CO-induced structural rearrangement of the C cluster in Carboxydothermus hydrogenoformans CO dehydrogenase-evidence from Ni K-edge X-ray absorption spectroscopy

We have examined the C cluster in type II CO dehydrogenase (CODH) from Carboxydothermus hydrogenformans using Ni K-edge X-ray absorption near edge spectroscopy and extended X-ray absorption fine structure (EXAFS) spectroscopy. The enzyme was studied under three conditions: "as-isolated" and after treatment with CO or Ti(III). The shape of the Ni K-edge changes slightly between the different conditions, but no significant edge shift is seen, suggesting that the C cluster contains Ni(II) in both forms. The Ni EXAFS of as-isolated CODH can be simulated with 4 Ni-S interactions at 2.20 A with a large spread in distances. A light atom (C, N, O) is not required to fit the spectrum. After CO treatment, significant changes are observed in the EXAFS. A new feature appears at approximately 2.7 A; this component is consistent with a Ni-Fe interaction. The average Ni-S distance also expands to approximately 2.25 A. The changes between the two forms suggest that the active site (C cluster) undergoes structural rearrangement after CO treatment, and the observed changes help reconcile the two different crystal structures. The implications of the structural change for the enzyme activation and mechanism are discussed.     

Structural examination of the nickel site in chromatium vinosum hydrogenase: redox state oscillations and structural changes accompanying reductive activation and CO binding

An X-ray absorption spectroscopic study of structural changes occurring at the Ni site of Chromatium vinosum hydrogenase during reductive activation, CO binding, and photolysis is presented. Structural details of the Ni sites for the ready silent intermediate state, SI(r), and the carbon monoxide complex, SI-CO, are presented for the first time in any hydrogenase. Analysis of nickel K-edge energy shifts in redox-related samples reveals that reductive activation is accompanied by an oscillation in the electron density of the Ni site involving formally Ni(III) and Ni(II), where all the EPR-active states (forms A, B, and C) are formally Ni(III), and the EPR-silent states are formally Ni(II). Analysis of XANES shows that the Ni site undergoes changes in the coordination number and geometry that are consistent with five-coordinate Ni sites in forms A, B, and SI(u); distorted four-coordinate sites in SI(r) and R; and a six-coordinate Ni site in form C. EXAFS analysis reveals that the loss of a short Ni-O bond accounts for the change in coordination number from five to four that accompanies formation of SI(r). A shortening of the Ni-Fe distance from 2.85(5) A in form B to 2.60(5) A also occurs at the SI level and is thus associated with the loss of the bridging O-donor ligand in the active site. Multiple-scattering analysis of the EXAFS data for the SI-CO complex reveals the presence of Ni-CO ligation, where the CO is bound in a linear fashion appropriate for a terminal ligand. The putative role of form C in binding H(2) or H(-) was examined by comparing the XAS data from form C with that of its photoproduct, form L. The data rule out the suggestion that the increase in charge density on the NiFe active site that accompanies the photoprocess results in a two-electron reduction of the Ni site [Ni(III) --> Ni(I)] [Happe, R. P., Roseboom, W., and Albracht, S. P. J. (1999) Eur. J. Biochem. 259, 602-608]; only subtle structural differences between the Ni sites were observed.     

Single crystal EPR study of electronic structure and exchange interactions for copper(II)(L-arginine)2(SO4).(H2O)6: a model system to study exchange interactions between unpaired spins in proteins

