, flanked by the two sodium ions. The non-bridging oxygens of all five phosphate groups make polar interactions with Lys 21 , Ser 22 (main chain and side chain), Thr 23 (main chain), Arg 47 and Arg 99 or with the metal ions. In addition, the side chain of Arg 18 has two alternate conformations, one of which hydrogen bonds to a phosphate oxygen of PC. The presence of this interaction is consistent with a role for Arg 18 in transition state stabilisation, as has been shown for the yeast and human enzymes. Two sodium ions were modelled in the structure, and lie roughly 0.7 Å from the equivalent ions in the dTMP-ADP complex. Superposition of PfTMPK-AP 5 dT with the equivalent complexes from other species (Figure 3b) confirms that the inhibitors bind in the same place, although only the thymidine moieties are truly coincident. The atoms of the five phosphate groups and the adenosine moieties are displaced by 0.3 -1.2 Å (one of the phosphates is ordered in the human complex). The Na (I) and Mg (I) ions lie 2.1 Å apart in the human and Plasmodium enzymes, although they carry out the same role, coordinating to two oxygen atoms of phosphates PC and PD (by PfTMPK naming of phosphates). The P-loops of the two structures overlay more closely than they do in the dTMP-ADP complex, although there are still main chain atom differences of 0.8 -1.7 Å. The side chains of Asp 17 (Asp 15 in hTMPK) overlap but the carboxylates make different hydrogen bonding interactions, to Tyr 107 in PfTMPK, and to Arg 97 in hTMPK. The P-loop arginine side chains are arranged much as they are in the dTMP-ADP complex, with the exception of the dual conformation of the side chain of Arg 18 in PfTMPK. The binding of AP 5 dT clearly varies in detail between species, but is in keeping with the accepted mechanism of the enzyme.

PfTMPK efficiently phosphorylates AZTMP

Another characteristic of PfTMPK highlighted by the present kinetic studies is its ability to phosphorylate AZTMP 200-fold more efficiently than hTMPK (Table 2). The conversion of AZTMP to AZTDP by the human enzyme is a crucial step in the production of the active drug AZTTP in anti-viral therapy. The anti-viral effect of the drug is achieved by its inhibition of nascent DNA chain growth during replication following its acceptance as a substrate by the viral DNA polymerase [START_REF] Lavie | Structural basis for efficient phosphorylation of 3'-azidothymidine monophosphate by Escherichia coli thymidylate kinase[END_REF].

Analysis of the PfTMPK-AZTMP-ADP complex shows that both nucleotide substrates are at full occupancy in each protomer in the asymmetric unit, however only in molecule A can the position of the azide group be defined with confidence (Figure 3c). The absence of electron density for azide in the other two protomers may reflect inherent disorder and is exacerbated by the modest (3.0 Å) resolution. The AZTMP azide moiety in molecule A is accommodated in a cavity surrounded by the side chains of Asp 17 , Leu 59 , Glu 151 and Glu 154 (Figure 3d). It causes a small reorientation of the dTMP phosphate group and minor conformational changes in the side chains of Glu 154 and Asp 17 , the latter retaining its hydrogen bond to the side chain of Tyr 107 . Owing to the limited resolution, metal ions were not modelled in this complex (Table 3). The structure superimposes very closely on the dTMP-ADP complex (rms∆ 0.27 Å over 208 C α atoms) and the positions of the two sets of nucleotide ligands are essentially identical.

Comparison with the structure of the equivalent human enzyme complex (PDB entry 1e99) shows differences in the P-loop organisation similar to those observed in the equivalent dTMP-ADP comparisons. The close proximity of the side chain of Asp 17 to the AZTMP in PfTMPK causes a reorientation of the azide group relative to that in the human complex. This reorientation does not appear to affect the availability of Asp 17 for catalysis. Indeed, given that it is likely that no further P-loop changes are required for transition state formation in the P. falciparum enzyme (cf. the structure of PfTMPK-AP 5 dT) it would appear to have a mechanistic advantage over the human enzyme, in which P-loop movements toward the transition state would result in a steric clash with the azide group of AZTMP. These structural observations provide a likely explanation for the higher activity towards AZTMP of PfTMPK relative to hTMPK (Table 2).

Poor AZTMP phosphorylation in the type I TMPKs (e.g. the yeast and human enzymes), which contain a basic residue in the P-loop has been proposed to be due to the azido moiety in AZTMP disrupting an important bidentate interaction between the 3′ hydroxyl group of TMP and the conserved P-loop carboxylic acid which results in a displacement of the P-loop [START_REF] Lavie | Structural basis for efficient phosphorylation of 3'-azidothymidine monophosphate by Escherichia coli thymidylate kinase[END_REF]. This is in contrast to type II TMPKs such as the E. coli enzyme where the analogous interaction with the P-loop carboxylic acid residue is side-on, permitting small conformational changes to accommodate the azido group of AZTMP without affecting significantly the position of the P-loop. It has been proposed that the P-loop could influence the phosphoryl transfer rate of type I TMPKs in different ways [START_REF] Ostermann | Potentiating AZT activation: Structures of wild-type and mutant Human thymidylate kinase suggest reasons for the mutants' improved kinetics with the HIV prodrug metabolite AZTMP[END_REF]. First, by shifting the position of ATP relative to the acceptor, catalysis would be impaired. Secondly, movement of the P-loop upon binding of AZTMP would result in the mis-positioning of P-loop arginine involved in catalysis (Arg 18 in PfTMPK). Third, mis-positioning of the P-loop carboxylic acid residue, a strictly conserved amino acid within the TMPK family, may hinder catalysis [START_REF] Brundiers | Modifying human thymidylate kinase to potentiate azidothymidine activation[END_REF]. The steric clash between the azide group and the P-loop as the latter closes over the substrate is not likely to occur in PfTMPK since hydrogen bonding between Asp 17 and Tyr 107 places the Ploop in the fully closed position at an earlier stage in the reaction, causing the azide group to adopt a different orientation (Figure 3c) which should not interfere with catalysis. This significance of this tyrosine residue for AZTMP activation is supported by kinetic data on the hTMPK F105Y mutant [START_REF] Brundiers | Modifying human thymidylate kinase to potentiate azidothymidine activation[END_REF][START_REF] Wöhrl | Expressing engineered thymidylate kinase variants in human cells to improve AZT phosphorylation and human immunodeficiency virus inhibition[END_REF] which is much more tolerant of AZTMP than the wild-type enzyme (Table 2).

