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The stability of iron–sulfur proteins during electrospray ionization in both positive ion and negative ion modes was investigated using Fourier transform ion cyclotron resonance mass spectrometry. Positive ion and negative ion mode mass spectra of iron and zinc rubredoxin from Clostridium pasteurianum and ferredoxins from Pyrococcus furiosus containing [3Fe–4S], [4Fe–4S], [3FeNi–4S], and [3FeMn–4S] clusters are compared. The results demonstrate that all clusters are stable as negative ions, whereas only the [4M–4S], (M = Fe, Mn, Ni) clusters are stable as positive ions. This is the first direct evidence of the existence of the [3FeMn–4S]-containing cluster from Pyrococcus furiosus. The formal oxidation state of each metal–sulfur cluster is determined by the mass-to-charge measurement, and is found to be the same for both positive and negative ions.

Electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry is used to determine the stoichiometry and oxidation states of the metal centers in several iron-sulfur proteins. Samples are introduced into the ESI source under nondenaturing conditions in order to observe intact metal-containing protein ions. The stoichiometry and oxidation state of the metal or metal-sulfur cluster in the protein ion can be derived from the mass spectrum. Mononuclear metal-containing proteins and [4Fe-4S] centers are very stable and yield the molecular ion with little or no fragmentation. Proteins that contain [2Fe-2S] clusters are less stable and yield loss of one or two sulfur atoms from the molecular species, although the molecular ion is more abundant than the fragment peaks. [3Fe-4S]-containing proteins are the least stable of the species investigated, yielding abundant peaks corresponding to the loss of one to four sulfur atoms in addition to a peak representing the molecular ion. Isotope labeling experiments show that the sulfur loss originates from the [3Fe-4S] center. Negative ion mode mass spectra were obtained and found to produce much more stable [3Fe-4S]-containing ions than obtained in positive ion mode. ESI analysis of the same proteins under denaturing conditions yields mass spectra of the apo form of the proteins. Disulfide bonds are observed in the apoprotein mass spectra that are not present in the holoprotein. These result from oxidative coupling of the cysteinyl sulfur atoms that are responsible for binding the metal center. In addition, inorganic sulfide is found to incorporate itself into the apoprotein by forming sulfur bridges between cysteine residues.

The hyperthermophilic bacterium, Thermotoga maritima , grows up to 90°C by fermenting carbohydrates and it disposes of excess reductant by H 2 production. The H 2 -evolving cytoplasmic hydrogenase of this organism was shown to consist of three different subunits of masses 73 (α), 68 (β) and 19 (γ) kDa and to contain iron as the only metal. The genes encoding the subunits were clustered in a single operon in the order hydC (γ), hydB (β), and hydA (α). Sequence analyses indicated that: (a) the enzyme is an Fe-S-cluster-containing flavoprotein which uses NADH as an electron donor; and (b) the catalytic Fe-S cluster resides within the α-subunit, which is equivalent to the single subunit that constitutes most mesophilic Fe-hydrogenases. The α- and β-subunits of the purified enzyme were separated by chromatography in the presence of 4 M urea. As predicted, the H 2 -dependent methyl viologen reduction activity of the holoenzyme (45–70 U mg −1 ) was retained in the α-subunit (130–160 U mg −1 ) after subunit separation. However, the holoenzyme did not contain flavin and neither it nor the α-subunit used NAD(P)(H) or T. maritima ferredoxin as an electron carrier. The holoenzyme, but not the α-subunit, reduced anthraquinone-2,6-disulfonate (apparent K m , 690 μM) with H 2 . The EPR properties of the reduced holoenzyme, when compared with those of the separated and reduced subunits, indicate the presence of a catalytic ‘H-cluster’ and three [4Fe-4S] and one [2Fe-2S] cluster in the α-subunit, together with one [4Fe-4S] and two [2Fe-2S] clusters in the β-subunit. Sequence analyses predict that the α-subunit should contain an additional [2Fe-2S] cluster, while the β-subunit should contain one [2Fe-2S] and three [4Fe-4S] clusters. The latter cluster contents are consistent with the measured Fe contents of about 32, 20 and 14 Fe mol −1 for the holoenzyme and the α- and β-subunits, respectively. The T. maritima enzyme is the first ‘complex’ Fe-hydrogenase to be purified and characterized, although the reason for its complexity remains unclear.

