Spectroscopic characterization of the nickel and iron-sulphur clusters of hydrogenase from the purple photosynthetic bacterium Thiocapsa roseopersicina. 1. Electron spin resonance spectroscopy

The thermostable hydrogenase from Thiocapsa roseopersicina was examined by low-temperature ESR spectroscopy. Two types of signals were detected, from an oxidized iron-sulphur cluster and a nickel centre (Ni-A). In the oxidized protein additional signals were observed due to spin-spin interaction between the two paramagnetic centres. This interaction could be reversibly abolished by reduction to a redox potential below 105 mV. This implies that an additional redox centre is involved in the interaction, for which an Fe3+ ion is suggested. Reduction with hydrogen induced a second type of nickel ESR signal (Ni-C), corresponding to an intermediate redox state seen in other nickel hydrogenases. The Ni-C species was light-sensitive at cryogenic temperatures. At temperatures near to 4.2 K the Ni-C signal showed evidence of interaction with another paramagnetic centre, presumably a second iron-sulphur cluster. On reoxidation a signal due to a third Ni(III) species, Ni-B, increased in amplitude. These results establish that metal centres in the hydrogenase from T. roseopersicina are closely similar to those of the well-studied hydrogenase from Chromatium vinosum.     

Distinct redox behaviour of prosthetic groups in ready and unready hydrogenase from Chromatium vinosum.

The redox behaviour of the Ni(III)/Ni(II) transition in hydrogenase from Chromatium vinosum is described and compared with the redox behaviour of the nickel ion in the F420-nonreducing hydrogenase from Methanobacterium thermoautotrophicum. Analogous to the situation in the oxidised hydrogenase of Desulfovibrio gigas (Fernandez, V.M., Hatchikian, E.C., Patil, D.S. and Cammack, R. (1986) Biochim. Biophys. Acta 883, 145-154), the C. vinosum enzyme can also exist in two forms: the 'unready' form (EPR characteristics of Ni(III): gx,y,z = 2.32, 2.24, 2.01) and the 'ready' form (EPR characteristics Ni(III): gx,y,z = 2.34, 2.16, 2.01). Like in the oxidised enzyme of M. thermoautotrophicum the Ni(III)/Ni(II) transition for the unready form titrated completely reversible (both at pH 6.0 and pH 8.0). In contrast, the reversibility of the Ni(III)/Ni(II) transition in the ready enzyme was strongly dependent on pH and temperature. At pH 6.0 and 2 degrees C reduction of Ni(III) in ready enzyme was completely irreversible, whereas at pH 8.0 and 30 degrees C Ni(III) in both ready and unready enzyme titrated with E0' = -115 mV (n = 1). Hampered redox equilibration between the ready enzyme and the mediating dyes is interpreted in terms of an obstruction of the electron transfer from nickel at the active site to the artificial electron acceptors in solution. The origin of this obstruction might be related to possible changes in the protein structure induced by the activation process. The E0'-value of the Ni(III)/Ni(II) equilibrium was pH sensitive (-60 mV/delta pH) indicating that reduction of nickel is coupled to a protonation. A similar pH-dependence was observed for the titration of the spin-spin interaction of Ni(III) and a special form of the [3Fe-4S]+ cluster (E0' = +150 mV, pH 8.0, 30 degrees C). Redox equilibration of this coupling was extremely sensitive to pH and temperature. The uncoupled [3Fe-4S]+ cluster titrated pH-independently with E0' = -10 mV (pH 8.0, 30 degrees C).     