In summary, despite the overall conservation of sequence and structure in the nucleotide binding site of human and P. falciparum TMPK, it is apparent that there are a number of subtle but seemingly significant differences in the substrate and inhibitor specificities. These are highlighted by differences in the reaction catalysed by the two enzymes with dGMP and AZTMP, evidence to suggest that P. falciparum TMPK may indeed be exploited as a target for anti-malarial drug discovery.

(a)

The dTMP binding site in the PfTMPK dTMP-ADP complex (protein and solvent coloured blue, dTMP by atom type) superimposed on the equivalent human complex (pdb code 1e2d, yellow).

(b)

The ADP binding site. The position of the ADP base is displaced somewhat, due to shifts in the lid and P-loops, the more tightly closed PfTMPK structure perhaps reflecting the binding of sodium ions.

(c) Superposition of magnesium and sodium ions in the human (yellow ribbon) and PfTMPK complexes (blue) respectively, with associated 2F o -F c electron density contoured at 1σ. Sodium ions are shown as grey spheres, magnesium ions in black and water molecules in red.

Figure 3.

(a) Stereo view of the binding of dGMP in the dGMP-ADP complex coloured by atom, with the main chain shown as a transparent blue ribbon. Superimposed on this are the PfTMPK-dTMP complex, (dTMP coloured red, protein side chains coral) and the human dTMP complex (protein side chains yellow, ligand omitted for clarity).

(b) Stereo view of the binding of the bisubstrate inhibitor AP 5 dT in the PfTMPK-TMP-AP 5 dT complex (coloured by atom). The PfTMPK main chain is shown as a transparent blue ribbon, and ligand-interacting side chains in ball and stick. Sodium ions (I and II) are shown as grey spheres; water molecules are red. Hydrogen bonding interactions are represented by dashed lines. Also shown are the same ligand in complexes for other species: human (1e2g, black), yeast (3tmk, coral) and E. coli (4tmk, blue). The Mg(I) ion from the human AP 5 dT-ADP complex is shown as a black sphere, the position of a second magnesium, Mg(II) is taken from the human TMP complex (1e2d).

(c) Stereo view of protomer A of the PfTMPK-AZTMP complex (ligands and side chains coloured by atom type, blue ribbon for main chain). There is electron density, albeit at a low level, to indicate the position of the azide in this protomer, but not in the other two molecules in the asymmetric unit. The AZTMP from the human complex is superimposed (yellow).

Biochemical

  Journal Immediate Publication. Published on 30 Mar 2010 as manuscript BJ20091880 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091880A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 30 Mar 2010 as manuscript BJ20091880 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091880 A c c e p t e d M a n u s c r i p t

Figure 1 .

 1 Figure 1.

Figure 2 .

 2 Figure 2.

( d )

 d Electrostatic surface around the ligand in the PfTMPK-AZTMP complex showing how it is accommodated in the binding site. 20 Biochemical Journal Immediate Publication. Published on 30 Mar 2010 as manuscript BJ20091880 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091880 A c c e p t e d M a n u s c r i p t

Table 1 . Kinetic parameters for Plasmodium falciparum TMPK Values

 1 obtained in this study were compared to parameters previously reported[START_REF] Kandeel | Mutational, inhibitory and microcalorimetric analyses of Plasmodium falciparum TMP kinase. Implications for drug discovery[END_REF]. Biochemical Journal Immediate Publication. Published on 30 Mar 2010 as manuscript BJ20091880 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091880 A c c e p t e d M a n u s c r i p t

	Substrate	k cat (s -1 )	K m (µM)	k cat / K m (s -1 mM -1 )	k cat / K m (s -1 mM -1 ) [20]
	dTMP	4.9 ± 0.1	10.7 ± 0.5	458	154
	AzTMP	1.8 ± 0.04	9.1 ± 0.5	198	-
	dGMP	4.7 ± 0.2	12.6 ± 0.6	373	94
	dUMP	6.2 ± 0.4	155 ± 10	40	5.2
	5FdUMP	11.3 ± 0.3	47 ± 4.5	240	-
	dIMP	3.0 ± 0.07	349 ± 25	9	0.77
	GMP	0.30 ± 0.01	253 ± 24	1	-

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Table 2 . Kinetic parameters of TMPKs from different organisms for dTMP and AZTMP

 2 Biochemical Journal Immediate Publication. Published on 30 Mar 2010 as manuscript BJ20091880 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091880 A c c e p t e d M a n u s c r i p t

	Organism	k cat (s -1 ) dTMP	K m (µM) dTMP	k cat / K m (s -1 mM -1 )	k cat (s -1 ) AZTMP	K m (µM) AZTMP	k cat / K m (s -1 mM -1 )	Ratio k cat / K m dTMP/AZTMP
	E. coli a	15	2.7	5555	6	30	200	28
	S. cerevisiae a	35	9	3889	0.175	6	29	134
	P. falciparum	4.9	10.7	458	1.8	9.1	198	2.3
	H. sapiens, WT b	0.73	6.3	116	0.012	12	1	116
	H. sapiens, F105Y b	0.17	4.2	40	0.25	3.8	66	0.6

a From reference

[14]

b From reference

[START_REF] Brundiers | Modifying human thymidylate kinase to potentiate azidothymidine activation[END_REF]

2 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Table 3 . Data processing and refinement statistics for the PfTMPK complexes

 3

	Protein complex	dTMP-ADP	AP 5 dT	AZTMP-ADP dGMP-ADP
	ESRF beamline / wavelength (Å)	ID23-1 / 0.9184	ID14-4 / 0.9765	ID14-4 / 0.9395	ID23-1 / 0.976
	Data Collection Statistics
	Resolution limits a (Å)	40.0 -1.90 (1.93 -1.90)	40.0 -2.70 (2.80 -2.70)	40.0 -3.00 (3.11 -3.00)	50.0 -2.4 (2.49 -2.40)
	P3 1 21 cell dimensions (Å)	a = 110.13 c = 120.10	a = 109.54 c = 119.35	a = 110.34 c = 119.11	a = 110.74 c = 119.36
	Number of unique reflections	67,190	23,265	17,385	33,557
	Completeness a (%)	94.3 (47.4)	99.2 (92.4)	99.9 (99.9)	100.0 (100.0)
	Multiplicity a	5.1 (2.0)	8.1 (3.7)	9.4 (5.6)	10.9 (10.4)
	R-merge a	0.083 (0.298)	0.121 (0.489)	0.115 (0.287)	0.150 (0.944)
	I/σI a	17.4 (2.2)	16.8 (1.7)	22.6 (5.0)	18.3 (3.3)
	Refinement Statistics
	R-cryst / R-free (%)	17.33 / 20.50	18.03 / 26.94	19.34 / 28.69	20.09 / 26.03
	rms∆ bond lengths (Å) b	0.012 (0.022)	0.021 (0.022)	0.020 (0.022)	0.014 (0.020)
	rms∆ angles (º) b	1.365 (1.979)	2.217 (1.977)	2.166 (1.972)	1.486 (1.922)
	rms∆ chiral volume (Å 3 ) b	0.091 (0.200)	0.130 (0.200)	0.137 (0.200)	0.108 (0.200)
	Ramachandran outliers (%)	1.8	1.8	4.5	1.6
	B average on all atoms (Å 2 )	19.3	39.0	31.8	43.5