Cellobiose dehydrogenases (CDH) were purified from cellulose-grown cultures of the fungi Phanerochaete chrysosporium and Humicola insolens. The pH optimum of the cellobiose-cytochrome c oxidoreductase activity ofP. chrysosporium CDH was acidic, whereas that of H. insolens CDH was neutral. The absorption spectra of the two CDHs showed them to be typical hemoproteins, but there was a small difference in the visible region. Limited proteolysis between the heme and flavin domains was performed to investigate the cofactors. There was no difference in absorption spectrum between the heme domains ofP. chrysosporium and H. insolens CDHs. The midpoint potentials of heme at pH 7.0 were almost identical, and no difference in pH dependence was observed over the range of pH 3–9. The pH dependence of cellobiose oxidation by the flavin domains was similar to that of the native CDHs, indicating that the difference in the pH dependence of the catalytic activity between the two CDHs is because of the flavin domains. The absorption spectrum of the flavin domain fromH. insolens CDH has absorbance maxima at 343 and 426 and a broad absorption peak at 660 nm, whereas that of P. chrysosporium CDH showed a normal flavoprotein spectrum. Flavin cofactors were extracted from the flavin domains and analyzed by high-performance liquid chromatography. The flavin cofactor fromH. insolens was found to be a mixture of 60% 6-hydroxy-FAD and 40% FAD, whereas that from P. chrysosporium CDH was normal FAD. After reconstitution of the deflavo-proteins it was found that flavin domains containing 6-hydroxy-FAD were clearly active but their cellobiose oxidation rates were lower than those of flavin domains containing normal FAD. Reconstitution of flavin cofactor had no effect on the optimum pH. From these results, it is concluded that the pH dependence is not because of the flavin cofactor but is because of the protein molecule.

The ferredoxin (7.5 kDa) of the hyperthermophilic archaeon, Pyrococcus furiosus, contains a single [4Fe-4S]1+,2+ cluster that is coordinated by three Cys and one Asp residue rather than the expected four Cys. The role of this Asp residue was investigated using a series of mutants, D14X, where X = C, S, H, N, V, and Y, prepared by heterologous gene expression in Escherichia coli. While the recombinant form of the wild-type and the D14S and D14C mutants contained a [4Fe-4S]1+,2+ cluster, the D14V, D14H, D14Y, and D14N proteins contained a [3Fe-4S]0,+ center, as determined by visible spectroscopy and electrochemistry. The redox potentials (at pH 7.0, 23 degrees C) of the D14C and D14S mutants were decreased by 58 and 133 mV, respectively, compared to those of the wild-type 4Fe-ferredoxin (Em -368 mV), while those of the 3Fe-protein mutants (including the 3Fe-form of the D14S, generated by chemical oxidation) were between 15 and 118 mV more positive than that of wild-type 3Fe-form (obtained by chemical oxidation, Em -203 mV). The reduction potentials of all of the 3Fe-forms, except the D14S mutant, showed a pH response over the range 3.0-10.0 with a pK of 3.3-4.7, and this was assigned to cluster protonation. The D14H mutant and the wild-type 3Fe-proteins showed an additional pK (both at 5.9) assumed to arise from protonation of the amino acid side chain. With the 4Fe-proteins, there was no dramatic change in the potentials of the wild-type or D14C form, while the pH response of the D14S mutant (pK 4.75) was ascribed to protonation of the serinate. While the ferredoxin variants exhibited a range of thermal stabilities (measured at 80 degrees C, pH 2.5), none of them showed any temperature-dependent transitions (0-80 degrees C) in their reduction potentials, and there was no correlation between the calculated DeltaS degrees' values and the absorbance maximum, reduction potential, or hydrophobicity of residue 14. In contrast, there was a linear correlation between the DeltaH degrees' value and reduction potential. Kinetic analyses were carried out at 80 degrees C using the ferredoxin as either an electron acceptor to pyruvate oxidoreductase (POR) or as an electron donor to ferredoxin:NADP oxidoreductase (FNOR, both from P. furiosus). The data showed that the reduction potential of the ferredoxin, rather than cluster type or the nature of the residue at position 14, appears to be the predominant factor in determining efficiency of electron transfer in both systems. However, compared to all the variants, the reduction potential of WT Fd makes it the most appropriate protein to both accept electrons from POR and donate them to FNOR.

