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The activation energy barrier of biochemical reactions is normally lowered by an enzyme catalyst, which directly helps the weakening of the bond(s) to be broken. In many metalloenzymes, this is a first coordination sphere effect. Besides having a direct catalytic action, enzymes can fix their reactive groups and substrates so that they are optimally positioned and also modify the water activity in the system. They can either activate substrates prior to their reaction or bind preactivated substrates, thereby drastically reducing local entropic effects. The latter type is well represented by some bisubstrate reactions, where they have been defined as "entropic traps". These can be described as " second coordination sphere " processes, but enzymes can also control the reactivity beyond this point through local conformational changes belonging to an " outer coordinate sphere " that can be modulated by substrate binding. We have chosen the [4Fe-4S] cluster-dependent enzyme quinolinate synthase to illustrate each one of these processes. In addition, this very old metalloenzyme shows low in vitro substrate binding specificity, atypical reactivity that produces dead-end products, and a unique modulation of its active site volume.

Quinolinate synthase, also called NadA, is a [4Fe-4S]-containing enzyme that uses what is probably the oldest pathway to generate quinolinic acid (QA), the universal precursor of the biologically essential cofactor nicotinamide adenine dinucleotide (NAD). Its synthesis comprises the condensation of dihydroxyacetone phosphate (DHAP) and iminoaspartate (IA), which involves dephosphorylation, isomerization, cyclization, and two dehydration steps. The convergence of the three homologous domains of NadA defines a narrow active site that contains a catalytically essential [4Fe-4S] cluster. A tunnel, which can be opened or closed depending on the nature (or absence) of the bound ligand, connects this cofactor to the protein surface. One outstanding riddle has been the observation that the so far characterized active site is too small to bind IA and DHAP simultaneously. Here, we have used site-directed mutagenesis, X-ray crystallography, functional analyses, and molecular dynamics simulations to propose a condensation mechanism that involves the transient formation of a second active site cavity to which one of the substrates can migrate before this reaction takes place.

The [Fe 2 S 2 ]-RsrR gene transcription regulator senses the redox status in bacteria by modulating DNA binding, while its cluster cycles between +1 and +2 states-only the latter binds DNA. We have previously shown that RsrR can undergo remarkable conformational changes involving a 100° rotation of tryptophan 9 between exposed ( Out ) and buried ( In ) states. Here, we have used the chemical modification of Trp9, site-directed mutagenesis, and crystallographic and computational chemical studies to show that (i) the Out and In states correspond to oxidized and reduced RsrR, respectively, (ii) His33 is protonated in the In state due to a change in its p K a caused by cluster reduction, and (iii) Trp9 rotation is conditioned by the response of its dipole moment to environmental electrostatic changes. Our findings illustrate a novel function of protonation resulting from electron transfer.

The recently discovered Rrf2 family transcriptional regulator RsrR coordinates a [2Fe-2S] cluster. Remarkably, binding of the protein to RsrR-regulated promoter DNA sequences is switched on and off through the facile cycling of the [2Fe-2S] cluster between +2 and +1 states. Here, we report high resolution crystal structures of the RsrR dimer, revealing that the [2Fe-2S] cluster is asymmetrically coordinated across the RsrR monomer-monomer interface by two Cys residues from one subunit and His and Glu residues from the other. To our knowledge, this is the first example of a protein bound [Fe-S] cluster with three different amino acid side chains as ligands, and of Glu acting as ligand to a [2Fe-2S] cluster. Analyses of RsrR structures revealed a conformational change, centered on Trp9, which results in a significant shift in the DNA-binding helix-turn-helix region.

NadA is a multifunctional enzyme that condenses dihydroxyacetone phosphate (DHAP) with iminoaspartate (IA) to generate quinolinic acid (QA), the universal precursor of the nicotinamide adenine dinucleotide (NAD(P)) cofactor. Using X-ray crystallography, we have (i) characterized two of the reaction intermediates of QA synthesis using a "pH-shift" approach and a slowly reacting Thermotoga maritima NadA variant and (ii) observed the QA product, resulting from the degradation of an intermediate analogue, bound close to the entrance of a long tunnel leading to the solvent medium. We have also used molecular docking to propose a condensation mechanism between DHAP and IA based on two previously published Pyrococcus horikoshi NadA structures. The combination of reported data and our new results provide a structure-based complete catalytic sequence of QA synthesis by NadA.