a Highest resolution shell statistics given in parentheses b Average ideal values given in parentheses.

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2010 The Authors Journal compilation © 2010 Portland Press Limited

© 2010 The Authors Journal compilation © 2010 Portland Press Limited

ACKNOWLEDGEMENTS

The authors thank the ESRF for access to beamlines and support by staff during visits, and Marek Brzozowski for assistance with crystal mounting. We thank the Diamond Light Source staff for help with use of the I24 microfocus beam line during initial solution of the dGMP complex.

FUNDING

Financial support for this research came from the Plan Nacional (SAF2007-62596), the RICET FIS Network (RD06/0021), the Junta de Andalucía (CVI-199), the European Union (ANTIMAL contract no. LSHP-CT-2005-018834) and the Wellcome Trust (grant no.


Borra Jp
email: jp.borra@pgp.u-psud.fr

Jidenko N Bourgeois

ATMOSPHERIC PRESSURE PLASMAS FOR AEROSOLS PROCESSES IN MATERIALS AND ENVIRONMENT

The paper highlights applications of some atmospheric pressure plasmas (dc-corona, streamer and spark and ac-Dielectric Barrier Discharges) to aerosol processes for Materials and Environment (filtration, diagnostics).

The production of vapor i.e. condensable gaseous species, leads to nano-sized particles by physical and chemical routes of nucleation in these AP Plasmas: (i) when dc streamer and spark filamentary discharges as well as ac filamentary Dielectric Barrier Discharges interact with metal or dielectric surfaces, and (ii) when Discharges induce reactions with gaseous precursors in volume. It is shown how composition, size and structure of primary nano-particles are related to plasma parameters (energy, number per unit surface and time and thermal gradients).

Then the growth by coagulation controls the final size of agglomerates versus plasma parameters and transit time in and after the plasma. Charging and electro-thermal collection are depicted to account for the related potential applications of controlled kinematics of charged aerosol.

1.

INTRODUCTION

Aerosol, defined as suspended liquid/solid particles (drop/powder) in gases, presents a large interfacial surface per unit volume used for heat exchanges, filtration and heterogeneous chemistry. More recently, nano-particles (1D min < 100nm) have been synthesized for their physical properties (e.g. for tribological and mechanical properties of inorganic and carbon fullerenes) and for their chemical and bio-functionalities (e.g. for lower melting temperature, catalysis and drug delivery).

In gas phase, nano-sized aerosols are formed either by evaporation of liquid droplets leading to crystallized solute (spray drying and pyrolysis), or by gas-to-particle conversion (nucleation) when the saturation vapor pressure is overcome (as for crystallization above a saturation solute concentration).

Gas-phase aerosol processes performed at atmospheric pressure provide an economic alternative for nano-technologies. They are cost-effective, environment friendly (no liquid by-products) and energy efficient. Since, high-purity nanometer particles, more easy to collect from gases than from liquids, are produced, some aerosol processes are scaled-up for daily production of hundreds of tons of tailored TiO 2 particles and others are integrated in chemistry, energy, environment and health processes.

Production processes control particle size, composition and structure. Indeed, if nano-powders are used to save raw materials, the size-dependant properties of nano-materials are reported for tailored nano-particles, smaller than 50 nm with geometric standard deviation of the size distribution below 1,2 [START_REF] Kodas | Aerosol Processing of Materials[END_REF].

A post-production processing of particles (e.g. size selection, shaping of aggregates, mixing and homogeneous/focused deposition) leads to powders, composite materials, suspensions and coatings. This paper focuses on the interest of dc to 100 kHz ac Atmospheric Pressure Electrical Discharges (hereafter referred as APED) for the production, the charging and the processing of aerosols. However, it does not cover processing of particles injected either in non-thermal Plasmas for functional coatings by post-discharge reactivity [START_REF] Tatoulian | [END_REF], nor in thermal plasmas for purification, spheroidisation, and surface coatings [3].

NANO-PARTICLE PRODUCTION BY NUCLEATION IN AP PLASMAS

Nucleation occurs when gaseous species are produced and saturated (partial pressure higher than the saturation vapor pressure). Two sources of vapor are depicted in AP Plasmas on Figure 1: (i) when dc filamentary streamer discharges as well as ac filamentary Dielectric Barrier Discharges interact with the metal or dielectric surface, and (ii) when the plasma induces reactions with gaseous precursors in volume. In both cases, condensable gaseous species lead to nanoparticles by physical and chemical routes of nucleation. It is shown how composition, size and structure of primary nanoparticles are related to plasma parameters (energy, number per unit surface and time and local thermal gradients).

Then the growth by agglomeration controlling the final size of agglomerates is also related to plasma parameters as well as to the transit time in the plasma.

Electrode with or without dielectric

Physical Nucleation

Vapors are produced by interaction of transient energy flux deposited by filamentary discharges on dielectric/electrode surface. Properties of primary nucleated nanoparticles can be controlled: -Nature of the surface and of the gas controls the final chemical composition of the nanoparticles.