Magnetic interaction of nickel(III) and the iron-sulphur cluster in hydrogenase from Chromatium vinosum

Upon partial reduction of hydrogenase from Chromatium vinosum with ascorbate plus phenazine methosulphate, EPR signals due to Ni(III) and a [3Fe-xS] cluster appear simultaneously and with equal intensities. Since the intact enzyme shows no $\text{S =}\text{1}\text{2}$ signals, it is concluded that Ni(III) and a [4Fe-4S]³⁺ cluster interact magnetically in such a way as to prevent the detection of the two paramagnets as individual $\text{S =}\text{1}\text{2}$ systems. This interaction is thought to be the origin of a signal in which Fe is involved and which is not due to an $\text{S =}\text{1}\text{2}$ system (Albracht, S.P.J., Albrecht-Ellmer, K.J., Schmedding, D.J.M. and Slater, E.C. (1982) Biochim. Biophys. Acta 681, 330–334). A variable fraction of the enzyme preparation shows signals due to Ni(III) and a [3Fe-xS] cluster with equal intensities without any further treatment. These are thought to be derived from irreversibly inactivated enzyme molecules. The enzyme contains no selenium.     

Spectroscopic characterization of the nickel and iron-sulphur clusters of hydrogenase from the purple photosynthetic bacterium Thiocapsa roseopersicina. 2. Electron spin-echo spectroscopy

Pulsed electron-spin-resonance techniques were applied to the hydrogenase of the purple photosynthetic bacterium Thiocapsa roseopersicina, an enzyme which contains nickel and iron-sulphur clusters but no flavin. The linear electric field effect profile of the spectrum in the region of g = 2.01 indicated that the strong ESR signal in the oxidized protein is due to a [3Fe-4S] cluster. The electron spin-echo envelope of this spectrum was modulated by hyperfine interactions with 1H and 14N nuclei, probably from the polypeptide chain. The ESR spectrum of this species shows a complex pattern arising from spin-spin interaction with another paramagnet. When the protein was partially reduced by ascorbate plus phenazine methosulphate, the complexity of the spectrum was abolished but the form of the electron spin-echo envelope modulation (ESEEM) pattern was unchanged. This indicates that the reversible disappearance of the spin-spin interaction pattern on partial reduction is not due to cluster interconversion to a [4Fe-4S] cluster. In the ESR spectrum of nickel(III), weak hyperfine interactions with 1H and 14N were also observed by ESEEM. The nature of the interacting nuclei is discussed.     

Characterization of the active site of catalytically inactive forms of [NiFe] hydrogenases by density functional theory

The inactive forms, unready (Ni-A, Ni-SU) and ready (Ni-B), of NiFe hydrogenases are modeled by examining the possibility of hydroxo, oxo, hydroperoxo, peroxo, and sulfenate groups in active-site models and comparing predicted IR frequencies and g tensors with those of the enzyme. The best models for Ni-A and Ni-SU have hydroxo (μ-OH) bridges between Fe and Ni and a terminal sulfenate [Ni–S(=O)Cys] group, although a hydroperoxo model for Ni-A is also quite viable, whereas the best model for Ni-B has only a μ-OH bridge. In addition, a mechanism for the activation of unready hydrogenase is proposed on the basis of the relative stabilities of sulfenate models versus peroxide models.     

Activation process of [NiFe] hydrogenase elucidated by high-resolution X-ray analyses: conversion of the ready to the unready state

Hydrogenases catalyze oxidoreduction of molecular hydrogen and have potential applications for utilizing dihydrogen as an energy source. [NiFe] hydrogenase has two different oxidized states, Ni-A (unready, exhibits a lag phase in reductive activation) and Ni-B (ready). We have succeeded in converting Ni-B to Ni-A with the use of Na2S and O2 and determining the high-resolution crystal structures of both states. Ni-B possesses a monatomic nonprotein bridging ligand at the Ni-Fe active site, whereas Ni-A has a diatomic species. The terminal atom of the bridging species of Ni-A occupies a similar position as C of the exogenous CO in the CO complex (inhibited state). The common features of the enzyme structures at the unready (Ni-A) and inhibited (CO complex) states are proposed. These findings provide useful information on the design of new systems of biomimetic dihydrogen production and fuel cell devices.     