The enzyme NadA catalyzes the synthesis of quinolinic acid (QA), the precursor of the universal nicotinamide adenine dinucleotide (NAD) cofactor. Here, we report the crystal structures of complexes between the Thermotoga maritima (Tm) NadA K219R/Y107F variant and (i) the first intermediate (W) resulting from the condensation of dihydroxyacetone phosphate (DHAP) with iminoaspartate and (ii) the DHAP analogue and triose-phosphate isomerase inhibitor phosphoglycolohydroxamate (PGH). In addition, using the TmNadA K219R/Y21F variant, we have reacted substrates and obtained a crystalline complex between this protein and the QA product. We also show that citrate can bind to both TmNadA K219R and its Y21F variant. The W structure indicates that condensation causes dephosphorylation. We propose that catalysis by the K219R/Y107F variant is arrested at the W intermediate because the mutated protein is unable to catalyze its aldo-keto isomerization and/or cyclization that ultimately lead to QA formation. Intriguingly, PGH binds to NadA with its phosphate group at the site where the carboxylate groups of W also bind. Our results shed significant light on the mechanism of the reaction catalyzed by NadA.

NosL is a member of a family of radical S-adenosylmethionine enzymes that catalyze the cleavage of the Cα-Cβ bond of aromatic amino acids. In this paper, we describe a set of experiments with substrate analogues and mutants for probing the early steps of the NosL mechanism. We provide biochemical evidence in support of the structural studies showing that the 5'-deoxyadenosyl radical abstracts a hydrogen atom from the amino group of tryptophan. We demonstrate that d-tryptophan is a substrate for NosL but shows relaxed regio control of the first β-scission reaction. Mutagenesis studies confirm that Arg323 is important for controlling the regiochemistry of the β-scission reaction and that Ser340 binds the substrate by hydrogen bonding to the indolic N1 atom.

In natural photosynthesis, light is used for the production of chemical energy carriers to fuel biological activity. The re-engineering of natural photosynthetic pathways can provide inspiration for sustainable fuel production and insights for understanding the process itself. Here, we employ a semiartificial approach to study photobiological water splitting via a pathway unavailable to nature: the direct coupling of the water oxidation enzyme, photosystem II, to the H2 evolving enzyme, hydrogenase. Essential to this approach is the integration of the isolated enzymes into the artificial circuit of a photoelectrochemical cell. We therefore developed a tailor-made hierarchically structured indium-tin oxide electrode that gives rise to the excellent integration of both photosystem II and hydrogenase for performing the anodic and cathodic half-reactions, respectively. When connected together with the aid of an applied bias, the semiartificial cell demonstrated quantitative electron flow from photosystem II to the hydrogenase with the production of H2 and O2 being in the expected two-to-one ratio and a light-to-hydrogen conversion efficiency of 5.4% under low-intensity red-light irradiation. We thereby demonstrate efficient light-driven water splitting using a pathway inaccessible to biology and report on a widely applicable in vitro platform for the controlled coupling of enzymatic redox processes to meaningfully study photocatalytic reactions.

Quinolinate synthase (NadA) is a Fe4S4 cluster-containing dehydrating enzyme involved in the synthesis of quinolinic acid (QA), the universal precursor of the essential nicotinamide adenine dinucleotide (NAD) coenzyme. A previously determined apo NadA crystal structure revealed the binding of one substrate analog, providing partial mechanistic information. Here, we report on the holo X-ray structure of NadA. The presence of the Fe4S4 cluster generates an internal tunnel and a cavity in which we have docked the last precursor to be dehydrated to form QA. We find that the only suitably placed residue to initiate this process is the conserved Tyr21. Furthermore, Tyr21 is close to a conserved Thr-His-Glu triad reminiscent of those found in proteases and other hydrolases. Our mutagenesis data show that all of these residues are essential for activity and strongly suggest that Tyr21 deprotonation, to form the reactive nucleophilic phenoxide anion, is mediated by the triad. NadA displays a dehydration mechanism significantly different from the one found in archetypical dehydratases such as aconitase, which use a serine residue deprotonated by an oxyanion hole. The X-ray structure of NadA will help us unveil its catalytic mechanism, the last step in the understanding of NAD biosynthesis.