We report EPR measurements at 9.77 and 34.1 GHz in powder and single crystal samples of the ternary copper amino acid complex Cu(L-arginine)(2)(SO(4)).(H(2)O)(6). The single crystal Electron Paramagnetic Resonance spectra display a single resonance for all magnetic field orientations in the ca and cb crystal planes. In the ab plane they display two resonances for most orientations of the magnetic field, and only one resonance for orientations close to the crystal axes. This behavior is a result of the selective collapse of the resonances corresponding to the four copper sites in the unit cell produced by the exchange interactions between copper ions. From the characteristics of the collapse and the angular dependences of the position and width of the resonances we evaluate the g-tensors of the copper molecules and estimate exchange interactions |J(1)/k(B)|=0.9 K and |J(2)/k(B)|=0.009 K between copper neighbors at 5.908 A and at 15.684 A, respectively. J(1) is assigned to a syn-anti equatorial-apical carboxylate bridge with a total bond length of 7.133 A. J(2) is assigned to a long bridge of 12 atoms with a total bond length of 19.789 A, that includes two hydrogen bonds. The results are discussed in terms of the crystal and electronic structure of Cu(L-arginine)(2)(SO(4)).(H(2)O)(6). We show that J(2) is in excellent agreement with the observed magnetic interaction between the reduced quinone acceptors in the photosynthetic reaction center protein of the bacterium Rb. sphaeroides, which is transmitted along a similar chemical path containing two hydrogen bonds. Our findings indicate that it is valid to estimate values for the exchange interactions between redox centers in proteins transmitted along long chemical paths containing sigma and H-bonds, from data obtained in model systems, and emphasize the importance of measuring exchange interactions in biologically relevant model systems.     

Characterization of the CO-induced, CO-tolerant hydrogenase from Rhodospirillum rubrum and the gene encoding the large subunit of the enzyme.

In the presence of carbon monoxide, the photosynthetic bacterium Rhodospirillum rubrum induces expression of proteins which allow the organism to metabolize carbon monoxide in the net reaction CO + H2O --> CO2 + H2. These proteins include the enzymes carbon monoxide dehydrogenase (CODH) and a CO-tolerant hydrogenase. In this paper, we present the complete amino acid sequence for the large subunit of this hydrogenase and describe the properties of the crude enzyme in relation to other known hydrogenases. The amino acid sequence deduced from the CO-induced hydrogenase large-subunit gene (cooH) shows significant similarity to large subunits of other Ni-Fe hydrogenases. The closest similarity is with HycE (58% similarity and 37% identity) from Escherichia coli, which is the large subunit of an Ni-Fe hydrogenase (isoenzyme 3). The properties of the CO-induced hydrogenase are unique. It is exceptionally resistant to inhibition by carbon monoxide. It also exhibits a very high ratio of H2 evolution to H2 uptake activity compared with other known hydrogenases. The CO-induced hydrogenase is tightly membrane bound, and its inhibition by nonionic detergents is described. Finally, the presence of nickel in the hydrogenase is addressed. Analysis of wild-type R. rubrum grown on nickel-depleted medium indicates a requirement for nickel for hydrogenase activity. However, analysis of strain UR294 (cooC insertion mutant defective in nickel insertion into CODH) shows that independent nickel insertion mechanisms are utilized by hydrogenase and CODH. CooH lacks the C-terminal peptide that is found in other Ni-Fe hydrogenases; in other systems, this peptide is cleaved during Ni processing.     

Removal of the bridging ligand atom at the Ni-Fe active site of [NiFe] hydrogenase upon reduction with H2, as revealed by X-ray structure analysis at 1.4 A resolution.

BACKGROUND: The active site of [NiFe] hydrogenase, a heterodimeric protein, is suggested to be a binuclear Ni-Fe complex having three diatomic ligands to the Fe atom and three bridging ligands between the Fe and Ni atoms in the oxidized form of the enzyme. Two of the bridging ligands are thiolate sidechains of cysteinyl residues of the large subunit, but the third bridging ligand was assigned as a non-protein monatomic sulfur species in Desulfovibrio vulgaris Miyazaki F hydrogenase. RESULTS: The X-ray crystal structure of the reduced form of D. vulgaris Miyazaki F [NiFe] hydrogenase has been solved at 1.4 A resolution and refined to a crystallographic R factor of 21.8%. The overall structure is very similar to that of the oxidized form, with the exception that the third monatomic bridge observed at the Ni-Fe site in the oxidized enzyme is absent, leaving this site unoccupied in the reduced form. CONCLUSIONS: The unusual ligand structure found in the oxidized form of D. vulgaris Miyazaki F [NiFe] hydrogenase was confirmed in the reduced form of the enzyme, with the exception that the electron density assigned to the monatomic sulfur bridge had almost disappeared. On the basis of this finding, as well as the observation that H2S is liberated from the oxidized enzyme under an atmosphere of H2 in the presence of its electron carrier, it was postulated that the monatomic sulfur bridge must be removed for the enzyme to be activated. A possible mechanism for the catalytic action of the hydrogenase is proposed.     

