Allochromatium vinosum DsrC: solution-state NMR structure, redox properties, and interaction with DsrEFH, a protein essential for purple sulfur bacterial sulfur oxidation

Sequenced genomes of dissimilatory sulfur-oxidizing and sulfate-reducing bacteria containing genes coding for DsrAB, the enzyme dissimilatory sulfite reductase, inevitably also contain the gene coding for the 12-kDa DsrC protein. DsrC is thought to have a yet unidentified role associated with the activity of DsrAB. Here we report the solution structure of DsrC from the sulfur-oxidizing purple sulfur bacterium Allochromatium vinosum determined with NMR spectroscopy in reducing conditions, and we describe the redox behavior of two conserved cysteine residues upon transfer to an oxidizing environment. In reducing conditions, the DsrC structure is disordered in the highly conserved carboxy-terminus. We present multiple lines of evidence that, in oxidizing conditions, a strictly conserved cysteine (Cys111) at the penultimate position in the sequence forms an intramolecular disulfide bond with Cys100, which is conserved in DsrC in all organisms with DsrAB. While an intermolecular Cys111-Cys111 disulfide-bonded dimer is rapidly formed under oxidizing conditions, the intramolecularly disulfide-bonded species (Cys100-Cys111) is the thermodynamically stable form of the protein under these conditions. Treatment of the disulfidic forms with reducing agent regenerates the monomeric species that was structurally characterized. Using a band-shift technique under nondenaturing conditions, we obtained evidence for the interaction of DsrC with heterohexameric DsrEFH, a protein encoded in the same operon. Mutation of Cys100 to serine prevented formation of the DsrC species assigned as an intramolecular disulfide in oxidizing conditions, while still allowing formation of the intermolecular Cys111-Cys111 dimer. In the reduced form, this mutant protein still interacted with DsrEFH. This was not the case for the Cys111Ser and Cys100Ser/Cys111Ser mutants, both of which also did not form protein dimers. Our observations highlight the central importance of the carboxy-terminal DsrC cysteine residues and are consistent with a role as a sulfur-substrate binding/transferring protein, as well as with an electron-transfer function via thiol-disulfide interchanges.     

Structural and molecular genetic insight into a widespread sulfur oxidation pathway

Many environmentally important photo- and chemolithoautotrophic bacteria accumulate globules of polymeric, water-insoluble sulfur as a transient product during oxidation of reduced sulfur compounds. Oxidation of this sulfur requires the concerted action of Dsr proteins. However, individual functions and interplay of these proteins are largely unclear. We proved with a ΔdsrE mutant experiment that the cytoplasmic α₂β₂γ₂-structured protein DsrEFH is absolutely essential for the oxidation of sulfur stored in the intracellular sulfur globules of the purple sulfur bacterial model organism Allochromatium vinosum. The ability to degrade stored sulfur was fully regained upon complementation with dsrEFH in trans. The crystal structure of DsrEFH was determined at 2.5 Å resolution to assist functional assignment in detail. In conjunction with phylogenetic analyses, two different types of putative active sites were identified in DsrE and DsrH and shown to be characteristic for sulfur-oxidizing bacteria. Conserved Cys78 of A. vinosum DsrE corresponds to the active cysteines of Escherichia coli YchN and TusD. TusBCD and the protein TusE are parts of sulfur relay system involved in thiouridine biosynthesis. DsrEFH interacts with DsrC, a TusE homologue encoded in the same operon. The conserved penultimate cysteine residue in the carboxy-terminus of DsrC is essential for the interaction. Here, we show that Cys78 of DsrE is strictly required for interaction with DsrC while Cys20 in the putative active site of DsrH is dispensable for that reaction. In summary, our findings point at the occurrence of sulfur transfer reactions during sulfur oxidation via the Dsr proteins.     

A novel octameric AMP-forming acetyl-CoA synthetase from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum

AMP-forming acetyl-CoA synthetases (ACSs) are ubiquitous in all three domains of life. Here, we report the first characterization of an ACS from a hyperthermophilic organism, from the archaeon Pyrobaculum aerophilum. The recombinant ACS, the gene product of ORF PAE2867, showed extremely high thermostability and thermoactivity at temperatures around 100 degrees C. In contrast to known monomeric or homodimeric mesophilic ACSs, the P. aerophilum ACS was a 610 kDa homooctameric protein, with a significant lower content of thermolabile (Cys, Asn, and Gln) and higher content of charged (Glu, Lys, and Arg) amino acids. Kinetic analyses revealed an unusual broad substrate spectrum for organic acids and an extremely high affinity for acetate (K(m) 3 microM).     