The most efficient catalysts for solar fuel production should operate close to reversible potentials, yet possess a bias for the fuel-forming direction. Protein film electrochemical studies of Ni-containing carbon monoxide dehydrogenase and [NiFeSe]-hydrogenase, each a reversible electrocatalyst, show that the electronic state of the electrode strongly biases the direction of electrocatalysis of CO2/CO and H(+)/H2 interconversions. Attached to graphite electrodes, these enzymes show high activities for both oxidation and reduction, but there is a marked shift in bias, in favor of CO2 or H(+) reduction, when the respective enzymes are attached instead to n-type semiconductor electrodes constructed from CdS and TiO2 nanoparticles. This catalytic rectification effect can arise for a reversible electrocatalyst attached to a semiconductor electrode if the electrode transforms between semiconductor- and metallic-like behavior across the same narrow potential range (<0.25 V) that the electrocatalytic current switches between oxidation and reduction.

"Hyd-1", produced by Escherichia coli , exemplifies a special class of [NiFe]-hydrogenase that can sustain high catalytic H(2) oxidation activity in the presence of O(2)-an intruder that normally incapacitates the sulfur- and electron-rich active site. The mechanism of "O(2) tolerance" involves a critical role for the Fe-S clusters of the electron relay, which is to ensure the availability-for immediate transfer back to the active site-of all of the electrons required to reduce an attacking O(2) molecule completely to harmless H(2)O. The unique [4Fe-3S] cluster proximal to the active site is crucial because it can rapidly transfer two of the electrons needed. Here we investigate and establish the equally crucial role of the high potential medial [3Fe-4S] cluster, located >20 Å from the active site. A variant, P242C, in which the medial [3Fe-4S] cluster is replaced by a [4Fe-4S] cluster, is unable to sustain steady-state H(2) oxidation activity in 1% O(2). The [3Fe-4S] cluster is essential only for the first stage of complete O(2) reduction, ensuring the supply of all three electrons needed to form the oxidized inactive state "Ni-B" or "Ready" (Ni(III)-OH). Potentiometric titrations show that Ni-B is easily reduced (E(m) ≈ +0.1 V at pH 6.0); this final stage of the O(2)-tolerance mechanism regenerates active enzyme, effectively completing a competitive four-electron oxidase cycle and is fast regardless of alterations at the proximal or medial clusters. As a consequence of all these factors, the enzyme's response to O(2), viewed by its electrocatalytic activity in protein film electrochemistry (PFE) experiments, is merely to exhibit attenuated steady-state H(2) oxidation activity; thus, O(2) behaves like a reversible inhibitor rather than an agent that effectively causes irreversible inactivation. The data consolidate a rich picture of the versatile role of Fe-S clusters in electron relays and suggest that Hyd-1 can function as a proficient hydrogen oxidase.

Ni-containing carbon monoxide dehydrogenases (CODH), present in many anaerobic microorganisms, catalyze the reversible oxidation of CO to CO(2) at the so-called C-cluster. This atypical active site is composed of a [NiFe(3)S(4)] cluster and a single unusual iron ion called ferrous component II or Fe(u) that is bridged to the cluster via one sulfide ion. After additional refinement of recently published high-resolution structures of COOH(x)-, OH(x)-, and CN-bound CODH from Carboxydothermus hydrogenoformans (Jeoung and Dobbek Science 2007, 318, 1461-1464; J. Am. Chem. Soc. 2009, 131, 9922-9923), we have used computational methods on the predominant resulting structures to investigate the spectroscopically well-characterized catalytic intermediates, C(red1) and the two-electron more-reduced C(red2). Several models were geometry-optimized for both states using hybrid quantum mechanical/molecular mechanical potentials. The comparison of calculated Mössbauer parameters of these active site models with experimental data allows us to propose that the C(red1) state has a Fe(u)-Ni(2+) bridging hydroxide ligand and the C(red2) state has a hydride terminally bound to Ni(2+). Using our combined structural and theoretical data, we put forward a revised version of an earlier proposal for the catalytic cycle of Ni-containing CODH (Volbeda and Fontecilla-Camps Dalton Trans. 2005, 21, 3443-3450) that agrees with available spectroscopic and structural data. This mechanism involves an abnormal CO(2) insertion into the Ni(2+)-H(-) bond., (© 2011 American Chemical Society)