The ground- and excited-state properties of heterometallic [CuFe(3)S(4)](2+,+), [CdFe(3)S(4)](2+,+), and [CrFe(3)S(4)](2+,+) cubane clusters assembled in Pyrococcus furiosus ferredoxin have been investigated by the combination of EPR and variable-temperature/variable-field magnetic circular dichroism (MCD) studies. The results indicate Cd(2+) incorporation into [Fe(3)S(4)](0,-) cluster fragments to yield S = 2 [CdFe(3)S(4)](2+) and S = (5)/(2) [CdFe(3)S(4)](+) clusters and Cu(+) incorporation into [Fe(3)S(4)](+,0) cluster fragments to yield S = (1)/(2) [CuFe(3)S(4)](2+) and S = 2 [CuFe(3)S(4)](+) clusters. This is the first report of the preparation of cubane type [CrFe(3)S(4)](2+,+) clusters, and the combination of EPR and MCD results indicates S = 0 and S = (3)/(2) ground states for the oxidized and reduced forms, respectively. Midpoint potentials for the [CdFe(3)S(4)](2+,+), [CrFe(3)S(4)](2+,+), and [CuFe(3)S(4)](2+,+) couples, E(m) = -470 +/- 15, -440 +/- 10, and +190 +/- 10 mV (vs NHE), respectively, were determined by EPR-monitored redox titrations or direct electrochemistry at a glassy carbon electrode. The trends in redox potential, ground-state spin, and electron delocalization of [MFe(3)S(4)](2+,+) clusters in P. furiosus ferredoxin are discussed as a function of heterometal (M = Cr, Mn, Fe, Co, Ni, Cu, Zn, Cd, and Tl).

The trivial name ‘rubr-erythrin’ is a contraction of two other trivial names: rubredoxin (ruber, red) and hemerythrin. It names a protein of undetermined biological function which putatively carries rubredoxin-like mononuclear iron and hemerythrin-like dinuclear iron. The name ‘nigerythrin’ (niger, black) is an analogy of rubrerythrin. It identifies a second protein of undetermined function which has prosthetic groups similar to rubrerythrin. Rubrerythrin was initially described [LeGall, J., Prickril, B. C., Moura, I., Xavier, A. V., Moura, J. J. G. 8 Huynh, B.-H. (1988) Biochemistry 27, 1636-16421 as a homodimer with four iron ions arranged into two rubredoxin sites and one inter-subunit dinuclear cluster. Nigerythrin is a novel protein. Here, we report that both proteins are homodimers, each dimer carrying not four but six iron ions in two mononuclear centers and two dinuclear clusters. Rubrerythrin and nigerythrin are probably both located in the cytoplasm; they are differentially characterized with respect to molecular mass, PI, N-terminal sequence, antibody cross-reactivity, optical absorption, EPR spectroscopy, and reduction potentials. All three reduction potentials in both proteins are > + 200 mV. These appear too high to be of practical relevance in the cytoplasm of the sulfate reducer Desulfovibrio vulgaris (Hildenborough). We suggest the possibility of a non-redox role for both proteins with all six iron ions in the ferrous state.

The electrochemical properties of flavine adenine dinucleotide (FAD) were studied using a glassy carbon electrode. The nature of the electrochemical reaction proved to be concentration dependent. A change from adsorbed to completely diffusion-controlled was observed when changing the FAD concentration from 1 μM to 1 mM at constant pH. The concentration dependence revealed a possible mechanism of self-mediation. In this mechanism adsorption causes formation of a stable monolayer of FAD on the electrode. These adsorbed molecules can act as mediators through which electron transfer from FAD molecules in solution to the electrode can occur. The heterogeneous rate constant of the surface charge transfer of the adsorbed film was unusually high, ksc° ≈ 219 s−1 and ksa° ≈ 191 s−1, for the cathodic and anodic reaction, respectively. The transfer coefficient for the same reaction gave a rather low value αc ≈ 0.31 and αa ≈ 0.37. The mechanism of self-mediation has some implications for the electrochemistry of biomolecules. Fast and reversible electron transfer between these molecules and solid electrodes requires compatible (self-mediating) surfaces.