Cytoplasmic sulfurtransferases in the purple sulfur bacterium Allochromatium vinosum: evidence for sulfur transfer from DsrEFH to DsrC

While the importance of sulfur transfer reactions is well established for a number of biosynthetic pathways, evidence has only started to emerge that sulfurtransferases may also be major players in sulfur-based microbial energy metabolism. Among the first organisms studied in this regard is the phototrophic purple sulfur bacterium Allochromatium vinosum. During the oxidation of reduced sulfur species to sulfate this Gammaproteobacterium accumulates sulfur globules. Low molecular weight organic persulfides have been proposed as carrier molecules transferring sulfur from the periplasmic sulfur globules into the cytoplasm where it is further oxidized via the "Dsr" (dissimilatory sulfite reductase) proteins. We have suggested earlier that the heterohexameric protein DsrEFH is the direct or indirect acceptor for persulfidic sulfur imported into the cytoplasm. This proposal originated from the structural similarity of DsrEFH with the established sulfurtransferase TusBCD from E. coli. As part of a system for tRNA modification TusBCD transfers sulfur to TusE, a homolog of another crucial component of the A. vinosum Dsr system, namely DsrC. Here we show that neither DsrEFH nor DsrC have the ability to mobilize sulfane sulfur directly from low molecular weight thiols like thiosulfate or glutathione persulfide. However, we demonstrate that DsrEFH binds sulfur specifically to the conserved cysteine residue DsrE-Cys78 in vitro. Sulfur atoms bound to cysteines in DsrH and DsrF were not detected. DsrC was exclusively persulfurated at DsrC-Cys111 in the penultimate position of the protein. Most importantly, we show that persulfurated DsrEFH indeed serves as an effective sulfur donor for DsrC in vitro. The active site cysteines Cys78 of DsrE and Cys20 of DsrH furthermore proved to be essential for sulfur oxidation in vivo supporting the notion that DsrEFH and DsrC are part of a sulfur relay system that transfers sulfur from a persulfurated carrier molecule to the dissimilatory sulfite reductase DsrAB.     

DsrC is involved in fermentative growth and interacts directly with the FlxABCD–HdrABC complex in Desulfovibrio vulgaris Hildenborough

DsrC is a key protein in dissimilatory sulfur metabolism, where it works as co-substrate of the dissimilatory sulfite reductase DsrAB. DsrC has two conserved cysteines in a C-terminal arm that are converted to a trisulfide upon reduction of sulfite. In sulfate-reducing bacteria, DsrC is essential and previous works suggested additional functions beyond sulfite reduction. Here, we studied whether DsrC also plays a role during fermentative growth of Desulfovibrio vulgaris Hildenborough, by studying two strains where the functionality of DsrC is impaired by a lower level of expression (IPFG07) and additionally by the absence of one conserved Cys (IPFG09). Growth studies coupled with metabolite and proteomic analyses reveal that fermentation leads to lower levels of DsrC, but impairment of its function results in reduced growth by fermentation and a shift towards more fermentative metabolism during sulfate respiration. In both respiratory and fermentative conditions, there is increased abundance of the FlxABCD–HdrABC complex and Adh alcohol dehydrogenase in IPFG09 versus the wild type, which is reflected in higher production of ethanol. Pull-down experiments confirmed a direct interaction between DsrC and the FlxABCD–HdrABC complex, through the HdrB subunit. Dissimilatory sulfur metabolism, where sulfur compounds are used for energy generation, is a key process in the ecology of anoxic environments, and is more widespread among bacteria than previously believed. Two central proteins for this type of metabolism are DsrAB dissimilatory sulfite reductase and its co-substrate DsrC. Using physiological, proteomic and biochemical studies of Desulfovibrio vulgaris Hildenborough and mutants affected in DsrC functionality, we show that DsrC is also relevant for fermentative growth of this model organism and that it interacts directly with the soluble FlxABCD-HdrABC complex that links the NAD(H) pool with dissimilatory sulfite reduction.     