-Energy per filament produced in DBD depends on the gap geometry, the dielectric properties (gap length, specific capacitance depending on thickness and permittivity of dielectric material [4] [5]), the relative humidity and controls the initial vapor flux from the surface and the subsequent diameter of the primary nucleated nanoparticle (cf. Figure 2a). The mass of material vaporized from the surface per filament is known to depend on the energy of the discharge filament [6]. That's the reason why we focus on the nucleation with « low energy » filamentary discharges to reduce vapor fluxes, local nucleated primary nanoparticles density and subsequent agglomeration, to prevent from larger size distributions and more dispersed related properties of such nano-powder produced in sparks (cf. § 2.3). Despite smaller energy per filament in DBD (~ µJ) than in spark, shorter duration (~ nanosecond) and smaller surface (~ 10 -5 cm 2 ) imply high surface power density (~ 10 12 W.m -2 ). The fluency threshold for vaporization (0.1 J.cm -2 ) is reached and nucleation of vapors occurs close to each filament but leads to smaller particles than with spark, at concentrations below 10 7 cm -3 , as expected from the smaller vapor flux than with more energetic spark (cf. Figure 2, 3, 4 and7).

-Thermal gradients around each filament, considered as a local and transient source of vapors, define the cooling rate of vapors by cold surrounding gas and the related structure of the primary nucleated nanoparticle (e.g. with spark in water [7]). Depending on the quenching rate, some unstable crystalline structures at STP can be produced (e.g. for fillers and low Temperature BaCO3 for NO x reduction [START_REF] Pratsinis | Journal of Aerosol Science invited conf[END_REF]).

Reactive Nucleation

The chemical route of nucleation from gas-phase reactions is similar in flame and in plasmas [6]. In both cases, (i) condensable species are formed by gas-phase reactions between a gaseous precursor and reactive species formed in APED (e-, photon, excited species, radicals and ions, O 3 , NO x , HNO x ), and (ii) the nucleated aerosol grows by simultaneous condensation and coagulation. Reactive nucleation in low frequency DBD is mentioned here for gas pollution control on Figure 2b [9][10] and film deposition (cf. § 4.3, [11]). The reader can refer to [12] for Carbon nano-tube production. The high selectivity and vapors production rate through controlled kinetics of reactions in plasmas lead to high purity nanoparticles when a gaseous precursor is injected in the plasma [START_REF] Kodas | Aerosol Processing of Materials[END_REF]

Dynamic of growth of primary nucleated nanoparticle into agglomerates by coagulation

Both filament-surface interaction in pure gases (no VOC traces) and gas-phase reactions with a precursor lead to intermediate transitory bimodal particle number size distributions corresponding to nucleation and agglomeration modes. The dynamics of particle formation by nucleation and of size evolution by coagulation are similar whatever the origin of the condensable species is. In both cases, nucleation occurs close to the vapor source leading to nanometer primary particles, which then coagulate within the plasma or in post-discharge maturation volume leading to agglomerates, as recently confirmed by [13]. Hence, the final size and properties (fractal dimensions and porosity) of agglomerates depend on the initial vapor flux and on the post-production growth and losses in the plasma reactor as well as in the eventual post-discharge maturation volume. Indeed, when aerosol measurements are performed after the reactor, the final size distribution depends on the size and density of primary nucleated nanoparticles, on the difference of gas and surface temperatures, on the transit times [11], and on the minimum size detected by the counter.

For instance in spark generators, the high energy per spark (from few mJ to J) leads to large particle diameter and high concentration (from 20 to 200 nm and 10 7 cm -3 ). Indeed, high particle concentration leads to particle growth by Brownian coagulation so that the initial bi-modal size distribution of nucleated and agglomerated nanoparticles turns into the so-called unimodal self preserving size distribution, as depicted on Figure 3 [14][15]. Hence, « low energy » filamentary discharges reduce local vapor fluxes, nucleation rates and related primary nucleated nanoparticles concentration to control coagulation leading to narrower size distributions than in sparks. Moreover, the porosity of agglomerates depends on the initial vapor flux and local thermal gradients, both related to the energy per discharge filament. Prevented sparks (from 0.5 to a few mJ per spark) at 200 Hz leads to similar aerosol size distribution and number concentration than the capacitive spark generator at 60 Hz with energy from 50 to 500 mJ per spark. Capacitive sparks produce mass concentration of Copper aerosol measured by Laser Induced Breakdown Spectroscopy above 100 µg.m -3 , while prevented sparks produce lower mass concentration below the detection limit. Hence, agglomerates from prevented sparks are more porous than with more energetic capacitive sparks. This tends to confirm that much higher vapor flux, nucleation rate, and local primary nucleated nanoparticles density lead to faster coagulation and less porous agglomerates closer to the "high energy" capacitive spark than with less energetic prevented sparks. Besides, the post-production densification could also be induced in hotter gas close to capacitive sparks. With "low energy" filaments in DBD, Figure 4a and b show that the post-discharge number of particles per filament also depends on maturation time and that the size distribution seems unimodal. Indeed, the counter detects particles bigger than 5 nm. The increasing concentration with maturation time proves that coagulation of primary nanoparticles smaller than 5 nm also occurs to produce a selfpreserving unimodal size distribution with modal diameter from a few to tens of nm, as shown on Figure 4b. For longer times, losses by diffusion reduce the number of particles per filament. Besides, despite higher power density at 60 kHz than at 50 Hz, the productions per filament show similar trends and ranges with different maturation times. On the one hand, this confirms that charged species that would be collected at 50 Hz (cf. §3.2), do not represent an important part of outcoming particles; On the other hand, similar filaments in colder surrounding gases at 50 Hz than at 60 kHz, lead to faster nucleation and to higher particle concentration bigger than 5 nm, sooner after the DBD.

AEROSOL CHARGING AND KINEMATICS OF CHARGED AEROSOLS

The analysis of forces on suspended particles shows that to deal with the sources of condensable gaseous species converted by nucleation into nanoparticles, the sinks of primary nanoparticles (losses to the wall -diffusion, thermophoresis and electro-collection-, as well as agglomeration) influences the final composition, structure and size of particles. Since charged aerosol kinematics depends mainly on electric forces, the charging mechanisms of submicron sized particles by collection of ions are presented in corona, post-corona and Dielectric Barrier Discharges. Indeed, in such defined electric field and ion densities profiles, both field and diffusion charging mechanisms account for the socontrolled kinematics of charged aerosols and for related applications (filtration, size distribution measurements, deposition).

Charging mechanisms

The charging mechanisms by collection of gaseous ions and the related charging laws are already defined. When particles get charged in gaseous ions/electrons densities "fine" particles smaller than 100 nm are mainly charged by diffusion of ions, "coarse" particles (>1 µm) are charged by the drift of ions on the field lines intersecting the surface of the particle, while in the intermediate size range (0.1 to 1 µm) particles are charged by combined field and diffusion charging. For a given particle size, the particle charge level depends on both the electric field and the product of ion density (Ni, cm -3 ) and transit time in this density (τ, s), as detailed in [6].