The hyperthermophilic archaeon Pyrococcus furiosus grows optimally at 100°C by the fermentation of peptides and carbohydrates. Growth of the organism was examined in media containing either maltose, peptides (hydrolyzed casein), or both as the carbon source(s), each with and without elemental sulfur (S 0 ). Growth rates were highest on media containing peptides and S 0 , with or without maltose. Growth did not occur on the peptide medium without S 0 . S 0 had no effect on growth rates in the maltose medium in the absence of peptides. Phenylacetate production rates (from phenylalanine fermentation) from cells grown in the peptide medium containing S 0 with or without maltose were the same, suggesting that S 0 is required for peptide utilization. The activities of 14 of 21 enzymes involved in or related to the fermentation pathways of P. furiosus were shown to be regulated under the five different growth conditions studied. The presence of S 0 in the growth media resulted in decreases in specific activities of two cytoplasmic hydrogenases (I and II) and of a membrane-bound hydrogenase, each by an order of magnitude. The primary S 0 -reducing enzyme in this organism and the mechanism of the S 0 dependence of peptide metabolism are not known. This study provides the first evidence for a highly regulated fermentation-based metabolism in P. furiosus and a significant regulatory role for elemental sulfur or its metabolites.

Hydrogenases catalyze the reversible reduction of protons to hydrogen gas. They are found in a wide variety of microorganisms and enable them to use H2 as a source of reductant under either aerobic or anaerobic conditions. Alternatively, fermentative-type organisms utilize hydrogenase to dispose of reductant without the need of terminal electron acceptors other than protons (1, 3). Hydrogenases can be divided into two major types, depending on the metals they contain (5). The so-called iron-only hydrogenases have high specific activities and usually function to evolve H2. Their catalytic site is comprised of a novel 6Fe cluster (26, 29). The active site of nickel- and iron-containing hydrogenases (NiFe-hydrogenases), on the other hand, consists of a binuclear NiFe center (12, 36). The NiFe-hydrogenases are less active than their Fe-only counterparts, and their physiological role is usually to oxidize H2. In aerobic H2-oxidizing bacteria, NiFe-hydrogenases can function both as cytoplasmic, NAD-reducing enzymes and as part of conventional membrane-bound (MB) respiratory chains where O2 is the terminal electron acceptor (4, 9). In contrast, in anaerobic respiratory systems, the role of hydrogenase is poorly understood. For example, the methanogen Methanosarcina barkeri contains an MB NiFe-hydrogenase as part of a multiprotein complex (18, 25), the components of which show high sequence similarity to a NiFe-hydrogenase-containing complex present in the photosynthetic bacterium Rhodospirillum rubrum (13, 14). Both of these MB systems are thought to be involved in energy conservation, but the pathways of electron transfer and the precise role of the hydrogenases and of the associated proteins are unclear. In addition, three of the four MB NiFe-hydrogenases present in Escherichia coli are also thought to be involved in energy conservation (7, 8, 34). In this study, we focused on the metabolism of H2 by the anaerobic archaeon Pyrococcus furiosus, an obligate organotroph that grows optimally near 100°C (11). This fermentative organism utilizes sugars via a modified ADP-dependent Embden-Meyerhof pathway (16), while amino acids derived from peptides are metabolized via transaminases and a suite of 2-keto acid oxidoreductases (2). In both pathways energy is conserved via substrate-level phosphorylation. The coenzyme A (CoA) derivatives that are generated are converted to organic acids directly by a novel pair of enzymes, acetyl-CoA synthetases I and II, which simultaneously convert ADP and phosphate to ATP (23). The major end products of fermentation are acetate, H2, and CO2; some other organic acids are also produced when peptides are the growth substrate. The oxidation of amino acid-derived 2-keto acids and of glyceraldehyde-3-phosphate and pyruvate in the glucose fermentation pathway are all carried out by ferredoxin-dependent oxidoreductases. It has been proposed (22) that the oxidation of reduced ferredoxin is coupled to the reduction of NADP via ferredoxin: NADP oxidoreductase (FNOR [19]) and that NADPH then serves as the electron donor to two cytoplasmic H2-evolving hydrogenases (I and II) (10, 21, 28). The reason why two such enzymes are present is not understood (21). P. furiosus also reduces elemental sulfur (S0) to H2S. This process decreases the amount of H2 produced and has a stimulatory effect on growth, as indicated by an increase in cell density and growth rate (11). Moreover, during growth on maltose, the cell yield per gram of substrate used is 50% higher if S0 is present in the medium (35). This suggests that the reduction of S0 by P. furiosus is not merely a means of disposing of excess reductant but rather is an energy-conserving process. To date three enzymes that are capable of reducing S0 to H2S have been purified from P. furiosus. These are the aforementioned FNOR, also referred to as sulfide dehydrogenase (19), and the H2-evolving hydrogenases, otherwise known as sulfhydrogenases (20, 21). However, all three of these enzymes are located in the cytoplasm, and it seems unlikely that they would be involved in energy conservation. In an effort to determine whether P. furiosus contains an MB sulfur reductase system analogous to that found in the S0-respiring mesophile Wolinella succinogenes (15, 30), we sought to obtain a membrane fraction from cell extracts that lacked the H2-dependent, S0 reduction activity of the cytoplasmic sulfhydrogenases. Surprisingly, even after repeated washings with buffers containing high salt concentrations, the membrane of P. furiosus still contained high hydrogenase (H2 evolution) activity. The purification and characterization of this integral MB hydrogenase is described herein. The enzyme is of the NiFe type, functions to evolve H2 but does not reduce S0, and is distinct from the well-characterized cytoplasmic enzyme. It appears to be part of a large multienzyme complex, the components of which show high sequence similarity to the respiratory-linked, MB NiFe hydrogenases found in some methanogens and photosynthetic bacteria and to the nonenergy-conserving formate hydrogen lyase system (hydrogenase 3) of E. coli (34).