Redox states of Desulfovibrio vulgaris DsrC,a key protein in dissimilatory sulfite reduction

Dissimilatory reduction of sulfite is carried out by the siroheme enzyme DsrAB, with the involvement of the protein DsrC, which has two conserved redox-active cysteines. DsrC was initially believed to be a third subunit of DsrAB. Here, we report a study of the distribution of DsrC in cell extracts to show that, in the model sulfate reducer Desulfovibrio vulgaris, the majority of DsrC is not associated with DsrAB and is thus free to interact with other proteins. In addition, we developed a cysteine-labelling gel-shift assay to monitor the DsrC redox state and behaviour, and procedures to produce the different redox forms. The oxidized state of DsrC with an intramolecular disulfide bond, which is proposed to be a key metabolic intermediate, could be successfully produced for the first time by treatment with arginine.     

Structural and functional features of an NDP kinase from the hyperthermophile crenarchaeon Pyrobaculum aerophilum

Nucleoside diphosphate (NDP) kinases are ubiquitous enzymes that transfer gamma-phosphates from nucleoside triphosphates to nucleoside diphosphates via a ping-pong mechanism. The important role of this large family of enzymes in controlling cellular functions and developmental processes along with their crystallizability has made them good candidates for structural studies. We recently determined the structure of an evolved version of an NDP kinase from Pyrobaculum aerophilum, an extreme thermophile. This NDP kinase has similarity to the 42 other NDP kinases deposited in the Protein Data Bank (PDB) but differs significantly in sequence, structure, and biophysical properties. The P. aerophilum NDP kinase sequence contains two unique segments not present in other NDP kinases, comprising residues 66-100 and 156-165. We show that deletion mutants of the P. aerophilum NDP kinase lacking either or both of these inserts have an altered substrate specificity, allowing dGTP as the phosphate donor. A structural analysis of the evolved NDP kinase in conjunction with mutagenesis experiments suggests that the substrate specificity of the P. aerophilum NDP kinase is related to the presence of these two inserts.     

Role of cysteine 306 in the catalytic mechanism of Ascaris suum phosphoenolpyruvate carboxykinase

Biochemical and metabolic data lead to the conclusion that the enzyme phosphoenolpyruvate carboxykinase (PEPCK) contributes to a critical point of divergence in energy conservation pathways between mammals and nematodes. The Ascaris suum PEPCK shares considerable homology with PEPCK from avian liver and is a good candidate for mutagenesis studies. The Cys306 substitution by Ser and Ala produced active enzymes and the two mutants are kinetically indistinguishable from each other. This substitution affects the catalytic affinity for the formation of the specific enzyme-nucleotide complex (k(cat)/K(m)) in the forward and reverse reactions. Studies with the substrate analogs 2(')dGDP and 2(')dGTP indicate that Cys306 in A. suum PEPCK is one of the residues important in nucleotide binding and may interact with the 2(')OH group in the ribose ring. Alternatively, mutation of this residue could cause protein changes that interfere with the proper conformation of the nucleotides for optimal catalysis to take place.     

Interplay between ion binding and catalysis in the thioredoxin-coupled arsenate reductase family

In the thioredoxin (Trx)-coupled arsenate reductase family, arsenate reductase from Staphylococcus aureus plasmid pI258 (Sa_ArsC) and from Bacillus subtilis (Bs_ArsC) are structurally related detoxification enzymes. Catalysis of the reduction of arsenate to arsenite involves a P-loop (Cys10Thr11Gly12Asn13Ser14Cys15Arg16) structural motif and a disulphide cascade between three conserved cysteine residues (Cys10, Cys82 and Cys89). For its activity, Sa_ArsC benefits from the binding of tetrahedral oxyanions in the P-loop active site and from the binding of potassium in a specific cation-binding site. In contrast, the steady-state kinetic parameters of Bs_ArsC are not affected by sulphate or potassium. The commonly occurring mutation of a histidine (H62), located about 6 A from the potassium-binding site in Sa_ArsC, to a glutamine uncouples the kinetic dependency on potassium. In addition, the binding affinity for potassium is affected by the presence of a lysine (K33) or an aspartic acid (D33) in combination with two negative charges (D30 and E31) on the surface of Trx-coupled arsenate reductases. In the P-loop of the Trx-coupled arsenate reductase family, the peptide bond between Gly12 and Asn13 can adopt two distinct conformations. The unique geometry of the P-loop with Asn13 in beta conformation, which is not observed in structurally related LMW PTPases, is stabilized by tetrahedral oxyanions and decreases the pK(a) value of Cys10 and Cys82. Tetrahedral oxyanions stabilize the P-loop in its catalytically most active form, which might explain the observed increase in k(cat) value for Sa_ArsC. Therefore, a subtle interplay of potassium and sulphate dictates the kinetics of Trx-coupled arsenate reductases.     