On the active site of hydrogenase from Chromatium vinosum

Isotope substitution of ⁵⁷Fe ($\text{I =}\text{1}\text{2}$) for ⁵⁶Fe has a pronounced effect on the two EPR signals of hydrogenase of Chromatium vinosum. It is proposed that signal 1, the intensity of which is increased several-fold by a deoxygenation-oxygenation cycle with a simultaneous increase of a signal from Fe³⁺, is due to a [3Fe-xS] cluster. It is further proposed that signal 2 is caused by a magnetic interaction of a [4Fe-4S]³⁺ cluster with an unidentified paramagnet. The addition of 10 μM Ni to the culture medium (already containing 1 μM Ni) increased the enzyme activity 3–6-fold, without effect on the growth of the bacterium. Addition of ⁶¹Ni ($\text{I =}\text{3}\text{2}$) to the medium did not change the EPR spectrum of hydrogenase. From a comparison of the EPR signal intensities and the enzyme activities it is concluded that, in the hydrogenase preparation as isolated, molecules containing a [3Fe-xS) cluster are not active, and that active molecules have a [4Fe-4S]^3+(3+,2+) cluster plus an as yet unidentified paramagnetic redox component. The latter is thought to be the primary site of interaction of the enzyme with H₂. Ni is considered as a possible candidate for this component.     

Evidence that centre 2 in Escherichia coli fumarate reductase is a [4Fe-4S]cluster

Redox titrations of the iron-sulphur clusters in fumarate reductase purified from Escherichia coli, monitored by ESR spectroscopy, identified three redox events, similar to those observed in other fumarate reductases and succinate dehydrogenases: Centre 1, a [2Fe-2S] cluster, at g = 2.03, 1.93, appeared on reduction with Em = -20 mV. Centre 3, probably a [3Fe-xS] cluster, at g = 2.02 appeared in the oxidized state with Em = -70 mV. Centre 2 has been observed as an increase in the electron-spin relaxation of Centre 1. It titrates as an n = 1 species with Em = -320 mV, but in our hands did not appear to contribute significant intensity to the g = 2.03, 1.93 signal. It therefore appears to be an additional centre which undergoes spin-spin interaction with Centre 1. The reduction of Centre 2 coincided with the appearance of an extremely broad ESR spectrum, observed at temperatures below 20 K, with features at g = 2.17, 1.9, 1.68. The broad signal was observed in both soluble and membrane-bound preparations. Its midpoint potential was -320 mV. Its integrated intensity was approximately equal to that of Centre 1, if its broad outer wings were taken into account. Consideration of the ESR properties of this signal, together with the amino acid sequence of the frdB subunit of the enzyme, indicates that Centre 2 is a [4Fe-4S] cluster which, in its reduced state, enhances the spin relaxation of the [2Fe-2S] Centre 1.     

Conformation, structure and activation of bovine cathepsin D. Unfolding and refolding studies.

Preparations of nitrate reductase in the resting state from Pseudomonas aeruginosa exhibit an Mo(V) e.p.r. signal. Progressive reduction of the enzyme results at first in the intensification and then in the disappearance of the signal. Three different species of Mo(V) were detected by e.p.r. These are the high-pH species (g1 = 1.9871; g2 = 1.9795; g3 = 1.9632) and nitrate and nitrite complexes of a low-pH species (respectively g1 = 2.0004; g2 = 1.9858; g3 = 1.9670; and g1 = 1.9975; g2 = 1.9848; g3 = 1.9652). These signals are closely analogous to those for the enzyme from Escherichia coli described by Vincent & Bray [(1978) Biochem. J. 171, 639-647]. Signals typical of iron-sulphur clusters were also detected. In the oxidized enzyme these are believed to arise from a [3Fe-4S] cluster (g = 2.01) and in the reduced enzyme from an unusual low-potential [4Fe-4S]+ cluster (g1 = 2.054; g2 = 1.952; g3 = 1.878). The iron-sulphur centres were also studied in a 'high-catalytic-activity' form of the enzyme. Reduction with Na2S2O4 resulted in the formation of a complex signal with g values at 2.054, 1.952, 1.928, 1.903 and 1.878. The signal could be deconvoluted by reductive titration of the enzyme into two species (g1 = 2.054; g2 = 1.952; g3 = 1.878; and g1 = 2.036; g2 = 1.928; g3 = 1.903). The degradation of a [4Fe-4S] into a [3Fe-4S] cluster in the enzyme is suggested by these studies, the process being dependent on the method used to purify the enzyme. The addition of nitrate to the reduced enzyme results in the oxidation of Mo(IV) to Mo(V) and of all the iron-sulphur centres.     