Superoxide reductase from the hyperthermophilic anaerobe Pyrococcus furiosus uses electrons from reduced nicotinamide adenine dinucleotide phosphate, by way of rubredoxin and an oxidoreductase, to reduce superoxide to hydrogen peroxide, which is then reduced to water by peroxidases. Unlike superoxide dismutase, the enzyme that protects aerobes from the toxic effects of oxygen, SOR does not catalyze the production of oxygen from superoxide and therefore confers a selective advantage on anaerobes. Superoxide reductase and associated proteins are catalytically active 80°C below the optimum growth temperature (100°C) of P. furiosus , conditions under which the organism is likely to be exposed to oxygen.

Pyruvate ferredoxin oxidoreductase (POR) from the hyperthermophilic archaeon Pyrococcus furiosus (Pf) catalyzes the final oxidative step in carbohydrate fermentation in which pyruvate is oxidized to acetyl-CoA and CO2, coupled to the reduction of ferredoxin (Fd). POR is composed of two 'catalytic units' of molecular mass approximately 120 kDa. Each unit consists of four subunits, alpha beta gamma delta, with masses of approximately 44, 36, 20, and 12 kDa, respectively, and contains at least two [4Fe-4S] clusters. The precise mechanism of catalysis and the role of the individual subunits are not known. The gene encoding the delta-subunit of Pf POR has been expressed in E. coli, and the protein was purified after reconstitution with iron and sulfide. The reconstituted delta-subunit (recPOR-delta) is monomeric with a mass of 11 879 +/- 1.2 Da as determined by mass spectrometry, in agreement with that predicted from the gene sequence. Purified recPOR-delta contains 8 Fe mol/mol and remained intact when incubated at 85 degreesC for 2 h, as judged by its visible absorption properties. The reduced form of the protein exhibited an EPR spectrum characteristic of two, spin-spin interacting [4Fe-4S]1+ clusters. When compared with the EPR properties of the reduced holoenzyme, the latter was shown to contain a third [4Fe-4S]1+ cluster in addition to the two within the delta-subunit. The reduction potential of the two 4Fe clusters in isolated recPOR-delta (-403 +/- 8 mV at pH 8.0 and 24 degreesC) decreased linearly with temperature (-1.55 mV/ degreesC) up to 82 degreesC. RecPOR-delta replaced Pf Fd as an in vitro electron carrier for two oxidoreductases from Pf, POR and Fd:NADP oxidoreductase, and the POR holoenzyme displayed a higher apparent affinity for its own subunit (apparent Km = 1.0 microM at 80 degreesC) than for Fd (apparent Km = 4.4 microM). The molecular and spectroscopic properties and amino acid sequence of the isolated delta-subunit suggest that it evolved from an 8Fe-type Fd by the addition of approximately 40 residues at the N-terminus, and that this extension enabled it to interact with additional subunits within POR.

The reduction potentials of bovine erythrocyte copper-zinc superoxide dismutase and Escherichia coli iron superoxide dismutase were determined in EPR-monitored redox titrations in homogeneous solution. The copper-zinc enzyme is reduced and reoxidized with a midpoint potential of + 120 mV versus standard hydrogen electrode (SHE) at pH 7.5. The iron enzyme can be reduced with an apparent midpoint potential of −67 mV versus SHE at pH 7.5. However, reaction with ferricyanide affords only slow, partial re-oxidation. Cyclic voltammetry of the copper-zinc enzyme in the presence of 50 MM Sc3+ at pH 4.0 using a glassy carbon electrode results in asymmetric voltammograms. The midpoint potential of the enzyme at this pH value, calculated as the average of the anodic and cathodic peak potentials, is +400 mV versus SHE. The physiological relevance of this value is limited, since EPR experiments indicated that reduction of the copper-zinc enzyme at pH 4.0 is not reversible. Consequences of the irreversible behavior of the two dismutases for the previously reported studies on their redox properties are discussed.