The crystal structure of Desulfovibrio vulgaris dissimilatory sulfite reductase bound to DsrC provides novel insights into the mechanism of sulfate respiration

Sulfate reduction is one of the earliest types of energy metabolism used by ancestral organisms to sustain life. Despite extensive studies, many questions remain about the way respiratory sulfate reduction is associated with energy conservation. A crucial enzyme in this process is the dissimilatory sulfite reductase (dSiR), which contains a unique siroheme-[4Fe4S] coupled cofactor. Here, we report the structure of desulfoviridin from Desulfovibrio vulgaris, in which the dSiR DsrAB (sulfite reductase) subunits are bound to the DsrC protein. The alpha(2)beta(2)gamma(2) assembly contains two siroheme-[4Fe4S] cofactors bound by DsrB, two sirohydrochlorins and two [4Fe4S] centers bound by DsrA, and another four [4Fe4S] centers in the ferredoxin domains. A sulfite molecule, coordinating the siroheme, is found at the active site. The DsrC protein is bound in a cleft between DsrA and DsrB with its conserved C-terminal cysteine reaching the distal side of the siroheme. We propose a novel mechanism for the process of sulfite reduction involving DsrAB, DsrC, and the DsrMKJOP membrane complex (a membrane complex with putative disulfide/thiol reductase activity), in which two of the six electrons for reduction of sulfite derive from the membrane quinone pool. These results show that DsrC is involved in sulfite reduction, which changes the mechanism of sulfate respiration. This has important implications for models used to date ancient sulfur metabolism based on sulfur isotope fractionations.     

Structure of coenzyme A-disulfide reductase from Staphylococcus aureus at 1.54 A resolution

Coenzyme A (CoASH) replaces glutathione as the major low molecular weight thiol in Staphylococcus aureus; it is maintained in the reduced state by coenzyme A-disulfide reductase (CoADR), a homodimeric enzyme similar to NADH peroxidase but containing a novel Cys43-SSCoA redox center. The crystal structure of S. aureus CoADR has been solved using multiwavelength anomalous dispersion data and refined at a resolution of 1.54 A. The resulting electron density maps define the Cys43-SSCoA disulfide conformation, with Cys43-S(gamma) located at the flavin si face, 3.2 A from FAD-C4aF, and the CoAS- moiety lying in an extended conformation within a cleft at the dimer interface. A well-ordered chloride ion is positioned adjacent to the Cys43-SSCoA disulfide and receives a hydrogen bond from Tyr361'-OH of the complementary subunit, suggesting a role for Tyr361' as an acid-base catalyst during the reduction of CoAS-disulfide. Tyr419'-OH is located 3.2 A from Tyr361'-OH as well and, based on its conservation in known functional CoADRs, also appears to be important for activity. Identification of residues involved in recognition of the CoAS-disulfide substrate and in formation and stabilization of the Cys43-SSCoA redox center has allowed development of a CoAS-binding motif. Bioinformatics analyses indicate that CoADR enzymes are broadly distributed in both bacterial and archaeal kingdoms, suggesting an even broader significance for the CoASH/CoAS-disulfide redox system in prokaryotic thiol/disulfide homeostasis.     

Alcohol dehydrogenase from the hyperthermophilic archaeon Pyrobaculum aerophilum: stability at high temperature

The structural and stability properties of a novel zinc-dependent alcohol dehydrogenase from the hyperthermophilic archaeon Pyrobaculum aerophilum (PyAeADHII) were investigated by Fourier transformed infrared spectroscopy (FTIR). This enzyme is a thermostable homo-tetramer belonging to the GroES-fold motif proteins characterized by an irregular β-barrel conformation. Our results revealed a protein with a secondary structure rich in β-sheet (32% of the total secondary elements) and it showed a three-step thermal unfolding pathway. The complete enzyme denaturation was preceded by the formation of a relaxed tertiary/quaternary structure and previously by an excited native-like conformation. Two-dimensional correlation spectroscopy analysis (2D-COS) and differential scanning calorimetry (DSC) experiments supported these data and allowed us to determine the protein melting temperature at 96.9 °C as well as to suggest the sequence of the events that occurred during the protein denaturation process.     