Inhibition of Desulfovibrio gigas hydrogenase with copper salts and other metal ions

The effect of several transition metals on the activity of Desulfovibrio gigas hydrogenase has been studied. Co(II) and Ni(II) at a concentration of 1 mM did not modify the activity of the enzyme; nor did they affect the pattern of activation/deactivation. Cu(II) inhibited the active hydrogenase, prepared by treatment with hydrogen, but had little effect on the 'unready' enzyme unless a reductant such as ascorbate was present, in which case inactivation took place either in air or under argon. Hg(II) also inactivated the enzyme irreversible in the 'unready' state without the requirement for reductants. The reaction of H2 uptake with methyl viologen was much more sensitive to inhibition than the H2/tritium exchange activity. EPR spectra of this preparation showed that the rates of decline were [3Fe-4S] signal greater than H2-uptake activity greater than Ni-A signal. Similar results were obtained when the protein was treated with Hg(II). The results demonstrate that the [3Fe-4S] cluster is not essential for H2-uptake activity with methyl viologen, but the integrity of [4Fe-4S] clusters is probably necessary to catalyze the reduction of methyl viologen with hydrogen. D. gigas hydrogenase was found to be highly resistant to digestion by proteases.     

EPR Spectrum at 4, 9 and 35 GHz of hydrogenase from Chromatium vinosum. Direct evidence for spin-spin interaction between Ni(III) and the ironsulphur cluster

EPR spectra at 4, 9 and 35 GHz of hydrogenase isolated from Chromatium vinosum have been compared. The spectra at 4 and 35 GHz confirmed our earlier conclusions, made from observations at 9 GHz (Albracht, S.P.J., Kalkman, M.L. and Slater, E.C. (1983) Biochim. Biophys. Acta 724, 309–316), that the irreversibly inactivated enzyme molecules in the preparation give rise to two EPR signals due to the independent non-interacting $\text{S =}\text{1}\text{2}$ systems of Ni(III) and a |3Fe-xS| cluster. It was observed that intact enzyme molecules show a complex EPR spectrum caused by a spin-coupled pair of Ni(III) and a |4Fe-4S|³⁺ cluster. The interaction energy is so weak (approx. 0.01 cm^?1) that the 35 GHz spectra of both the Ni(III) and the |4Fe-4S|³⁺ cluster have the appearance of rather normal $\text{S =}\text{1}\text{2}$ spectra with additional splittings as a result of the spin-spin interaction. At lower microwave frequencies, the spectra become increasingly complex but phenomenologically they behave as expected for an exchange-coupled pair of dissimilar ions. The distance between the two spin systems is estimated to be at the most 1.2 nm. The spin-relaxation rate of the Ni(III) ion is dramatically enhanced as a result of the coupling to the rapidly relaxing Fe-S cluster. The g values and so presumably also the ligand fields of Ni in intact and irreversibly inactivated enzyme molecules are identical. This suggests that the specific coordination of the nickel in the enzyme is not the only requirement for activity with artificial electron donors or acceptors, and that the presence of a nearby, intact |4Fe-4S|^3+(3+,2+) cluster might be another essential factor. From the g values and the probable function of Ni in the enzyme we propose, as a working hypothesis, that the nickel ion has five ligands provided by the protein in a square-pyramidal coordination.     

