Mossbauer and electron paramagnetic resonance (EPR) spectroscopies were used to characterize the diheme cytochrome c peroxidase from Paracoccus denitrificans (L.M.D. 52.44). The spectra of the oxidized enzyme show two distinct spectral components characteristic of low spin ferric hemes (S = 1/2), revealing different heme environments for the two heme groups. The Paracoccus peroxidase can be non-physiologically reduced by ascorbate. Mossbauer investigation of the ascorbate-reduced peroxidase shows that only one heme (the high potential heme) is reduced and that the reduced heme is diamagnetic (S = 0). The other heme (the low potential heme) remains oxidized, indicating that the enzyme is in a mixed valence, half-reduced state. The EPR spectrum of the half-reduced peroxidase, however, shows two low spin ferric species with gmax = 2.89 (species I) and gmax = 2.78 (species II). This EPR observation, together with the Mossbauer result, suggests that both species are arising from the low potential heme. More interestingly, the spectroscopic properties of these two species are distinct from that of the low potential heme in the oxidized enzyme, providing evidence for heme-heme interaction induced by the reduction of the high potential heme. Addition of calcium ions to the half-reduced enzyme converts species II to species I. Since calcium has been found to promote peroxidase activity, species I may represent the active form of the peroxidatic heme.

The periplasmic hydrogenase of Desulfovibrio vulgaris (Hildenbourough) is an all Fe-containing hydrogenase. It contains two ferredoxin type [4Fe-4S] clusters, termed the F clusters, and a catalytic H cluster. Recent X-ray crystallographic studies on two Fe hydrogenases revealed that the H cluster is composed of two sub-clusters, a [4Fe-4S] cluster ([4Fe-4S](H)) and a binuclear Fe cluster ([2Fe](H)), bridged by a cysteine sulfur. The aerobically purified D. vulgaris hydrogenase is stable in air. It is inactive and requires reductive activation. Upon reduction, the enzyme becomes sensitive to O(2), indicating that the reductive activation process is irreversible. Previous EPR investigations showed that upon reoxidation (under argon) the H cluster exhibits a rhombic EPR signal that is not seen in the as-purified enzyme, suggesting a conformational change in association with the reductive activation. For the purpose of gaining more information on the electronic properties of this unique H cluster and to understand further the reductive activation process, variable-temperature and variable-field Mossbauer spectroscopy has been used to characterize the Fe-S clusters in D. vulgaris hydrogenase poised at different redox states generated during a reductive titration, and in the CO-reacted enzyme. The data were successfully decomposed into spectral components corresponding to the F and H clusters, and characteristic parameters describing the electronic and magnetic properties of the F and H clusters were obtained. Consistent with the X-ray crystallographic results, the spectra of the H cluster can be understood as originating from an exchange coupled [4Fe-4S]-[2Fe] system. In particular, detailed analysis of the data reveals that the reductive activation begins with reduction of the [4Fe-4S](H) cluster from the 2+ to the 1+ state, followed by transfer of the reducing equivalent from the [4Fe-4S](H) subcluster to the binuclear [2Fe](H) subcluster. The results also reveal that binding of exogenous CO to the H cluster affects significantly the exchange coupling between the [4Fe-4S](H) and the [2Fe](H) subclusters. Implication of such a CO binding effect is discussed.

To characterise the NADH oxidase activity of both xanthine dehydrogenase (XD) and xanthine oxidase (XO) forms of rat liver xanthine oxidoreductase (XOR) and to evaluate the potential role of this mammalian enzyme as an O2*- source, kinetics and electron paramagnetic resonance (EPR) spectroscopic studies were performed. A steady-state kinetics study of XD showed that it catalyses NADH oxidation, leading to the formation of one O2*- molecule and half a H(2)O(2) molecule per NADH molecule, at rates 3 times those observed for XO (29.2 +/- 1.6 and 9.38 +/- 0.31 min(-1), respectively). EPR spectra of NADH-reduced XD and XO were qualitatively similar, but they were quantitatively quite different. While NADH efficiently reduced XD, only a great excess of NADH reduced XO. In agreement with reductive titration data, the XD specificity constant for NADH (8.73 +/- 1.36 microM(-1) min(-1)) was found to be higher than that of the XO specificity constant (1.07 +/- 0.09 microM(-1) min(-1)). It was confirmed that, for the reducing substrate xanthine, rat liver XD is also a better O2*- source than XO. These data show that the dehydrogenase form of liver XOR is, thus, intrinsically more efficient at generating O2*- than the oxidase form, independently of the reducing substrate. Most importantly, for comparative purposes, human liver XO activity towards NADH oxidation was also studied, and the kinetics parameters obtained were found to be very similar to those of the XO form of rat liver XOR, foreseeing potential applications of rat liver XOR as a model of the human liver enzyme.