Liberation of hydrogen sulfide during the catalytic action of Desulfovibrio hydrogenase under the atmosphere of hydrogen

The active site of [NiFe] hydrogenase from Desulfovibrio species is composed of a binuclear Ni-Fe complex bearing three diatomic nonprotein ligands to Fe and three bridges between the two metals, two of which are thiolate side chains of the protein moiety. The third bridging atom in the enzyme isolated from D. vulgaris Miyazaki F was suggested to be sulfur species, but was suggested to be oxygen species in D. gigas enzyme. When the hydrogenase from D. vulgaris Miyazaki F was incubated under the atmosphere of H2, H2S was liberated from the enzyme only in the presence of its electron carrier, cytochrome c3 or methylviologen. The amount of H2S liberation was little in the absence of electron carrier or essentially null when the enzyme was incubated under N2. The amount of H2S liberated was about 37% of the hydrogenase contained in the reaction vial in molar basis. These observations are in agreement with the recent observation that the third bridging site at the Ni-Fe active site is vacant in the reduced form of the enzyme revealed by X-ray crystallography.     

Enzymes of hydrogen metabolism in Pyrococcus furiosus.

The genome of Pyrococcus furiosus contains the putative mbhABCDEFGHIJKLMN operon for a 14-subunit transmembrane complex associated with a Ni-Fe hydrogenase. Ten ORFs (mbhA-I and mbhM) encode hydrophobic, membrane-spanning subunits. Four ORFs (mbhJKL and mbhN) encode putative soluble proteins. Two of these correspond to the canonical small and large subunit of Ni-Fe hydrogenase, however, the small subunit can coordinate only a single iron-sulfur cluster, corresponding to the proximal [4Fe-4S] cubane. The structural genes for the small and the large subunits, mbhJ and mbhL, are separated in the genome by a third ORF, mbhK, encoding a protein of unknown function without Fe/S binding. The fourth ORF, mbhN, encodes a 2[4Fe-4S] protein. With P. furiosus soluble [4Fe-4S] ferredoxin as the electron donor the membranes produce H2, and this activity is retained in an extracted core complex of the mbh operon when solubilized and partially purified under mild conditions. The properties of this membrane-bound hydrogenase are unique. It is rather resistant to inhibition by carbon monoxide. It also exhibits an extremely high ratio of H2 evolution to H2 uptake activity compared with other hydrogenases. The activity is sensitive to inhibition by dicyclohexylcarbodiimide, an inhibitor of NADH dehydrogenase (complex I). EPR of the reduced core complex is characteristic for interacting iron-sulfur clusters with Em approximately -0.33 V. The genome contains a second putative operon, mbxABCDFGHH'MJKLN, for a multisubunit transmembrane complex with strong homology to the mbh operon, however, with a highly unusual putative binding motif for the Ni-Fe-cluster in the large hydrogenase subunit. Kinetic studies of membrane-bound hydrogenase, soluble hydrogenase and sulfide dehydrogenase activities allow the formulation of a comprehensive working hypothesis of H2 metabolism in P. furiosus in terms of three pools of reducing equivalents (ferredoxin, NADPH, H2) connected by devices for transduction, transfer, recovery and safety-valving of energy.     

A proton-NMR investigation of the fully reduced cytochrome c7 from Desulfuromonas acetoxidans. Comparison between the reduced and the oxidized forms.

The solution structure via 1H NMR of the fully reduced form of cytochrome c7 has been obtained. The protein sample was kept reduced by addition of catalytic amounts of Desulfovibrio gigas iron hydrogenase in H2 atmosphere after it had been checked that the presence of the hydrogenase did not affect the NMR spectrum. A final family of 35 conformers with rmsd values with respect to the mean structure of 8.7 +/- 1.5 nm and 12.4 +/- 1.3 nm for the backbone and heavy atoms, respectively, was obtained. A highly disordered loop involving residues 54-61 is present. If this loop is ignored, the rmsd values are 6.2 +/- 1.1 nm and 10.2 +/- 1.0 nm for the backbone and heavy atoms, respectively, which represent a reasonable resolution. The structure was analyzed and compared with the already available structure of the fully oxidized protein. Within the indetermination of the two solution structures, the result for the two redox forms is quite similar, confirming the special structural features of the three-heme cluster. A useful comparison can be made with the available crystal structures of cytochromes c3, which appear to be highly homologous except for the presence of a further heme. Finally, an analysis of the factors affecting the reduction potentials of the heme irons was performed, revealing the importance of net charges in differentiating the reduction potential when the other parameters are kept constant.     