Direct, unmediated electrochemistry has been used to compare the redox properties of [2Fe-2S] clusters in spinach ferredoxin, Spirulina platensis ferredoxin and the water soluble fragment of the Rieske protein. The use of electrochemistry enabled, for the first time, the observation of the second reduction step, [Fe(III),Fe(II)] to [Fe(II),Fe(II)], in a biological [2Fe-2S] system. A water-soluble fragment of the Rieske protein from bovine heart bc1 complex exhibits two subsequent quasi-reversible responses in cyclic voltammetry on activated glassy carbon. In contrast the ferredoxins from spinach and Spirulina platensis only show one single reduction potential. These results support a seniority scheme for biological iron-sulfur clusters relating cluster size to electron transfer versatility. Electrochemical reduction of spinach ferredoxin in the presence of NADP+ and ferredoxin: NADP+ oxidoreductase results in the generation of NADPH. The second order rate constant for the reaction between the ferredoxin and the reductase was estimated from cyclic voltammetry experiments to be >3 · 105 M−1 · s−1.

A spectroelectrochemical study is described of the sixteen hemes in the high-molecular-mass, monomeric cytochrome c (Hmc) from the periplasmic space of Desulfovibrio vulgaris, strain Hildenborough. One of the hemes has special properties. In the oxidized state at pH 7 it is predominantly high-spin, S= 5/2, with a g⊥ value of less than 6 indicative of quantum-mechanical mixing with a low-lying (800 cm−1) S= 3/2 state; the balance is probably a low-spin derivative. The high-spin heme has an Em,7.5 value of +61 mV. The fifteen other hemes are low-spin bis-histidine coordinated with Em,7.5 values of approximately –0.20 V. Two of these hemes exhibit very anisotropic EPR spectra with a g1 value of 3.65 characteristic for strained bis-histidine coordination. A previous proposal, namely that methionine is coordinated to one of the hemes [Pollock, W. B. R., Loufti, M. Bruschi, M. Rapp-Giles, B. J., Wall, J. & Voordouw, G. (1991) J. Bacteriol. 173, 220] is disproved using spectroscopic evidence. Contrasting electrochemical data sets from two previous studies [Tan, J. & Cowan, J. A. (1990) Biochemistry 29, 4886; Bruschi, M., Bertrand, P., More, C., Leroy, G., Bonicel, J., Haladjian, J., Chottard, G., Pollock, W. B. R. & Voordouw, G. (1992) Biochemistry 31, 3281] are not consistent with our EPR titration results and are not reproducible. Hmc can be reduced by D. vulgaris Fe-hydrogenase in the presence of molecular hydrogen.

An electrochemical study of the periplasmic cytochrome c553 of Desulfovibrio vulgaris (Hildenborough) is presented. The dependence of the midpoint potential on temperature and pH was studied with cyclic voltammetry. The voltammograms obtained were reversible and revealed that this cytochrome showed fast electron transfer on a bare glassy carbon electrode. The midpoint potential at pH 7.0 and 25 degrees C was found to be 62 mV versus the normal hydrogen electrode. It was observed that the temperature dependence was discontinuous with a transition temperature at 32 degrees C. The standard reaction entropy at the growth temperature of the organism (37 degrees C) was calculated to be delta S degree ' = -234 J mol-1 K-1. The pH dependence of the midpoint potential could be described with one pK of the oxidized form with a value of 10.6. The second-order rate constant for electron transfer between cytochrome c553 and the Fe-hydrogenase from D. vulgaris (H) was also determined with cyclic voltammetry. The equivalent rate constant for cytochrome c3 and hydrogenase was measured for comparison. The second-order rate constants are 2 x 10(7) M-1 s-1 for cytochrome c553 and 2 x 10(8) M-1 s-1 for cytochrome c3. The kinetic parameters of the hydrogenase for both cytochromes were determined using the spectrophotometric hydrogen consumption assay. With cytochrome c553 this resulted in a Km of 46 microM and a maximum turnover number of 7.1 x 10(2) s-1 in the H2 consumption assay. The values with cytochrome c3 were 17 microM and 6.4 x 10(2) s-1, respectively. The importance of the different kinetic parameters for contrasting models proposed to describe the function of the Fe-hydrogenase are discussed.