Interactions between Copper-binding Sites Determine the Redox Status and Conformation of the Regulatory N-terminal Domain of ATP7B

Copper-transporting ATPase ATP7B is essential for human copper homeostasis and normal liver function. ATP7B has six N-terminal metal-binding domains (MBDs) that sense cytosolic copper levels and regulate ATP7B. The mechanism of copper sensing and signal integration from multiple MBDs is poorly understood. We show that MBDs communicate and that this communication determines the oxidation state and conformation of the entire N-terminal domain of ATP7B (N-ATP7B). Mutations of copper-coordinating Cys to Ala in any MBD (2, 3, 4, or 6) change the N-ATP7B conformation and have distinct functional consequences. Mutating MBD2 or MBD3 causes Cys oxidation in other MBDs and loss of copper binding. In contrast, mutation of MBD4 and MBD6 does not alter the redox status and function of other sites. Our results suggest that MBD2 and MBD3 work together to regulate access to other metal-binding sites, whereas MBD4 and MBD6 receive copper independently, downstream of MBD2 and MBD3. Unlike Ala substitutions, the Cys-to-Ser mutation in MBD2 preserves the conformation and reduced state of N-ATP7B, suggesting that hydrogen bonds contribute to interdomain communications. Tight coupling between MBDs suggests a mechanism by which small changes in individual sites (induced by copper binding or mutation) result in stabilization of distinct conformations of the entire N-ATP7B and altered exposure of sites for interactions with regulatory proteins.     

The asymmetric trimeric architecture of [2Fe-2S] IscU: implications for its scaffolding during iron-sulfur cluster biosynthesis

IscU is a key component of the ISC machinery and is involved in the biogenesis of iron-sulfur (Fe-S) proteins. IscU serves as a scaffold for assembly of a nascent Fe-S cluster prior to its delivery to an apo protein. Here, we report the first crystal structure of IscU with a bound [2Fe-2S] cluster from the hyperthermophilic bacterium Aquifex aeolicus, determined at a resolution of 2.3 Å, using multiwavelength anomalous diffraction of the cluster. The holo IscU formed a novel asymmetric trimer that harbored only one [2Fe-2S] cluster. One iron atom of the cluster was coordinated by the S^γ atom of Cys36 and the N^ε atom of His106, and the other was coordinated by the S^γ atoms of Cys63 and Cys107 on the surface of just one of the protomers. However, the cluster was buried inside the trimer between the neighboring protomers. The three protomers were conformationally distinct from one another and associated around a noncrystallographic pseudo-3-fold axis. The three flexible loop regions carrying the ligand-binding residues (Cys36, Cys63, His106 and Cys107) and the N-terminal α1 helices were positioned at the interfaces and underwent substantial conformational rearrangement, which stabilized the association of the asymmetric trimer. This unique trimeric A. aeolicus holo-IscU architecture was clearly distinct from other known monomeric apo-IscU/SufU structures, indicating that asymmetric trimer organization, as well as its association/dissociation, would be involved in the scaffolding function of IscU.     

Comparison between the nitric oxide reductase family and its aerobic relatives,the cytochrome oxidases

The denitrification pathway has been studied in the hyperthermophilic archaeon Pyrobaculum aerophilum. In contrast with Gram-negative bacteria, all four denitrification enzymes are membrane-bound. P. aerophilum is also the only denitrifyer identified so far in which menaquinol is the electron donor to all four denitrification reductases. The NO reductase (NOR) of P. aerophilum belongs to the superfamily of haem-copper oxidases and is of the qNOR (quinol-dependent) type. Three types of NOR have been purified so far: cNOR (cytochrome c/pseudoazurin-dependent), qNOR and qCu(A)NOR (qNOR that contains Cu(A) at the electron entry site). It is proposed that the NORs and the various cytochrome oxidases have evolved by modular evolution, in view of the structure of their electron donor sites. qNOR is further proposed to be the ancestor of all NORs and cytochrome oxidases belonging to the superfamily of haem-copper oxidases.     

Development of an optimized refolding process for recombinant Ala-Glu-IGF-1.