Solid state NMR studies of hydrogen bonding in a citrate synthase inhibitor complex.

The ionization state and hydrogen bonding environment of the transition state analogue (TSA) inhibitor, carboxymethyldethia coenzyme A (CMX), bound to citrate synthase have been investigated using solid state NMR. This enzyme-inhibitor complex has been studied in connection with the postulated contribution of short hydrogen bonds to binding energies and enzyme catalysis: the X-ray crystal structure of this complex revealed an unusually short hydrogen bond between the carboxylate group of the inhibitor and an aspartic acid side chain [Usher et al. (1994) Biochemistry 33, 7753-7759]. To further investigate the nature of this short hydrogen bond, low spinning speed 13C NMR spectra of the CMX-citrate synthase complex were obtained under a variety of sample conditions. Tensor values describing the chemical shift anisotropy of the carboxyl groups of the inhibitor were obtained by simulating MAS spectra (233 +/- 4, 206 +/- 5, and 105 +/- 2 ppm vs TMS). Comparison of these values with our previously reported database and ab initio calculations of carbon shift tensor values clearly indicates that the carboxyl is deprotonated. New data from model compounds suggest that hydrogen bonds in a syn arrangement with respect to the carboxylate group have a pronounced effect upon the shift tensors for the carboxylate, while anti hydrogen bonds, regardless of their length, apparently do not perturb the shift tensors of the carboxyl group. Thus the tensor values for the enzyme-inhibitor complex could be consistent with either a very long syn hydrogen bond or an anti hydrogen bond; the latter would agree very well with previous crystallographic results. Two-dimensional 1H-13C heteronuclear correlation spectra of the enzyme-inhibitor complex were obtained. Strong cross-peaks were observed from the carboxyl carbon to proton(s) with chemical shift(s) of 22 +/- 5 ppm. Both the proton chemical shift and the intensity of the cross-peak indicate a very short hydrogen bond to the carboxyl group of the inhibitor, the C.H distance based upon the cross-peak intensity being 2.0 +/- 0.4 A. This proton resonance is assigned to Hdelta2 of Asp 375, on the basis of comparison with crystal structures and the fact that this cross-peak was absent in the heteronuclear correlation spectrum of the inhibitor-D375G mutant enzyme complex. In summary, our NMR studies support the suggestion that a very short hydrogen bond is formed between the TSA and the Asp carboxylate.     

Further characterization of the [Fe]-hydrogenase from Desulfovibrio desulfuricans ATCC 7757.

The properties of the periplasmic hydrogenase from Desulfovibrio desulfuricans ATCC 7757, previously reported to be a single-subunit protein [Glick, B. R., Martin, W. G., and Martin, S. M. (1980) Can. J. Microbiol. 26, 1214-1223] were reinvestigated. The pure enzyme exhibited a molecular mass of 53.5 kDa as measured by analytical ultracentrifugation and was found to comprise two different subunits of 42.5 kDa and 11 kDa, with serine and alanine as N-terminal residues, respectively. The N-terminal amino acid sequences of its large and small subunits, determined up to 25 residues, were identical to those of the Desulfovibrio vulgaris Hildenborough [Fe]-hydrogenase. D. desulfuricans ATCC 7757 hydrogenase was free of nickel and contained 14.0 atoms of iron and 14.4 atoms of acid-labile sulfur/molecule and had E400, 52.5 mM-1.cm-1. The purified hydrogenase showed a specific activity of 62 kU/mg of protein in the H2-uptake assay, and the H2-uptake activity was higher than H2-evolution activity. The enzyme isolated under aerobic conditions required incubation under reducing conditions to express its maximum activity both in the H2-uptake and 2H2/1H2 exchange reaction. The ratio of the activity of activated to as-isolated hydrogenase was approximately 3. EPR studies allowed the identification of two ferredoxin-type [4Fe-4S]1+ clusters in hydrogenase samples reduced by hydrogen. In addition, an atypical cluster exhibiting a rhombic signal (g values 2.10, 2.038, 1.994) assigned to the H2-activating site in other [Fe]-hydrogenases was detected in partially reduced samples. Molecular properties, EPR spectroscopy, catalytic activities with different substrates and sensitivity to hydrogenase inhibitors indicated that D. desulfuricans ATCC 7757 periplasmic hydrogenase is a [Fe]-hydrogenase, similar in most respects to the well characterized [Fe]-hydrogenase from D. vulgaris Hildenborough.     