The final step of bacterial denitrification, the two-electron reduction of N(2)O to N(2), is catalyzed by a multi-copper enzyme named nitrous oxide reductase. The catalytic centre of this enzyme is a tetranuclear copper site called CuZ, unique in biological systems. The in vitro reconstruction of the activity requires a slow activation in the presence of the artificial electron donor, reduced methyl viologen, necessary to reduce CuZ from the resting non-active state (1Cu(II)/3Cu(I)) to the fully reduced state (4Cu(I)), in contrast to the turnover cycle, which is very fast. In the present work, the direct reaction of the activated form of Pseudomonas nautica nitrous oxide reductase with stoichiometric amounts of N(2)O allowed the identification of a new reactive intermediate of the catalytic centre, CuZ degrees , in the turnover cycle, characterized by an intense absorption band at 680 nm. Moreover, the first mediated electrochemical study of Ps. nautica nitrous oxide reductase with its physiological electron donor, cytochrome c-552, was performed. The intermolecular electron transfer was analysed by cyclic voltammetry, under catalytic conditions, and a second-order rate constant of (5.5 +/- 0.9) x 10(5) M(-1 )s(-1) was determined. Both the reaction of stoichiometric amounts of substrate and the electrochemical studies show that the active CuZ degrees species, generated in the absence of reductants, can rearrange to the resting non-active CuZ state. In this light, new aspects of the catalytic and activation/inactivation mechanism of the enzyme are discussed.

SORs (superoxide reductases) are enzymes involved in bacterial resistance to reactive oxygen species, catalysing the reduction of superoxide anions to hydrogen peroxide. So far three structural classes have been identified. Class I enzymes have two iron-centre-containing domains. Most studies have focused on the catalytic iron site (centre II), yet the role of centre I is poorly understood. The possible roles of this iron site were approached by an integrated study using both classical and fast kinetic measurements, as well as direct electrochemistry. A new heterometallic form of the protein with a zinc-substituted centre I, maintaining the iron active-site centre II, was obtained, resulting in a stable derivative useful for comparison with the native all-iron from. Second-order rate constants for the electron transfer between reduced rubredoxin and the different SOR forms were determined to be 2.8 x 10 M(1) . s(1) and 1.3 x 10 M(1) . s(1) for SORFe(IIII)-Fe(II) and for SORFe(IIII)-Fe(III) forms respectively, and 3.2 x 10 M(1) . s(1) for the SORZn(II)-Fe(III) form. The results obtained seem to indicate that centre I transfers electrons from the putative physiological donor rubredoxin to the catalytic active iron site (intramolecular process). In addition, electrochemical results show that conformational changes are associated with the redox state of centre I, which may enable a faster catalytic response towards superoxide anion. The apparent rate constants calculated for the SOR-mediated electron transfer also support this observation.

Adenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate nucleotides and contributes to the maintenance of their constant cellular level, necessary for energy metabolism and nucleic acid synthesis. The AK were purified from crude extracts of two sulfate-reducing bacteria (SRB), Desulfovibrio (D.) gigas NCIB 9332 and Desulfovibrio desulfuricans ATCC 27774, and biochemically and spectroscopically characterised in the native and fully cobalt- or zinc-substituted forms. These are the first reported adenylate kinases that bind either zinc or cobalt and are related to the subgroup of metal-containing AK found, in most cases, in Gram-positive bacteria. The electronic absorption spectrum is consistent with tetrahedral coordinated cobalt, predominantly via sulfur ligands, and is supported by EPR. The involvement of three cysteines in cobalt or zinc coordination was confirmed by chemical methods. Extended X-ray absorption fine structure (EXAFS) indicate that cobalt or zinc are bound by three cysteine residues and one histidine in the metal-binding site of the "LID" domain. The sequence 129Cys-X5-His-X15-Cys-X2-Cys of the AK from D. gigas is involved in metal coordination and represents a new type of binding motif that differs from other known zinc-binding sites of AK. Cobalt and zinc play a structural role in stabilizing the LID domain.