Denatured and reduced N-terminal extended insulin-like growth factor-1 (AE-IGF-1) was purified from Escherichia coli extracts and subjected to in vitro folding. The renaturation process was shown to be a function of the redox potential of the solution. Folding by different methods had no significant effect on the renaturation. A maximal yield of 60% (w/w) was obtained. The folded AE-IGF-1 was enzymatically converted to IGF-1. The major by-product (20% w/w) was identified as scrambled IGF-1. Enzymatic digestion at alkaline and acidic pH suggested two possible disulphide bond arrangements; (i) Cys6-Cys47, Cys18-Cys61, Cys48-Cys52; or (ii) Cys6-Cys52, Cys18-Cys61, Cys47 and Cys48 being in their reduced forms. Energy minimization and molecular modelling suggested that the scrambled IGF-1, having reduced cysteines at positions 47 and 48, was the energetically most stable conformation of the two.     

Conformation NMR analysis of the spatial structure of Buthus eupeus insectotoxin I5A

1H NMR spectroscopy has been used to collect data related to the spatial structure of insectotoxin I5A Buthus eupeus: pH-dependence of the chemical shifts, deuterium exchange rates of individual amide hydrogens, spin-spin coupling of the H-N-C alpha-H and H-C alpha-C beta-H protons, and nuclear Overhauser effect between distinct protons belonging to amino acid residues remote in the sequence. Molecular conformation in the regions from Asp9 to Cys19 (beta-turn 9-12 and right-hand alpha-helix 12-19) and from Asn23 to Asn34 (antiparallel beta-sheet with the beta-turn 27-30) directly follows from the observed parameters. Pseudoatomic approach of distance geometry algorithm was used to solve the overall folding of the molecule and propose the most probable set of disulfide bridges: Cys2-Cys19, Cys5-Cys31, Cys16-Cys26 and Cys20-Cys33. The spatial structure of insectotoxin I5A B. eupeus demonstrates remarkable similarity with that of a "long" type scorpion neurotoxin V-3 Centruroides sculpturatus.     

Copper‐chaperones with dicoordinated Cu(I)—Unique protection mechanism

Cu(I) dicoordination with thiolate ligands is not common. Yet, different from its homologue proteins, human copper chaperone is known to bind Cu(I) using this low coordination number while binding Cu(I) only via the two conserved Cysteine residues, Cys12 and Cys15. Based on structural analysis, this work determines that the protein possesses two distinct conformations referred to as “in” and “out” due to the relative positioning of Cys12 (one of Cu(I) binding residues). The “out” conformation, with Cys12 pointing out, imposes a buried Cu(I) position, whereas the “in” conformation with Cys12 pointing inwards results in a more exposed Cu(I) thus, available for transfer. Using QM/MM methods along with thermodynamic cycles these two conformations are shown to exhibit different coordination preference, suggesting that the protein has evolved to have a unique Cu(I) protection mechanism. It is proposed that the “out” conformation with a preference to dicoordination prevents Cu(I) interaction with external ligands and/or Cu(I) release to the solvent, whereas the “in” conformation with preference to tricoordinated Cu(I), facilitates Cu(I) transfer to target proteins, where additional ligands are involved. Proteins 2013; 81:1411–1419. © 2013 Wiley Periodicals, Inc.     

The R163K mutant of human thymidylate synthase is stabilized in an active conformation: structural asymmetry and reactivity of cysteine 195

Loop 181-197 of human thymidylate synthase (hTS) populates two conformational states. In the first state, Cys195, a residue crucial for catalytic activity, is in the active site (active conformer); in the other conformation, it is about 10 A away, outside the active site (inactive conformer). We have designed and expressed an hTS variant, R163K, in which the inactive conformation is destabilized. The activity of this mutant is 33% higher than that of wt hTS, suggesting that at least one-third of hTS populates the inactive conformer. Crystal structures of R163K in two different crystal forms, with six and two subunits per asymmetric part of the unit cells, have been determined. All subunits of this mutant are in the active conformation while wt hTS crystallizes as the inactive conformer in similar mother liquors. The structures show differences in the environment of catalytic Cys195, which correlate with Cys195 thiol reactivity, as judged by its oxidation state. Calculations show that the molecular electrostatic potential at Cys195 differs between the subunits of the dimer. One of the dimers is asymmetric with a phosphate ion bound in only one of the subunits. In the absence of the phosphate ion, that is in the inhibitor-free enzyme, the tip of loop 47-53 is about 11 A away from the active site.