Dihydrogen (H(2)) production by [FeFe]-hydrogenases is strongly inhibited by formaldehyde (methanal) in a reaction that is rapid, reversible, and specific to this type of hydrogenase. This discovery, using three [FeFe]-hydrogenases that are homologous about the active site but otherwise structurally distinct, was made by protein film electrochemistry, which measures the activity (as electrical current) of enzymes immobilized on an electrode; importantly, the inhibitor can be removed after addition. Formaldehyde causes rapid loss of proton reduction activity which is restored when the solution is exchanged. Inhibition is confirmed by conventional solution assays. The effect depends strongly on the direction of catalysis: inhibition of H(2) oxidation is much weaker than for H(2) production, and formaldehyde also protects against CO and O(2) inactivation. By contrast, inhibition of [NiFe]-hydrogenases is weak. The results strongly suggest that formaldehyde binds at, or close to, the active site of [FeFe]-hydrogenases at a site unique to this class of enzyme--highly conserved lysine and cysteine residues, the bridgehead atom of the dithiolate ligand, or the reduced Fe(d) that is the focal center of catalysis.

A study of hybrid, enzyme-modified nanoparticles able to produce H(2) using visible light as the energy source has been carried out to establish per-site performance standards for H(2) production catalysts able to operate under ambient conditions. The [NiFeSe]-hydrogenase from Desulfomicrobium baculatum (Db [NiFeSe]-H) is identified as a particularly proficient catalyst. The optimized system consisting of Db [NiFeSe]-H attached to Ru dye-sensitized TiO(2), with triethanolamine as a sacrificial electron donor, produces H(2) at a turnover frequency of approximately 50 (mol H(2)) s(-1) (mol total hydrogenase)(-1) at pH 7 and 25 degrees C, even under the typical solar irradiation of a northern European sky. The system shows high electrocatalytic stability not only under anaerobic conditions but also after prolonged exposure to air, thus making it sufficiently robust for benchtop applications.

A major obstacle for future biohydrogen production is the oxygen sensitivity of [FeFe]-hydrogenases, the highly active catalysts produced by bacteria and green algae. The reactions of three representative [FeFe]-hydrogenases with O(2) have been studied by protein film electrochemistry under conditions of both H(2) oxidation and H(2) production, using CO as a complementary probe. The hydrogenases are DdHydAB and CaHydA from the bacteria Desulfovibrio desulfuricans and Clostridium acetobutylicum , and CrHydA1 from the green alga Chlamydomonas reinhardtii . Rates of inactivation depend on the redox state of the active site 'H-cluster' and on transport through the protein to reach the pocket in which the H-cluster is housed. In all cases CO reacts much faster than O(2). In the model proposed, CaHydA shows the most sluggish gas transport and hence little dependence of inactivation rate on H-cluster state, whereas DdHydAB shows a large dependence on H-cluster state and the least effective barrier to gas transport. All three enzymes show a similar rate of reactivation from CO inhibition, which increases upon illumination: the rate-determining step is thus assigned to cleavage of the labile Fe-CO bond, a reaction likely to be intrinsic to the atomic and electronic state of the H-cluster and less sensitive to the surrounding protein.

Ni-dependent acetyl-CoA synthase (ACS) and CO dehydrogenase (CODH) constitute the central enzyme complex of the Wood-Ljungdahl pathway of acetyl-CoA formation. The crystal structure of a recombinant bacterial ACS lacking the N-terminal domain that interacts with CODH shows a large reorganization of the remaining two globular domains, producing a narrow cleft of suitable size, shape, and nature to bind CoA. Sequence comparisons with homologous archaeal enzymes that naturally lack the N-terminal domain show that many amino acids lining this cleft are conserved. Besides the typical [4Fe-4S] center, the A-cluster contains only one proximal metal ion that, according to anomalous scattering data, is most likely Cu or Zn. Incorporation of a functional Ni(2)Fe(4)S(4) A-cluster would require only minor structural rearrangements. Using available structures, a plausible model of the interaction between CODH and the smaller ACS in archaeal multienzyme complexes is presented, along with a discussion of evolutionary relationships of the archaeal and bacterial enzymes.