The hydrogenase from Desulfovibrio baculatus (DSM 1743) was purified from each of three different fractions: soluble periplasmic (wash), soluble cytoplasmic (cell disruption) and membrane-bound (detergent solubilization). Plasma-emission metal analysis detected in all three fractions the presence of iron plus nickel and selenium in equimolecular amounts. These hydrogenases were shown to be composed of two non-identical subunits and were distinct with respect to their spectroscopic properties. The EPR spectra of the native (as isolated) enzymes showed very weak isotropic signals centered around g approximately 2.0 when observed at low temperature (below 20 K). The periplasmic and membrane-bound enzymes also presented additional EPR signals, observable up to 77 K, with g greater than 2.0 and assigned to nickel(III). The periplasmic hydrogenase exhibited EPR features at 2.20, 2.06 and 2.0. The signals observed in the membrane-bound preparations could be decomposed into two sets with g at 2.34, 2.16 and approximately 2.0 (component I) and at 2.33, 2.24, and approximately 2.0 (component II). In the reduced state, after exposure to an H2 atmosphere, all the hydrogenase fractions gave identical EPR spectra. EPR studies, performed at different temperatures and microwave powers, and in samples partially and fully reduced (under hydrogen or dithionite), allowed the identification of two different iron-sulfur centers: center I (2.03, 1.89 and 1.86) detectable below 10 K, and center II (2.06, 1.95 and 1.88) which was easily saturated at low temperatures. Additional EPR signals due to transient nickel species were detected with g greater than 2.0, and a rhombic EPR signal at 77 K developed at g 2.20, 2.16 and 2.0. This EPR signal is reminiscent of the Ni-signal C (g at 2.19, 2.14 and 2.02) observed in intermediate redox states of the well characterized Desulfovibrio gigas hydrogenase (Teixeira et al. (1985) J. Biol. Chem. 260, 8942]. During the course of a redox titration at pH 7.6 using H2 gas as reductant, this signal attained a maximal intensity around -320 mV. Low-temperature studies of samples at redox states where this rhombic signal develops (10 K or lower) revealed the presence of a fast-relaxing complex EPR signal with g at 2.25, 2.22, 2.15, 2.12, 2.10 and broad components at higher field. The soluble hydrogenase fractions did not show a time-dependent activation but the membrane-bound form required such a step in order to express full activity.(ABSTRACT TRUNCATED AT 400 WORDS)

A dihaem cytochrome (Mr 37 400) with cytochrome c peroxidase activity was purified from Pseudomonas stutzeri (ATCC 11 607). The haem redox potentials are far apart: one of the haems is completely ascorbate-reducible and the other is only reduced by dithionite. The coordination, spin states and redox properties of the covalently bound haems were probed by visible, NMR and electron paramagnetic resonance (EPR) spectroscopies in three oxidation states. In the oxidized state, the low-temperature EPR spectrum of the native enzyme is a complex superimposition of three components: (I) a low-spin haem indicating a histidinyl-methionyl coordination; (II) a low-spin haem indicating a histidinyl-histidinyl coordination; and (III) a minor high-spin haem component. At room temperature, NMR and optical studies indicate the presence of high-spin and low-spin haems, suggesting that for one of the haems a high-spin to low-spin transition is observed when temperature is decreased. In the half-reduced state, the component I (high redox potential) of the EPR spectrum disappears and induces a change in the g-values and linewidth of component II; the high-spin component II is no longer detected at low temperature. Visible and NMR studies reveal the presence of a high-spin ferric and a low-spin (methionyl-coordinated) ferrous state. The NMR data fully support the haem-haem interaction probed by EPR. In the reduced state, the NMR spectrum indicates that the low-potential haem is high-spin ferrous.

A c-type monoheme ferricytochrome c550 (9.6 kDa) was isolated from cells of Bacillus halodenitrificans sp.nov., grown anaerobically as a denitrifier. The visible absorption spectrum indicates the presence of a band at 695 nm characteristic of heme–methionine coordination. The mid-point redox potential was determined at several pH values by visible spectroscopy. The redox potential at pH 7.6 is 138 mV. When studied by 1H-NMR spectroscopy as a function of pH, the spectrum shows a pH dependence with pKa values of 6.0 and 11.0. According to these pKa values, three forms designated as I, II and III can be attributed to cytochrome c550. The first pKa is probably associated with protonation of the propionate groups. The second pKa value introduces a larger effect in the 1H-NMR spectrum and is probably due to the ionisation of the axial histidine. Studies of temperature variation of the 1H-NMR spectra for both the ferrous and ferri forms of the cytochrome were performed. Heme meso protons, the heme methyl groups, the thioether protons, two protons from a propionate and the methylene protons from the axial methionine were identified in the reduced form. The heme methyl resonances of the ferri form were also assigned. EPR spectroscopy was also used to probe the ferric heme environment. A signal at gmax∼ 3.5 at pH 7.5 was observed indicating an almost axial heme environment. At higher pH values the signal at gmax∼ 3.5 converts mainly to a signal at g∼ 2.96. The pKa associated with this change is around 11.3. The N-terminal sequence of this cytochrome was determined and compared with known amino acid sequences of other cytochromes.

We report the 98% assignment of the apo-form of an orange protein, containing a novel Mo-Cu cluster isolated from Desulfovibrio gigas. This protein presents a region where backbone amide protons exchange fast with bulk solvent becoming undetectable. These residues were assigned using 13C-detection experiments.