Characterization of hydrogenase II from the hyperthermophilic archaeon Pyrococcus furiosus and assessment of its role in sulfur reduction

The fermentative hyperthermophile Pyrococcus furiosus contains an NADPH-utilizing, heterotetrameric (alphabetagammadelta), cytoplasmic hydrogenase (hydrogenase I) that catalyzes both H(2) production and the reduction of elemental sulfur to H(2)S. Herein is described the purification of a second enzyme of this type, hydrogenase II, from the same organism. Hydrogenase II has an M(r) of 320,000 +/- 20,000 and contains four different subunits with M(r)s of 52,000 (alpha), 39,000 (beta), 30,000 (gamma), and 24,000 (delta). The heterotetramer contained Ni (0.9 +/- 0.1 atom/mol), Fe (21 +/- 1.6 atoms/mol), and flavin adenine dinucleotide (FAD) (0.83 +/- 0.1 mol/mol). NADPH and NADH were equally efficient as electron donors for H(2) production with K(m) values near 70 microM and k(cat)/K(m) values near 350 min(-1) mM(-1). In contrast to hydrogenase I, hydrogenase II catalyzed the H(2)-dependent reduction of NAD (K(m), 128 microM; k(cat)/K(m), 770 min(-1) mM(-1)). Ferredoxin from P. furiosus was not an efficient electron carrier for either enzyme. Both H(2) and NADPH served as electron donors for the reduction of elemental sulfur (S(0)) and polysulfide by hydrogenase I and hydrogenase II, and both enzymes preferentially reduce polysulfide to sulfide rather than protons to H(2) using NADPH as the electron donor. At least two [4Fe-4S] and one [2Fe-2S] cluster were detected in hydrogenase II by electron paramagnetic resonance spectroscopy, but amino acid sequence analyses indicated a total of five [4Fe-4S] clusters (two in the beta subunit and three in the delta subunit) and one [2Fe-2S] cluster (in the gamma subunit), as well as two putative nucleotide-binding sites in the gamma subunit which are thought to bind FAD and NAD(P)(H). The amino acid sequences of the four subunits of hydrogenase II showed between 55 and 63% similarity to those of hydrogenase I. The two enzymes are present in the cytoplasm at approximately the same concentration. Hydrogenase II may become physiologically relevant at low S(0) concentrations since it has a higher affinity than hydrogenase I for both S(0) and polysulfide.     

Characterization of hydrogenase from the hyperthermophilic archaebacterium, Pyrococcus furiosus

The archaebacterium, Pyrococcus furiosus, grows optimally at 100 degrees C by a fermentative type metabolism in which H2 and CO2 are the only detectable products. The organism also reduces elemental sulfur (S0) to H2S. Cells grown in the absence of S0 contain a single hydrogenase, located in the cytoplasm, which has been purified 350-fold to apparent homogeneity. The yield of H2 evolution activity from reduced methyl viologen at 80 degrees C was 40%. The hydrogenase has a Mr value of 185,000 +/- 15,000 and is composed of three subunits of Mr 46,000 (alpha), 27,000 (beta), and 24,000 (gamma). The enzyme contains 31 +/- 3 g atoms of iron, 24 +/- 4 g atoms of acid-labile sulfide, and 0.98 +/- 0.05 g atoms of nickel/185,000 g of protein. The H2-reduced hydrogenase exhibits an electron paramagnetic resonance (EPR) signal at 70 K typical of a single [2Fe-2S] cluster, while below 15 K, EPR absorption is observed from extremely fast relaxing iron-sulfur clusters. The oxidized enzyme is EPR silent. The hydrogenase is reversibly inhibited by O2 and is remarkably thermostable. Most of its H2 evolution activity is retained after a 1-h incubation at 100 degrees C. Reduced ferredoxin from P. furiosus also acts as an electron donor to the enzyme, and a 350-fold increase in the rate of H2 evolution is observed between 45 and 90 degrees C. The hydrogenase also catalyzes H2 oxidation with methyl viologen or methylene blue as the electron acceptor. The temperature optimum for both H2 oxidation and H2 evolution is greater than 95 degrees C. Arrhenius plots show two transition points at approximately 60 and approximately 80 degrees C independent of the mode of assay. That occurring at 80 degrees C is associated with a dramatic increase in H2 production activity. The enzyme preferentially catalyzes H2 production at all temperatures examined and appears to represent a new type of "evolution" hydrogenase.     