Comparison of corona and DBD chargers

APED are efficient aerosol chargers. Coronae are used on large voltage ranges due to high ion density in air/Nitrogen (<10 9 cm -3 ). Diffusion charging [START_REF] White | [END_REF][17], field charging [18], and combined charging laws [START_REF] Cochet | Colloque Int. la Phys. Forces Electrostat. et Leurs Application[END_REF] have been validated in stationary ion densities, especially in DC corona [START_REF] Alonso | [END_REF][21] [22]. However, dc-corona chargers produce ozone and particles and leads to electro-collection [START_REF] Alonso | [END_REF][21] [22] with related destabilization of the discharge. One way of limiting the losses is to work with lower voltage and lower related charge level; another is to use a post-discharge diffusion charging to reduce losses down to 50 % by self-electrostatic repulsion only, without discharge modification.

Figure 5a presents the measured mean number of charges per particle (points) versus particle diameter and theoretical diffusion and field charging laws (lines). In corona chargers, the mean Ni•τ product (3.10 6 to 10 7 s.cm -3 ) is validated by Pauthenier and White or Fuchs' charging laws, respectively for field charging of particles above 0.1 µm and for diffusion charging of smaller ones. Higher numbers of charges per particle are reached in corona than in post-corona charger. Indeed both mechanisms occur in the first one whereas only diffusion charging leads to smaller charge levels in post-discharge. Indeed, in post-discharge diffusion chargers, the ion density is lower than in the corona gap and decreases from 10 6 cm -3 by self-repulsion and electro-collection, inducing N i •τ product around 10 6 s .cm -3 . Taking advantage of tunable frequency, different DBD have been tested as aerosol chargers to reduce electro-collection [23][24][25], compared to corona chargers. The mean charge per particle (extrapolated from the collection efficiencies in the intermediate size range from 0.1 to 1 µm) is positive and higher in DBD (20 kV peak-to-peak and 1-60 kHz frequency) than in stationary ion density profiles of dc corona working at N i •τ product around 10 7 s.cm -3 [23]. However, our first charge distribution on 102 nm particles of Figure 5b shows that for such small particles only charged by diffusion, the mean charge per particle extrapolated from electro-collection efficiency is overestimated. Indeed, in this case, higher collection efficiency can result from turbulent mixing and/or additional charging mechanism for particles bigger than 30 nm, as well as thermophoresis from hotter gas to surface between filaments [6][26]. DBD is an alternative to corona discharges to charge particles efficiently with minimal losses. Indeed, the oxidation of metal electrodes as well as sparking, both producing particles and unstable discharges can be prevented. Moreover, electro-collection can be reduced in DBD, by working at ac-frequency above a few tens of kHz with similar charge than in coronae for particles charged by diffusion only (up to 100 nm), but with expected higher charge for intermediate and coarse particles (from 0.1 to 1 µm) also charged by field charging. Low frequency DBD with transit times shorter than the charging time (~ 5 periods, at least up to 9 kHz [23]), also leads to reduced charge and electro-collection [25].

NANO-MATERIALS PRODUCTION AND PROCESSING BY AP-PLASMA

For the production of nanoparticles from plasmas as well as for deposition in or after plasmas, charging and electro-thermal deposition have to be controlled.

Interest of plasma for applications of controlled kinematics of charged particles

Aerosol charging is involved in many applications such as electrostatic precipitation, coating, selfrepulsion to preserve high interfacial areas, post-production particle assembly by Coulombian coagulation of bipolar aerosols and measurements based on electrostatic techniques. Depending on the application, high charge level, or reduced losses in the charger is required. Recent advances in nanotechnology imply increasing efforts to control the charge distribution of aerosols for size measurement, sampling, transport, and material processing, especially for particles smaller than 100 nm. In this size range, only diffusion charging can account for the charging of nucleated particles. As in dc coronae, the charging of particles by repetitive ion sources in DBD is effective down to 10-30 nm, but partial charging of only a fraction of incoming particles is observed below 10 nm [11]. However, particle size measurement and film deposition including nanoparticles of controlled properties (size, composition and structure) are promising at least down to 10 nm.

Electro-collection may be prevented for aerosol size distribution measurements and for nano-materials production (cf. 4.2). Many aerosol instruments are based on defined charge-diameter relations with coronae, radioactive, UV or X-ray sources. Then particles are selected versus the size-dependant mobility [START_REF] Biskos | [END_REF]. DBD may be used as aerosol chargers for aerosol characterization; all the more that frequency of DBD can be tuned above a few kHz to limit electro-collection of particles compared to coronae. Electro-deposition of such ultra-fine nucleated nanoparticles (< 100nm) is prevented in dc and ac filamentary discharges due to poor charging efficiency with short transit time (< 0.1 ms) and to poor collection efficiency in DBD at high frequency (10-100 kHz). On the contrary, when electro-collection is required for film deposition, the charging by collection of ions is efficient only for particles bigger than a few nanometers. Hence, electro-collection of particles on surfaces is used for thin film deposition in 2.5 kHz DBD, with transit times in the DBD longer than the times of vapors formation, nucleation and growth above the minimum size to get charged [11].

Post-discharge collection: when particles have to be extracted from the plasma, thermophoretic and diffusion deposition on cold post-discharge tubes and/or cooled substrates as well as space charge repulsion, have to be considered for accurate prediction of deposition positions and rates, as well as to calculate the real initial size distribution from post-discharge mobility selection and current measurements of collected charged particles (or particle concentration), for each mobility range.

Calibrated nanoparticle generator by AP plasma for standards

reference aerosols are required to study size-dependant properties of nano-materials, as well as the indoor/outdoor transport to define the dispersion and deposition rates (e.g. for security norms of manipulation of nano-powders, exposition of people in industrial environment and for material aging). Besides, such low cost size-calibrated standard aerosols generator is also needed for the calibration of size measurement tools, which are under development. Figure 7 shows that « low energy » filament developing in DBD can be used to produce unimodal narrow size-distribution of nanoparticles.

Particle diameter (nm)

Focused/homogeneous deposition by controlled nucleation, growth and charging

Focused/ homogeneous deposition is achieved in AP Plasmas by controlling the charge, either directly in the plasma where particles are produced by nucleation or by post-production Plasma chargers [6]. Deposition of nucleated particles from plasma-gas interaction is used for pollution control (cf. 2.2) and coating, depicted here, while aerosol injection in plasma chargers is used for filtration by electrostatic precipitation (ESP) as well as for post-production sampling and assembly on surfaces [6].