Pseudomonas perfectomarinus (ATCC 14405) dihaem cytochrome c552 was studied by 300-MHz proton magnetic resonance. Some of the haem resonances were assigned in the fully reduced and fully oxidized states. No evidence was found for methionine haem axial coordination. The oxidation-reduction equilibrium was studied in detail. Due to the large difference in mid-point redox potential between the two haems (+174 mV, for haem II and -180 mV for haem I) an intermediate oxidation state could be obtained containing reduced haem I and oxidized haem II. In this way the total paramagnetic shift at different oxidation levels could be decomposed in the intrinsic and extrinsic contributions. It was found that the two haems interact. The rate of electron exchange is slow on the NMR time scale. The redox equilibria are discussed for four possible redox species in solution.

Nitrous oxide (N20) is a greenhouse gas, the third most significant contributor to global warming. As a key process for N20 elimination from the biosphere, N20 reductases catalyze the two-electron reduction of N20 to N2. These 2 x 65 kDa copper enzymes are thought to contain a CuA electron entry site, similar to that of cytochrome c oxidase, and a CuZ catalytic center. The copper anomalous signal was used to solve the crystal structure of N20 reductase from Pseudomonas nautica by multiwavelength anomalous dispersion, to a resolution of 2.4 A. The structure reveals that the CuZ center belongs to a new type of metal cluster, in which four copper ions are liganded by seven histidine residues. N20 binds to this center via a single copper ion. The remaining copper ions might act as an electron reservoir, assuring a fast electron transfer and avoiding the formation of dead-end products.

1H nuclear magnetic resonance (NMR) spectroscopy has been used to examine cytochrome c551.5 (c7) from the sulfur reducer, Desulfuromonas acetoxidans. This protein contains three hemes. Two stable oxidation states (the fully oxidized and the fully reduced) as well as intermediate oxidation states were studied. The axial ligands of the iron were found to be neutral histidines. The redox properties of cytochrome c7 were examined and good quantitative agreement found between the NMR results and previously reported redox potential measurements. The properties of cytochrome c7 are discussed together with those of the homologous tetraheme cytochromes c3 isolate from sulfate-reducing bacteria.

1H nuclear magnetic resonance (NMR) spectroscopy has been used to examine cytochrome c551.5 (c7) from the sulfur reducer, Desulfuromonas acetoxidans. This protein contains three hemes. Two stable oxidation states (the fully oxidized and the fully reduced) as well as intermediate oxidation states were studied. The axial ligands of the iron were found to be neutral histidines. The redox properties of cytochrome c7 were examined and good quantitative agreement found between the NMR results and previously reported redox potential measurements. The properties of cytochrome c7 are discussed together with those of the homologous tetraheme cytochromes c3 isolate from sulfate-reducing bacteria.

The [NiFe] hydrogenase isolated from Desulfovibrio gigas was poised at different redox potentials and studied by Mossbauer spectroscopy. The data firmly establish that this hydrogenase contains four prosthetic groups: one nickel center, one [3Fe-xS], and two [4Fe-4S] clusters. In the native enzyme, both the nickel and the [3Fe-xS] cluster are EPR-active. At low temperature (4.2 K), the [3Fe-xS] cluster exhibits a paramagnetic Mossbauer spectrum typical for oxidized [3Fe-xS] clusters. At higher temperatures (greater than 20 K), the paramagnetic spectrum collapses into a quadrupole doublet with parameters magnitude of delta EQ magnitude of = 0.7 +/- 0.06 mm/s and delta = 0.36 +/- 0.06 mm/s, typical of high-spin Fe(III). The observed isomer shift is slightly larger than those observed for the three-iron clusters in D. gigas ferredoxin II (Huynh, B. H., Moura, J. J. G., Moura, I., Kent, T. A., LeGall, J., Xavier, A. V., and Munck, E. (1980) J. Biol. Chem. 255, 3242-3244) and in Azotobacter vinelandii ferredoxin I (Emptage, M. H., Kent, T. A., Huynh, B. H., Rawlings, J., Orme-Johnson, W. H., and Munck, E. (1980) J. Biol. Chem. 255, 1793-1796) and may indicate a different iron coordination environment. When D. gigas hydrogenase is poised at potentials lower than -80 mV (versus normal hydrogen electrode), the [3Fe-xS] cluster is reduced and becomes EPR-silent. The Mossbauer data indicate that the reduced [3Fe-xS] cluster remains intact, i.e. it does not interconvert into a [4Fe-4S] cluster. Also, the electronic properties of the reduced [3Fe-xS] cluster suggest that it is magnetically isolated from the other paramagnetic centers.