Spectroscopic and kinetic characterization of active site mutants of Desulfovibrio fructosovorans Ni-Fe hydrogenase

Site-directed mutagenesis of amino acid residues proximate to the active site of the Ni-Fe hydrogenase of Desulfovibrio fructosovorans has been done. The different mutants have been analyzed by FTIR spectroscopy and compared with wild type enzyme. The changes observed in the spectra confirm that hydrogen bonds between the CN(-) ligands of the active site's Fe atom and certain neighbor amino acid residues stabilize the active center within the protein matrix. However, kinetic analysis of the mutants indicates that none of the replaced residues have an important role in the catalytic mechanism of the hydrogenase.     

The mechanisms of H2 activation and CO binding by hydrogenase I and hydrogenase II of Clostridium pasteurianum

Hydrogenase I (bidirectional) and hydrogenase II (uptake) of Clostridium pasteurianum have been investigated by electron paramagnetic resonance (EPR) spectroscopy, in the presence and absence of the inhibitor, CO. These hydrogenases contain both a novel type of iron-sulfur cluster (H), which is the proposed site of H2 catalysis, and ferredoxin-type [4Fe-4S] clusters (F). The results show that the H clusters of these two hydrogenases have very different properties. The H cluster of oxidized hydrogenase II (Hox-II) exhibits three distinct EPR signals, two of which are pH-dependent. Hox-II binds CO reversibly to give a single, pH-independent species with a novel, rhombic EPR spectrum. The H cluster of reduced hydrogenase II (Hred-II) does not react with CO. In contrast, the EPR spectrum of Hox-I appears homogeneous and independent of pH. Hox-I has a much lower affinity for CO than Hox-II, and binds CO irreversibly to give an axial EPR signal. Hred-I also binds CO irreversibly. The EPR spectra of Fred-I and Fred-II show little or no change after CO treatment. Prior exposure to CO does not affect the catalytic activity of the reduced or oxidized hydrogenases when assayed in the absence of CO, but both enzymes are irreversibly inactivated if CO is present during catalysis. Mechanisms for H2 activation by hydrogenase I and hydrogenase II are proposed from the determined midpoint potentials (Em, pH 8.0) of H-I and H-II (Em approximately -400 mV, -CO; approximately -360 mV, +CO), F-I (Em = -420 mV, +/- CO), and F-II (Em = -180 mV, +/- CO). These allow one to rationalize the different modes of CO binding to the two hydrogenases and suggest why hydrogenase II preferentially catalyzes H2 oxidation. The results are discussed in light of recent spectroscopic data on the structures of the two H clusters.     

Extended X-ray absorption fine structure study of Rhodospirillum rubrum and Rhodospirillum molischianum cytochromes c': relationship between heme stereochemistry and spin state

An EXAFS study on the oxidized and reduced forms of cytochromes c' from Rhodospirillum rubrum and Rhodospirillum molischianum was performed at pH 7. The cytochromes c' have an apparent coordination number of 5 in both oxidation states. Average Fe-ligand bond lengths of 2.02 +/- 0.025 and 2.06 +/- 0.025 A are obtained in their oxidized and reduced forms, respectively. By use of suitable values for the Fe-NHis bond length and Fe out-of-plane displacement, as determined by small molecule crystallographic techniques, the Fe-Npyrrole bond lengths and the porphyrin center-to-Npyrrole distance have been estimated for cytochrome c' in both of its oxidation states. With this model, estimates of the Fe-Npyrrole bond lengths are 2.01 +/- 0.03 and 2.05 +/- 0.03 A, for the oxidized and reduced cytochromes c', respectively. The center-to-Npyrrole distance is estimated to be 1.99 +/- 0.03 A for oxidized cytochrome c' and 2.03 +/- 0.03 A for reduced cytochrome c'.     