Adenosine phosphosulfate reductase from Desulfovibrio vulgaris Hildenborough has been purified to homogeneity and was found to consist of two subunits. The alpha and beta subunits have molecular masses of 67.8 kDa and 25.6 kDa, respectively. The apparent molecular mass of the protein is dependent on the ionic strength of the buffer. At low ionic strength, a high molecular-mass multimer is formed, which reversibly changes into smaller units upon addition of salt. The smallest catalytically active unit of the enzyme has a molecular-mass of 186 kDa, as determined by gel-filtration chromatography and, therefore, an alpha 2 beta 2 stoichiometry is proposed. The protein was found to contain 5.6 +/- 1.1 iron and 4.4 +/- 0.6 acid-labile sulfur atoms/alpha beta heterodimer. The reduced protein exhibits a single, rhombic S = 1/2 signal with g values 2.070, 1.932 and 1.891. Lowering the ionic strength of the buffer reversibly changes this spectrum into a complex EPR spectrum, indicating intermolecular, dipolar magnetic coupling. Spin quantification of the reduced protein either at low or at high ionic strength never resulted in more than 1 spin/alpha beta heterodimer. Hence, it follows that the iron and sulfur atoms are arranged in one single cluster. The reduction potential of the iron sulfur cluster, measured in an EPR-monitored redox titration, was found to be -19 mV versus the normal hydrogen electrode (NHE) at pH 7.5. The reduction potential of the flavin measured in an optical titration was found to be -59 mV against NHE at pH 7.5. The flavin behaves as a two-electron-transferring group; no evidence was obtained for a stabilization of the intermediate semiquinone state in the enzyme. Determination of the kinetic parameters of adenosine 5'-phosphosulfate (Ado-PSO4) reductase for its substrates resulted in Km values for sulfite and AMP of 130 microM and 50 microM, respectively. It is proposed that AdoPSO4 reductase contains a single novel Fe/S structure, possibly with an iron-nuclearity greater than four.

The active site of Escherichia coli NADPH-sulfite reductase has previously been modeled as a siroheme with its iron bridged to a nearby iron-sulfur cubane, resulting in antiferromagnetic exchange coupling between all iron atoms. The model has been suggested to hold also for other sulfite reductases and nitrite reductases. We have recently challenged the generality of the model with the finding that the EPR of Fe/S in dissimilatory sulfite reductase (desulfoviridin) from Desulfovibrio vulgaris indicates that an S = 9/2 system is not subject to coupling. Siroheme in desulfoviridin is to a large extent demetalated, and therefore coupling is physically impossible. We have now studied examples from a second class of dissimilatory sulfite reductases, desulforubidins, which have their siroporphyrins fully metalated. Desulforubidin from Desulfosarcina variabilis is a 208-kDa alpha 2 beta 2 gamma 2 hexamer. The alpha- and beta-subunits are immunologically active with antibodies raised against the corresponding subunits from D. vulgaris desulfoviridin, whereas the gamma-subunit is not. The desulforubidin contains two fully metalated sirohemes and a total of approximately 15 Fe and approximately 19 S2-. Quantification of high-spin plus low-spin heme EPR signals accounts for all sirohydrochlorin. The frequency-independent (9-35 GHz) effective perpendicular g-values of the high-spin S = 5/2 siroheme (6.33, 5.19) point to quantum mixing with an excited (approximately 770 cm-1) S = 3/2 multiplet. Similar anomalous g-values are observed with sulfite reductases from Desulfovibrio baarsii and Desulfotomaculum acetoxidans. The D. variabilis enzyme exhibits very approximately stoichiometric S = 9/2 EPR (g = 16).(ABSTRACT TRUNCATED AT 250 WORDS)