To account for the size of particles included in the SiO x film deposition from Silicon precursor, all smaller than 50 nm, similar size-dependent particle charging and electro-collection are involved in both filamentary and homogeneous DBD. Indeed, despite different charging conditions, similar films are formed closer to the precursor injection in filamentary than in homogeneous DBD. The higher energy density leads to faster formation, growth and charging of particles in filamentary than in homogeneous DBD, due to higher densities of active species for reactions producing vapors and nanoparticles, and of ions for particle charging [11]. Below 10 kHz, electro-collection reduces the production of vapors and nucleated particles from DBD, either by initial collection of high mobility clusters formed by ion-induced nucleation or by postproduction charging by ion diffusion and photo-emission for 30 nm and bigger particles, for transit times above a few ms. At higher frequencies, turbulent mixing, additional charging mechanism and thermophoretic losses lead to higher collection efficiency in DBD than expected by electro-collection.

Composites

Finally, any material can be associated into composites by co-evaporation in sparks with different electrodes. Moreover, bi-metallic agglomerates made of mono-metallic nucleated primary nanoparticles from each electrode are produced in spark with two different electrodes. Hence, rapid and local nucleation occurs close to vapor sources, while coagulation leading to agglomerates occurs later. Resulting bi-metallic agglomerates could be melted to reach composite materials [START_REF] Kodas | Aerosol Processing of Materials[END_REF][3] [7].

CONCLUSION

The interest of plasmas in aerosol processes lies on tunable properties (composition, size, structure and density) of aerosol that can be produced and processed in Atmospheric Pressure Electrical Discharges:

-Both energy density in the spot of discharge filaments and gas flow rate control the vapor flux from each filament (with voltage in asymmetric gap and with gap length in DBD), the subsequent particle concentration and growth by coagulation to produce nucleated nano-powders with tailored size.

-Any material can be produced by filamentary discharges (streamer and spark) on different surfaces -High selectivity and vapors production rate lead to high purity particles from gaseous precursors.

-Controlled thermal gradient and cooling rate around the vapor source controls the particle structure, -Fast coagulation occurs for Np > 10 7 cm -3 , so that nano-materials with size dependant properties can only be produced if coagulation is prevented either by immediate in line post-production processing or at concentration lower than 10 7 cm -3 , both possible with AP Plasmas. Hence, aerosol with narrow size distribution can be produced to reach size-dependant properties of nano-materials with high specific surface area of any composition (polymer, multi-metal oxides and non-oxide Nitrides, Carbides and Borides) that can, then, be processed in powders, thin film or associated in composites by AP plasmas. New non-thermal APED, such as the Micro-hollow Cathode, have been developed in the last decade and may be used to reach higher production rates in more homogeneous and larger volume plasmas. APED can thus be considered as one of the missing links between the production goals and means in the challenging field of aerosol processes for new structural and functional materials either in powders for off-line processing, or in films, or in composite materials from integrated in-line processing.

Figure 1 :

 1 photo, thermo & ion diffusion CHARGING

  [3][8][12].

Figure 2 :

 2 Figure 2: Size distributions of (a) SiOx particles vs energy per filament for 1 or 2 mm DBD (5 mm glass at 70 kHz, 28 kV, 0.66 lpm N2 with 150 ms maturation) ; (b) Gas-phase reaction with gaseous combustion by-product and particles in ac dbd treatment of 2000 ppmv toluene in air for 90 W injected in 300 l/h gas flow [6].

Figure 3 :

 3 Figure 3 : Particles size distributions produced by APFD (a) in point-to-plane dc+ streamer and spark FD in clean and dry air, d=1cm and r point = 50µm

Figure 4 :

 4 Figure 4 : (a) Number of particles per filament, for different post-discharge maturation time at 60kHz (V pp =15 kV) and 50Hz (V pp =20 kV); (b) Al 2 O 3 particles vs maturation time (70 kHz, Vpp=14 kV, 0.5mm Al 2 O 3 , 10 lpm)

Figure 5 :

 5 Figure 5: (a) Comparison of theoretical and measured mean number of charges by diffusion (White and Fuchs) and field (Pauthenier and Smith et al.) charging versus particle diameter [6], (b) Charge distribution in DBD charger (F=20 kHz, Vpp=17.5 kV for + and 20 kV for -, with 15 ms transit in the DBD).

Figure 6 :

 6 Figure 6: Filament development and ion distribution around a single filament during half a period

  lpm, σ = 1.43 10 lpm, σ = 1.36 8.3 lpm, σ = 1.35 7.2 lpm, σ = 1.39 10.6 lpm, σ = 1.43 10 lpm, σ = 1.36 8.3 lpm, σ = 1.35 7.2 lpm, σ = 1.39

Figure 7 :

 7 Figure 7: Size distribution and related geometric standard deviation (σ) produced by our DBD aerosol generator for different N 2 flow rates from 2 mm thick Alumina DBD (gap = 1 mm at 7 kV, 70 kHz), in collaboration with Prof. Weber from Clausthal University.

b-Attachment & recombinationc-Ion dispersion for a fraction up to few µs

ACKNOWLEDGMENTS

LIBS measurements have been performed by Frejafon E, Dutouquet C. and Amodeo T from INERIS

In DBD, thin and brief filaments (diameter ~100 µm, duration ~20 ns) are homogeneously distributed in space during two quarters of period. Each filament can be considered as a local brief and confined ion/electron source (cf. Figure 6, [6]). Indeed, charging by ions mainly occurs outside the bipolar plasma confined in the filamentary discharge i.e. in the ion clouds remaining in the gap after each filament due to surface polarization, for a time depending on geometry, voltage and frequency. In DBD, the net space charge as well as the mean of the resulting charge distribution (Figure 5b) is positive, due to faster collection of electrons and negative ions on the anode side of the filament.


Karl-Heinz Schmidtf
email: k.h.schmidt@gsi.de

Beatriz Juradof
email: jurado@cenbg.in2p3.fr

Entropy-driven excitation-energy sorting in superfluid fission dynamics

Keywords: numbers: 05.30.Fk, 05.70.-a, 05.70.Fh, 21.10.Ma, 24.75.+i

It is shown that the constant-temperature behaviour of nuclei in the superfluid regime leads to an energy-sorting process if two nuclei are in thermal contact, as is the case in the fission process. This effect explains for the first time why an increase of the initial excitation energy leads an increase of the number of emitted neutrons from the heavy fission fragment, only. The observed essentially complete energy sorting may be seen as a new counter-intuitive manifestation of quantum-mechanical properties of microscopic systems.