Content and localization of FMN, Fe-S clusters and nickel in the NAD-linked hydrogenase of Nocardia opaca 1b

By preparative polyacrylamide gel electrophoresis at pH 8.5, and in the absence of nickel ions, two types of subunit dimers of the NAD-linked hydrogenase from Nocardia opaca 1b were separated and isolated, and their properties were compared with each other as well as with the properties of the native enzyme. The intact hydrogenase contained 14.3 +/- 0.4 labile sulphur, 13.6 +/- 1.1 iron and 3.8 +/- 0.1 nickel atoms and approximately 1 FMN molecule per enzyme molecule. The oxidized hydrogenase showed an absorption spectrum with maxima (shoulders) at 380 nm and 420 nm and an electron spin resonance (ESR) spectrum with a signal at g = 2.01. The midpoint redox potential of the Fe-S cluster giving rise to this signal was +25 mV. In the reduced state, hydrogenase gave characteristic low-temperature (10-20 K) and high-temperature (greater than 40 K) ESR spectra which were interpreted as due to [4Fe-4S] and [2Fe-2S] clusters, respectively. The midpoint redox potentials of these clusters were determined to be -420 mV and -285 mV, respectively. The large hydrogenase dimer, consisting of subunits with relative molecular masses Mr, of 64000 and 31000, contained 9.9 +/- 0.4 S2- and 9.3 +/- 0.5 iron atoms per protein molecule. This dimer contained the FMN molecule, but no nickel. The absorption and ESR spectra of the large dimer were qualitatively similar to the spectra of the whole enzyme. This dimer did not show any hydrogenase activity, but reduced several electron acceptors with NADH as electron donor (diaphorase activity). The small hydrogenase dimer, consisting of subunits with Mr of 56000 and 27000, was demonstrated to have substantially different properties. For iron and labile sulphur average values of 3.9 and 4.3 atoms/dimer molecule have been determined, respectively. The dimer contained, in addition, about 2 atoms of nickel and was free of flavins. In the oxidized state this dimer showed an absorption spectrum with a broad band in the 400-nm region and a characteristic ESR signal at g = 2.01. The reduced form of the dimer was ESR-silent. The small dimer alone was diaphorase-inactive and did not reduce NAD with H2, but it displayed high H2-uptake activities with viologen dyes, methylene blue and FMN, and H2-evolving activity with reduced methyl viologen. Hydrogen-dependent NAD reduction was fully restored by recombining both subunit dimers, although the reconstituted enzyme differed from the original in its activity towards artificial acceptors and the ESR spectrum in the oxidized state.     

Single crystal EPR studies of the oxidized active site of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F

The Ni-A and the Ni-B forms of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F have been studied in single crystals by continuous wave and pulsed EPR spectroscopy at different temperatures (280?K, 80?K, and 10?K). For the first time, the orientation of the g-tensor axes with respect to the recently published atomic structure of the active site at 1.8?Å resolution was elucidated for Ni-A and Ni-B. The determined g-tensors have a similar orientation. The configuration of the electronic ground state is proposed to be Ni(III) 3d ¹ _z2 for Ni-A and Ni-B. The g _z principal axis is close to the Ni-S(Cys549) direction; the g _x and the g _y axes are approximately along the Ni-S(Cys546) and Ni-S(Cys81) bonds, respectively. It is proposed that the difference between the Ni-A and Ni-B states lies in a protonation of the bridging ligand between the Ni and the Fe.     