The periplasmic Fe-hydrogenase from Desulfovibrio vulgaris (Hildenborough) contains three iron-sulfur prosthetic groups: two putative electron transferring [4Fe-4S] ferredoxin-like cubanes (two F-clusters), and one putative Fe/S supercluster redox catalyst (one H-cluster). Combined elemental analysis by proton-induced X-ray emission, inductively coupled plasma mass spectrometry, instrumental neutron activation analysis, atomic absorption spectroscopy and colorimetry establishes that elements with Z21 (except for 12-15 Fe) are present in 0.001-0.1 mol/mol quantities, not correlating with activity. Isoelectric focussing reveals the existence of multiple charge conformers with pI in the range 5.7-6.4. Repeated re-chromatography results in small amounts of enzyme of very high H2-production activity determined under standardized conditions (approximately 7000 U/mg). The enzyme exists in two different catalytic forms: as isolated the protein is 'resting' and O2-insensitive; upon reduction the protein becomes active and O2-sensitive. EPR-monitored redox titrations have been carried out of both the resting and the activated enzyme. In the course of a reductive titration, the resting protein becomes activated and begins to produce molecular hydrogen at the expense of reduced titrant. Therefore, equilibrium potentials are undefined, and previously reported apparent Em and n values [Patil, D. S., Moura, J. J. G., He, S. H., Teixeira, M, Prickril, B. C., DerVartanian, D. V., Peck, H. D. Jr, LeGall, J.Huynh, B.-H. (1988) J. Biol. Chem. 263, 18,732-18,738] are not thermodynamic quantities. In the activated enzyme an S = 1/2 signal (g = 2.11, 2.05, 2.00; 0.4 spin/protein molecule), attributed to the oxidized H cluster, exhibits a single reduction potential, Em,7 = -307 mV, just above the onset potential of H2 production. The midpoint potential of the two F clusters (2.0 spins/protein molecule) has been determined either by titrating active enzyme with the H2/H+ couple (E,m = -330 mV) or by dithionite-titrating a recombinant protein that lacks the H-cluster active site (Em,7.5 = -340 mV). There is no significant redox interaction between the two F clusters (n approximately 1).

Desulfoferrodoxin from Desulfovibrio vulgaris, strain Hildenborough, is a homodimer of 28 kDa; it contains two Fe atoms per 14.0 kDa subunit. The N-terminal amino-acid sequence is homogeneous and corresponds to the previously described Rbo gene, which encodes a highly charged 14 kDa polypeptide without a leader sequence. Although one of the two iron centers, FeA, has previously been described as a ‘strained rubredoxin-like’ site, EPR of the ferric form proves very similar to that of the pentagonal bipyramidally coordinated iron in ferric complexes of DTPA, diethylenetriaminepentaacetic acid: both systems have spin S = 5 2 and rhombicity E/D = 0.08. Unlike the Fe site in rubredoxin the FeA site in desulfoferrodoxin has a pH dependent midpoint potential with pKox= 9.2 and pKred = 5.3. Upon reduction (Em,7.5 = +2 mV) FeA exhibits an unusually sharp S = 2 resonance in parallel-mode EPR. The second iron, FeB, has S = 5 2 and E/D = 0.33; upon reduction (Em,7.5 = +90 mV) FeB turns EPR-silent.

Thermotoga maritima is a hyperthermophilic bacterium that contains a complex, heterotrimeric (alpha(beta)gamma) Fe-only hydrogenase. Sequence analysis indicates that the gene encoding the smallest subunit (gamma), hydC, contains a predicted iron-sulfur cluster binding motif. However, characterization of the native gamma-subunit has been hampered by interference from and the inability to separate intact gamma-subunit from the other two subunits (alpha and beta). To investigate the function and properties of the isolated gamma-subunit, the gene encoding HydG was expressed in Escherichia coli. Two forms of the recombinant protein were obtained with molecular masses of 10 and 18 kDa, respectively. Both contained a single [2Fe-2S] cluster based on metal analysis, EPR and UV-visible spectroscopy. NH2-terminal sequencing revealed that the 10 kDa protein is a truncated form of the intact gamma-subunit and lacks the first 65 amino acid residues. The midpoint potential of the 18 kDa form was -356 mV at pH 7.0 and 25 degrees C, as measured by direct electrochemistry, and was pH dependent with a pK(ox) of 7.5 and a pK(red) of 7.7. The oxidized, recombinant gamma-subunit was stable at 80 degrees C under anaerobic conditions with a half-life greater than 24 h, as judged by the UV-visible spectrum of the [2Fe-2S] cluster. In the presence of air the protein was less stable and denatured with a half-life of approx. 2.5 h. The recombinant gamma-subunit was electron transfer competent and was efficiently reduced by pyruvate ferredoxin oxidoreductase from Pyrococcus furiosus, with a Km of 5microM and a Vmax of 9 U/mg. In contrast, native T. maritima hydrogenase holoenzyme and its separated alpha-subunit were much less effective electron donors for the gamma-subunit, with a V(max) of 0.01 U/mg and 0.1 U/mg, respectively.