Hydrogenases catalyze the conversion between 2H(+) + 2e(-) and H(2)(1). Most of these enzymes are inhibited by O(2), which represents a major drawback for their use in biotechnological applications. Improving hydrogenase O(2) tolerance is therefore a major contemporary challenge to allow the implementation of a sustainable hydrogen economy. We succeeded in improving O(2) tolerance, which we define here as the ability of the enzyme to resist for several minutes to O(2) exposure, by substituting with methionines small hydrophobic residues strongly conserved in the gas channel. Remarkably, the mutated enzymes remained active in the presence of an O(2) concentration close to that found in aerobic solutions in equilibrium with air, while the wild type enzyme is inhibited in a few seconds. Crystallographic and spectroscopic studies showed that the structure and the chemistry at the active site are not affected by the mutations. Kinetic studies demonstrated that the inactivation is slower and reactivation faster in these mutants. We propose that in addition to restricting O(2) diffusion to the active site of the enzyme, methionine may also interact with bound peroxide and provide an assisted escape route for H(2)O(2) toward the gas channel. These results show for the first time that it is possible to improve O(2)-tolerance of [NiFe] hydrogenases, making possible the development of biohydrogen production systems.

Heme has been recently described as a regulating ligand for the activity of the human nuclear receptors (NR) REV-ERBalpha and REV-ERBbeta and their Drosophila homologue E75. Here, we report the cloning, expression in Escherichia coli, purification, and screening for the heme-binding ability of 11 NR ligand-binding domains of Drosophila melanogaster (DHR3, DHR4, DHR39, DHR51, DHR78, DHR83, HNF4, TLL, ERR, FTZ-F1, and E78), of unknown structure. One of these NRs, DHR51, homologous to the human photoreceptor cell-specific nuclear receptor (PNR), specifically binds heme and exhibits a UV-visible spectrum identical to that of heme-bound E75-LBD. EPR and UV-visible absorption spectroscopy indicates that, like in E75, the heme contains a hexa-coordinated low spin ferric iron. One of its axial ligands is a tightly bound cysteine, while the other one is a histidine. A dissociation constant of 0.5 microM for the heme was measured by isothermal titration calorimetry. We show that DHR51 binds NO and CO and discuss the possibility that DHR51 may be either a gas or a heme sensor.

Protein film voltammetry studies of the [NiFeSe]-hydrogenase from Desulfomicrobium baculatum show it to be a highly efficient H2 cycling catalyst. In the presence of 100% H2, the ratio of H2 production to H2 oxidation activity is higher than for any conventional [NiFe]-hydrogenases (lacking a selenocysteine ligand) that have been investigated to date. Although traces of O2 (<< 1%) rapidly and completely remove H2 oxidation activity, the enzyme sustains partial activity for H2 production even in the presence of 1% O2 in the atmosphere. That H2 production should be partly allowed, whereas H2 oxidation is not, is explained because the inactive product of O2 attack is reductively reactivated very rapidly, but this requires a potential that is almost as negative as the thermodynamic potential for the 2H(+)/H2 couple. The study provides further encouragement and clues regarding the feasibility of microbial/enzymatic H2 production free from restrictions of anaerobicity.

Escherichia coli and related bacteria require nickel for the synthesis of hydrogenases, enzymes involved in hydrogen oxidation and proton reduction. Nickel transport to the cytoplasm depends on five proteins, NikA-E. We have previously reported the three-dimensional structure of the soluble periplasmic nickel transporter NikA in a complex with FeEDTA(H 2O) (-). We have now determined the structure of EDTA-free NikA and have found that it binds a small organic molecule that contributes three ligands to the coordination of a transition metal ion. Unexpectedly, His416, which was far from the metal-binding site in the FeEDTA(H 2O) (-)-NikA complex, becomes the fourth observed ligand to the metal. The best match to the omit map electron density is obtained for butane-1,2,4-tricarboxylate (BTC). Our attempts to obtain a BTC-Ni-NikA complex using apo protein and commercial reagents resulted in nickel-free BTC-NikA. Overall, our results suggest that nickel transport in vivo requires a specific metallophore that may be BTC.