In fission, the energy difference between the ground-state masses of the initial fissioning system and the final fission fragments, given by the Q value, and the initial excitation energy of the fissioning nucleus E* CN , ends up either in the total excitation energy (TXE) or in the total kinetic energy (TKE) of the fragments. The TXE is available for particle evaporation and gamma emission either before scission or from the separated fragments. In this work, we consider low-energy fission with initial excitation energies E* CN up to a few MeV where evaporation and gamma emission on the fission path is considered to be weak. The same is true for neck emission of neutrons. Since fission fragments are neutron-rich, evaporation proceeds almost exclusively by neutrons. We assume that already somewhat before the scission configuration the two nascent fragments have acquired their individual properties concerning shell effects [D 3D , D4D, D5D] and pairing correlations [D 6D ] and can be treated as two well defined nuclei set in thermal contact through the neck. Theoretical investigations of the gradual transition from the mononucleus regime to the di-nuclear system [X 3X , X 4X , distinguish three classes of energy, which add up to the final TXE of the fission fragments, according to their appearance at scission: (i) Collective excitations stored in normal modes. (ii) Intrinsic excitations by single-particle or quasi-particle excitations. (iii) Deformation energy. The deformation energy ends up as part of the E* available when the fission fragments recover their ground-state deformations. The deformation induced in the two nascent fragments can be considered as a superposition of a macroscopic trend, caused by the mutual Coulomb repulsion of the nascent fragments, which favours a large prolate deformation around β = 0.5 [D 11D ] and a structural influence due to shell effects. Different fission modes correspond to substantially different deformations at scission and thus to different amounts of deformation energy of the individual fragments. Theoretical arguments on the deformation of the fragments at scission can be deduced from shell-model calculations [X 11X ,D 12D ], while experimental information can be extracted from the saw-tooth-like behaviour of the neutron yields, which is thought to be mostly caused by the variation of the contribution of the deformation energy to the E* of the fragments. The division of collective excitations among the two fragments is intimately related to the nature of the specific collective mode considered. As an example, the division of E* stored in angular-momentumbearing modes is governed by the momenta of inertia of the fragments and the conservation of total angular momentum. If the fissioning nucleus has zero angular momentum, and orbital angular momentum is neglected, both fragments must carry the same amount of angular momentum (in opposite direction), and, thus, the E* is inversely proportional to their moment of inertia. For these specific modes, the lighter fragment tends to carry the larger portion of E*.

The division of intrinsic excitations can be derived when thermal equilibrium is assumed among the intrinsic degrees of freedom in each fragment. As said above, the nuclear level density at low E* is very well described by the constant-temperature formula:

  * exp( * / ) E E T   (1)
In a recent work,

This leads to a very interesting situation for the two nascent fragments at the scission-point configuration: The level density of each fragment is represented by the constant-temperature formula (1) with a specific value of T for each fragment. As a consequence, there is no solution for the division of intrinsic E* with T 1 =T 2 . As long as some excitation energy remains in the fragment with the higher temperature, its E* is transferred to the fragment with the lower temperature. That means, a process of E* sorting takes place where all E* accumulates in the fragment with the lower value of the T parameter, while the other fragment looses its entire E*. According to formula (2) the heavy fragment generally has the lower T and thus attracts all the E*. Some deviations from the constant-temperature behaviour appear only in the range of the first quasi particle excitations [D 13D ], which might influence the energy-sorting mechanism in its final phase.

Due to the influence of shell corrections on T, see eq. ( 2), the direction of the energy transfer may be reversed if the heavy fragment is stabilized by a strong shell effect. This may be expected in the standard I (SI) fission channel, which is characterised by the formation of a heavy fragment close to the doubly magic 132 Sn. The flow of E* from the hot fragment to the cold fragment can be seen as a way for the entire system made of the two nascent fragments in contact to maximize the number of occupied states or its entropy. The number of available states of the light nucleus or closed-shell nucleus is small compared to that of the complementary fragment. Therefore, the situation in which the light nucleus or the closed-shell nucleus has part of the E* leads to a smaller entropy than the situation in which the entire E* is transferred to the heavy or the non-closed-shell nucleus which has considerable more available states.

The number of evaporated neutrons as a function of the fragment mass is directly proportional to the E* of the fragment -except an offset of a few MeV that is taken away by gamma emission-and therefore should clearly reflect the peculiar situation of the full transfer of the intrinsic E* to the cold fragment. Moreover, since the neutron yield of the fission fragments for a fixed mass fluctuates over several neutrons, the mean value is a very sensitive measure of the fragment excitation energy.

If the mean energy available changes by only a fraction of the neutron separation energy, the contribution on one or the other wing of the neutron-multiplicity distribution decreases respectively increases, and thus the mean value is shifted. The neutron-induced fission of 237 Np has been studied very carefully at two different neutron energies [D 14D ]. Fig. 1 shows the average number of evaporated neutrons as a function of the fragment mass. As mentioned above, the well known sawtooth-like behaviour of this curve is attributed to the deformation energy. The minimum close to A=130 is due to the shell closures N=82, Z=50 that lead to spherical fission fragments. An increase of incident neutron energy translates into an increase of E* CN . The increase of the emitted neutrons near symmetry for 110<A<130 with incident neutron energy is caused by the increase of the yield of the super long (SL) mode which is related to well deformed fission fragments. For more asymmetric mass splits outside this range, we observe a very peculiar feature: Interestingly, Fig. 1 shows that the increase of E* leads to an increase of the number of evaporated neutrons for the heavy fragment, only. Actually, a quantitative analysis of the data reveals that all of the increased E* appears in the heavy fragmentF a F.