The cis-Pro touch-turn: a rare motif preferred at functional sites

A new motif of three-dimensional (3D) protein structure is described, called the cis-Pro touch-turn. In this four-residue, three-peptide motif, the central peptide is cis. Residue 2, which precedes the proline, has phi, psi values either in the "prePro region" of the Ramachandran plot near -130 degrees, 75 degrees or in the Lalpha region near +60 degrees, +60 degrees. The Calpha(1)-Calpha(4) distance is 4-5 A and the two flanking peptides lie parallel to one another, making van der Waals contact rather than a hydrogen bond. Apparently, this arrangement is locally unfavorable and therefore rare, usually occurring only if needed for biological function. Of the 12 examples in a 500-protein database, cis-Pro touch-turns are found at the catalytic sites of pectate lyase, Ni-Fe hydrogenase, glucoamylase, xylanase, and opine dehydrogenase and at the primary binding sites of ribonuclease H, type I DNA polymerase, ribotoxin, and phage gene 3 protein. In each of these protein families, the touch-turns serve different roles; their functional importance is supported by conservation and mutagenesis data. In analyzing the conservation patterns of these 3D motifs, new methods for in-depth quality evaluation of the structural bioinformatic data are employed to distinguish between significant exceptions and errors     

Purification and properties of membrane-bound hydrogenase from Azotobacter vinelandii

Uptake hydrogenase (EC 1.12) from Azotobacter vinelandii has been purified 250-fold from membrane preparations. Purification involved selective solubilization of the enzyme from the membranes, followed by successive chromatography on DEAE-cellulose, Sephadex G-100, and hydroxylapatite. Freshly isolated hydrogenase showed a specific activity of 110 mumol of H2 uptake (min X mg of protein)-1. The purified hydrogenase still contained two minor contaminants that ran near the front on sodium dodecyl sulfate-polyacrylamide gels. The enzyme appears to be a monomer of molecular weight near 60,000 +/- 3,000. The pI of the protein is 5.8 +/- 0.2. With methylene blue or ferricyanide as the electron acceptor (dyes such as methyl or benzyl viologen with negative midpoint potentials did not function), the enzyme had pH optima at pH 9.0 or 6.0, respectively, It has a temperature optimum at 65 to 70 degrees C, and the measured half-life for irreversible inactivation at 22 degrees C by 20% O2 was 20 min. The enzyme oxidizes H2 in the presence of an electron acceptor and also catalyzes the evolution of H2 from reduced methyl viologen; at the optimal pH of 3.5, 3.4 mumol of H2 was evolved (min X mg of protein)-1. The uptake hydrogenase catalyzes a slow deuterium-water exchange in the absence of an electron acceptor, and the highest rate was observed at pH 6.0. The Km values varied widely for different electron acceptors, whereas the Km for H2 remained virtually constant near 1 to 2 microM, independent of the electron acceptors.     

Displacement of iron by zinc at the diiron site of Desulfovibrio vulgaris rubrerythrin: X-ray crystal structure and anomalous scattering analysis

X-ray crystal structures of recombinant Desulfovibrio (D.) vulgaris rubrerythrin (Rbr) have shown a diiron site, whereas the crystal structure of Rbr "as-isolated" from D. vulgaris was reported to contain a mixed Zn,Fe binuclear site. To investigate the possibility that zinc had displaced iron during isolation or crystallization of the "as-isolated" D. vulgaris Rbr, the X-ray crystal structure of recombinant D. vulgaris all-iron Rbr that had been incubated with excess zinc sulfate prior to crystallization, yielding a protein labeled Zn,FeRbr, was solved. Analysis of the anomalous scattering data obtained at two different wavelengths showed that zinc had displaced a significant proportion of iron from both iron centers of the diiron site, and that no iron had been displaced from the [Fe(SCys)(4)] site. UV-visible absorption spectra of the redissolved Zn,FeRbr crystals showed 30-40% retention of oxo-bridged diferric sites, and the redissolved crystals had 37% of the peroxidase specific activity of the starting all-iron Rbr, which, together with the crystallographic results, indicate a predominant mixture of Fe1,Fe2 and Zn1,Zn2 sites. The structure of the Zn(Fe)1,Fe(Zn)2 binuclear site in the Zn,FeRbr crystals was very similar to that of the Zn,Fe binuclear site reported for the "as-isolated" D. vulgaris Rbr, including tetrahedral four-coordination at the Zn(Fe)1 site. The diiron sites in the recombinant Zn,FeRbr crystals were likely at least partially reduced during synchrotron irradiation. Our results suggest that the mixed-metal binuclear site reported for the "as-isolated" D. vulgaris Rbr could be due to displacement of iron from a native diiron site by adventitious zinc during isolation and/or crystallization, and that reduced diiron and dizinc sites can adopt very similar structures in Rbr.