Studies of the catalytic properties of the [FeFe]-hydrogenase from Desulfovibrio desulfuricans by protein film voltammetry, under a H2 atmosphere, reveal and establish a variety of interesting properties not observed or measured quantitatively with other techniques. The catalytic bias (inherent ability to oxidize hydrogen vs reduce protons) is quantified over a wide pH range: the enzyme is proficient at both H2 oxidation (from pH > 6) and H2 production (pH < 6). Hydrogen production is inhibited by H2, but the effect is much smaller than observed for [NiFe]-hydrogenases from Allochromatium vinosum or Desulfovibrio fructosovorans. Under anaerobic conditions and positive potentials, the [FeFe]-hydrogenase is oxidized to an inactive form, inert toward reaction with CO and O2, that rapidly reactivates upon one-electron reduction under 1 bar of H2. The potential dependence of this interconversion shows that the oxidized inactive form exists in two pH-interconvertible states with pK(ox) = 5.9. Studies of the CO-inhibited enzyme under H2 reveals a strong enhancement of the rate of activation by white light at -109 mV (monitoring H2 oxidation) that is absent at low potential (-540 mV, monitoring H+ reduction), thus demonstrating photolability that is dependent upon the oxidation state.

Drosophila E75 is a member of the nuclear receptor superfamily. These eukaryotic transcription factors are involved in almost all physiological processes. They regulate transcription in response to binding of rigid hydrophobic hormone ligands. As it is the case for many nuclear receptors, the E75 hormone ligand was originally unknown. Recently, however, it was shown that the ligand binding domain (LBD) of E75 contains a tightly bound heme prosthetic group and is gas responsive. Here we have used site-directed mutagenesis along with UV-visible and electron paramagnetic resonance (EPR) spectroscopies to characterize and assign the heme iron axial ligands in E75. The F370Y mutation and addition of hemin to the growth medium during expression of the protein in Escherichia coli were necessary to produce good yields of heme-enriched E75 LBD. EPR studies revealed the presence of several species containing a strongly iron bound thiolate. The involvement of cysteines 396 and 468 in heme binding was subsequently shown by single and double mutations. Using a similar approach, we have also established that the sixth iron ligand of a well-defined coordination conformation, which accounts for approximately half of the total species, is histidine 574. The other iron coordination pairs are discussed. We conclude that E75 is a new example of a thiolate hemoprotein and that it may be involved in hormone synthesis regulation.

A new strategy is described for comparing, quantitatively, the ability of hydrogenases to tolerate exposure to O2 and anoxic oxidizing conditions. Using protein film voltammetry, the inherent sensitivities to these challenges (thermodynamic potentials and rates of reactions) have been measured for enzymes from a range of mesophilic microorganisms. In the absence of O2, all the hydrogenases undergo reversible inactivation at various potentials above that of the H+/H2 redox couple, and H2 oxidation activities are thus limited to characteristic "potential windows". Reactions with O2 vary greatly; the [FeFe]-hydrogenase from Desulfovibrio desulfuricans ATCC 7757, an anaerobe, is irreversibly damaged by O2, surviving only if exposed to O2 in the anaerobically oxidized state (which therefore affords protection). In contrast, the membrane-bound [NiFe]-hydrogenase from the aerobe, Ralstonia eutropha, reacts reversibly with O2 even during turnover and continues to catalyze H2 oxidation in the presence of O2.

Because nickel is both essential and toxic to a great variety of organisms, its detection and transport is highly regulated. In Escherichia coli and other related Gram-negative bacteria, high affinity nickel transport depends on proteins expressed by the nik operon. A central actor of this process is the periplasmic NikA transport protein. A previous structural report has proposed that nickel binds to NikA as a pentahydrate species. However, both stereochemical considerations and X-ray absorption spectroscopic results are incompatible with that interpretation. Here, we report the 1.8 A resolution structure of NikA and show that it binds FeEDTA(H2O)- with very high affinity. In addition, we provide crystallographic evidence that a metal-EDTA complex was also bound to the previously reported NikA structure. Our observations strongly suggest that nickel transport in E. coli requires the binding of this metal ion to a metallophore that bears significant resemblance to EDTA. They also provide a basis for the potential use of NikA in the bioremediation of toxic transition metals and the design of artificial metalloenzymes.