This observation is rather general as it was also found for other fissioning systems such as 233 U and 238 U and other incident particles like protons [D 15D , D16D, D17D, D18D]. However, no clear explanation has yet been found for this effect. The reason is that all the work [D 19D , D20D, D21D] done to study the partition of intrinsic excitation energy between fission fragments is based on a wrong description of the level density of the fragments. In fact, up to now one has assumed that the level density at low E* is well described by the analytical formula of Bethe [D 22D ]:

  ( *) exp 2 * E a E   ( 3
)
where a is the level-density parameter which is proportional to the mass number A of the nucleus. The latter formula is based on an equidistant single-particle level scheme. The temperature T is given by the inverse logarithmic slope of ρ(E*)

1 ln( ( *)) * d E T dE   (4)
which leads to the well-known relation

E*= aT 2 (5)
between E* and the temperature T of the system. If the temperatures of the two nascent fragments are required to be equal, we obtain an intrinsic E* division in proportion of the mass ratio of the fragments a Based on the experimental uncertainties, we estimated that the measurement shown in figure 1 is sensitive to a difference in the excitation energy in the order of 200 keV between the two measurements. The difference between the average number of neutrons (weighted over the corresponding mass yields) emitted by the light fragments at 5.5 and 0.8 MeV neutron energy is -0.02. Assuming a neutron separation energy of 6.5 MeV leads to an excitation energy difference of -0.02*6.5=-0.13±0.20 MeV. On the other hand, the average neutron yield for the heavy fragments increases by about 0.74 units at 5.5 MeV neutron energy. This implies an increase in excitation energy of 0.74*6.5=4.8±0.2 MeV for the heavy fragments which exploits fully the 4.7 MeV increase in neutron energy.

* 1 1 * 2 2 E A E A  (6)
The tendency to realize relation ( 6) has been confirmed empirically in many binary reactions involving relatively high E* [D 23D ], although also deviations from full equilibration were observed due to insufficient reaction time [D 24D ]. However, this expression is not applicable at low E* and fails to explain the observation presented in Fig. 1 that all the increase in E* is found in the heavy fragment. Actually, this effect is a direct consequence of the different constant temperatures of the two fragments at scission. According to eq. ( 2), the temperature of the heavy fragment, in the absence of strong shell effects, is always lower than the temperature of the light fragment. Therefore, the heavy fragment will absorb the entire available intrinsic E* and evaporate more neutrons. When the heavy fragment is close to the 132 Sn shell closures, its temperature is increased. According to eq. ( 2) the value amounts to T heavy =0.83 MeV (with A heavy =130 and S heavy = -5MeV [D 25D ]). If the shell effect of the complementary light fragment is zero, its temperature amounts to only T light =0.77 MeV (with A light =108 and S light = 0), and, thus, the direction of the energy-sorting mechanism will be reversed. In this case, the light fragment receives the entire intrinsic E*. The dip around A=130 and the peak related to the complementary fragment around A=108 may be an indication for this feature. Note that the magnitude with which the effect of the shell closure is reflected by the data depends also on the relative yield at A=130 of the S1 fission channel with respect to the SL and the Standard 2 (S2) channels. The deformed shell closure around N=88, assumed to be responsible for the S2 fission channel, is weaker [X 11X , X 12X ] than the 132 Sn shell closure and related to more asymmetric splits than the S1 channel. If we tentatively assume S heavy = -1 MeV (-4 MeV), this leads to T heavy = 0.68 MeV (0.75MeV) and T light = 0.82 MeV where we have taken A heavy = 140, A light = 98 and S light = 0. Therefore, in this case the shell closure is not strong enough to reverse the direction of the flow of E*. As for the symmetric fission channel SL, for S2 the total E* is found in the heavy fragment. We would like to stress that our argumentation is based on the same assumptions as other work that investigates the sharing of intrinsic E* at scission [X 19X , X 20X , X 21X ]. That is, we have assumed independent fission fragments and thermal equilibration between the fragments at scission. What is substantially different in our approach is that we use the constant-temperature level density which correctly describes the behaviour of nuclei at moderate E* and not the commonly used Fermi gas level density of eq. ( 3) derived by Bethe [X 22X ] which is only valid at high E*.

In conclusion, at low excitation energy E* nuclei are peculiar systems, characterised by a phase transition from superfluidity to normal-liquid behaviour. As is typical for first-order phase transitions, their temperature remains constant with increasing E*. In this sense, the nuclear superfluid to normal-liquid phase transition seems to behave like a first-order phase transition. The very special feature of this phenomenon in nuclei is that the constant-temperature regime reaches down to zero energy. The scission configuration of the fission process offers the unique possibility to investigate how two different nuclei in this special regime of constant temperature share the available intrinsic excitation when they are in thermal contact. We have shown for the first time that in this regime we reach a peculiar state of thermal equilibrium at scission in which the temperatures of the nascent fragments remain different in spite of the flow of E* from the hot to the cold fragment. Rather unexpectedly, this implies that the total amount of intrinsic E* available at scission is found in the fragment with the lower temperature. Our discovery of the energy-sorting mechanism may be considered as a new counter-intuitive manifestation of quantum-mechanical properties of microscopic systems. This entropy-driven E*-sorting process appears to have similarities with Maxwell's demon [D 26D ] on the nucleonic level. However, there is an essential difference: While sorting the energy between two containers of ideal gas reduces the entropy of the system, this is not the case for a system of two nuclei in the superfluid regime that are in thermal contact. Here the entropy is a linear function of the energy partition, which is maximum if all the available excitation energy is concentrated on the nucleus with the lower temperature. Therefore, the phenomenon of nuclear energy sorting is fully compatible with the second law of thermodynamics. This E*-sorting effect explains very easily an issue that remained unsolved up to present when comparing the number of emitted neutrons as a function of fragment mass for different initial excitation energies. It was observed in asymmetric mass splits that the increase of intrinsic E* of the fissioning nucleus appears as an increase of E* in the heavy fission fragments, only. Indeed, the temperature of the heavy fission fragments is generally lower than that of the light ones. Therefore, all the intrinsic E* is cumulated in the heavy fragment. Our work shows that the behaviour of highly excited systems according to the Fermi-gas level density, where the total E* is shared by the fragments in proportion of their masses, is strongly violated at moderate E*. Our finding has important consequences for the understanding of fission. Many conclusions that result from a wrong assumption on how the total intrinsic excitation energy is shared between the fission fragments should be revisited.

Figure 1 :

 1 Figure 1 : (Colour online) Average number of prompt neutrons as a function of the primary fragment mass for the neutron-induced fission of 237 Np at two incident neutron energies, data taken from ref. [X 14X ].

J. Earman, J. D. Norton, Stud. Hist. Phil. Mod. Phys. 29, 435 (1998).

Acknowledgments

This work was supported by the EURATOM 6. Framework Programme "European Facilities for Nuclear Data Measurements" (EFNUDAT), contract number FP6-036434. We thank F. Käppeler for providing us with the numerical values of the neutron-yield data.