Recent experimental and theoretical studies have focused on the mechanism of the A-cluster active site of acetyl-CoA synthase that produces acetyl-CoA from a methyl group, carbon monoxide, and CoA. Several proposals have been made concerning the redox states of the (Ni-Ni) bimetallic center and the iron-sulfur cluster connected to one of the metals. Using hybrid density functional theory, we have investigated putative intermediate states from the catalytic cycle. Among our conclusions are the following: (i) the zerovalent state proposed for the proximal metal is unlikely if the charge on the iron-sulfur cluster is +2; (ii) a mononuclear mechanism in which both CO and CH(3) bind the proximal nickel is favored over the binuclear mechanism in which CO and CH(3) bind the proximal and distal nickel ions, respectively; (iii) the formation of a disulfide bond in the active site could provide the two electrons necessary for the reaction but only if methylation occurs simultaneously; and (iv) the crystallographic closed form of the active site needs to open to accommodate ligands in the equatorial site.

ZORA relativistic DFT calculations are presented which aim to model the geometric and electronic structure of the active site of NiFe hydrogenases in its EPR-active oxidized states Ni-A (unready state) and Ni-B (ready state). Starting coordinates are taken from the X-ray structure of a mutant of Desulfovibrio fructosovorans hydrogenase refined at 1.81 A resolution. Nine possible candidates for Ni-A and Ni-B are analyzed in terms of their geometric and electronic structure. Comparison of calculated geometric and magnetic resonance parameters with available experimental data indicates that both oxidized states have a micro-hydroxo bridge between the two metal centers. The different electronic structures of both forms can be explained by a modification of a terminal cysteine in Ni-B, best modeled by protonation of the sulfur atom. A possible mechanism for the activation of both oxidized forms is presented.

Fe-only hydrogenases, as well as their NiFe counterparts, display unusual intrinsic high-frequency IR bands that have been assigned to CO and CN(-) ligation to iron in their active sites. FTIR experiments performed on the Fe-only hydrogenase from Desulfovibrio desulfuricans indicate that upon reduction of the active oxidized form, there is a major shift of one of these bands that is provoked, most likely, by the change of a CO ligand from a bridging position to a terminal one. Indeed, the crystal structure of the reduced active site of this enzyme shows that the previously bridging CO is now terminally bound to the iron ion that most likely corresponds to the primary hydrogen binding site (Fe2). The CO binding change may result from changes in the coordination sphere of Fe2 or its reduction. Superposition of this reduced active site with the equivalent region of a NiFe hydrogenase shows a remarkable coincidence between the coordination of Fe2 and that of the Fe ion in the NiFe cluster. Both stereochemical and mechanistic considerations suggest that the small organic molecule found at the Fe-only hydrogenase active site and previously modeled as 1,3-propanedithiolate may, in fact, be di-(thiomethyl)-amine.

C1s is a multidomain serine protease that is responsible for the enzymatic activity of C1, the first component of the classical pathway of complement. Its catalytic region (gamma-B) comprises two contiguous complement control protein (CCP) modules, IV and V (about 60 residues each), a 15-residue intermediary segment, and the B chain (251 residues), which is the serine protease domain. With a view to identify domain-domain interactions within this region, the gamma-B fragment of C1s, obtained by limited proteolysis with plasmin, was chemically cross-linked with the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide; then cross-linked peptides were isolated after CNBr cleavage and thermolytic digestion. N-Terminal sequence and mass spectrometry analyses allowed us to identify two cross-links between Lys 405 of module V and Glu 672 of the B chain and between Glu 418 of the intermediary segment and Lys 608 of the B chain. Three-dimensional modeling of the CCP modules IV and V and of the catalytic B chain was also carried out on the basis of their respective homology with the 16th and 5th CCP modules of complement factor H and type I serine proteases. The information provided by both the chemical cross-linking studies and the homology modeling enabled us to construct a three-dimensional model for the assembly of the C-terminal part of the gamma-B region, comprising module V, the intermediary segment, and the B chain. This model shows that module V interacts with the serine protease B chain on the side opposite to both the activation site and the catalytic site. Functional implications of this interaction are discussed in terms of the possible role of module V in the specific recognition and positioning of C4, one of the two substrates of C1s.