Bias from H2 cleavage to production and coordination changes at the Ni-Fe active site in the NAD+-reducing hydrogenase from Ralstonia eutropha

The soluble NAD+-reducing Ni-Fe hydrogenase (SH) from Ralstonia eutropha H16 is remarkable because it cleaves hydrogen in the presence of dioxygen at a unique Ni-Fe active site (Burgdorf et al. (2005) J. Am. Chem. Soc. 127, 576). By X-ray absorption (XAS), FTIR, and EPR spectroscopy, we monitored the structure and oxidation state of its metal centers during H2 turnover. In NADH-activated protein, a change occurred from the (CN)O2Ni(II)(mu-S)2Fe(II)(CN)3(CO) site dominant in the wild-type SH to a standard-like S2Ni(II)(mu-S)2Fe(II)(CN)2(CO) site as the prevailing species in a specific mutant protein, HoxH-H16L. The wild-type SH primarily was active in H2 cleavage. The nonstandard reaction mechanism does not involve stable EPR-detectable trivalent Ni oxidation states, namely, the Ni-A,B,C states as observed in standard hydrogenases. In the HoxH-mutant protein H16L, H2 oxidation was impaired, but H2 production occurred via a stable Ni-C state (Ni(III)-H(-)-Fe(II)), suggesting a reaction sequence similar to that of standard hydrogenases. It is proposed that reductive activation by NADH of both wild-type and H16L proteins causes the release of an oxygen species from Ni and is initiated by electron transfer from a [2Fe-2S] cluster in the HoxU subunit that at first becomes reduced by electrons from NADH. Electrons derived from H2 cleavage, on the other hand, are transferred to NAD+ via a different pathway involving a [4Fe-4S] cluster in HoxY, which is reducible only in wild-type SH but not in the H16L variant.     

Photosensitivity of the Ni-A state of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F with visible light

[NiFe] hydrogenase catalyzes reversible oxidation of molecular hydrogen. Its active site is constructed of a hetero dinuclear Ni–Fe complex, and the oxidation state of the Ni ion changes according to the redox state of the enzyme. We found that the Ni-A state (an inactive unready, oxidized state) of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F (DvMF) is light sensitive and forms a new state (Ni-AL) with irradiation of visible light. The Fourier transform infrared (FT-IR) bands at 1956, 2084 and 2094 cm^?1 of the Ni-A state shifted to 1971, 2086 and 2098 cm^?1 in the Ni-AL state. The g-values of g_x = 2.30, g_y = 2.23 and g_z = 2.01 for the signals in the electron paramagnetic resonance (EPR) spectrum of the Ni-A state at room temperature varied for ?0.009, +0.012 and +0.010, respectively, upon light irradiation. The light-induced Ni-AL state converted back immediately to the Ni-A state under dark condition at room temperature. These results show that the coordination structure of the Fe site of the Ni-A state of [NiFe] hydrogenase is perturbed significantly by light irradiation with relatively small coordination change at the Ni site.     

Spectroscopic studies of the nature of the iron clusters in the soluble hydrogenase from Desulfovibrio desulfuricans (strain Norway 4)

57Fe-enriched samples of the soluble hydrogenase from Desulfovibrio desulfuricans (Norway) have been investigated in both the native (oxidized) and the dithionite-reduced states using M?ssbauer spectroscopy. The data clearly show that the iron in this enzyme is predominantly in the form of iron-sulphur clusters which are closely similar to the [4Fe-4S] clusters found in a large number of ferredoxins, such as that from Bacillus stearothermophilus. There appear to be two [4Fe-4S] clusters. The iron-sulphur clusters in the oxidized protein are virtually diamagnetic, as indicated by M?ssbauer, electron spin resonance and magnetic circular dichroic spectroscopy. On reduction by dithionite + methyl viologen, M?ssbauer spectroscopy showed that only 50% of the [4Fe-4S] clusters were reduced. Even reduction with hydrogen up to a pressure of 23 GPa did not reduce the iron-sulphur clusters completely. An ESR signal due to a rapidly relaxing species with g = 2.03, 1.89 was observed in the reduced protein, together with a weaker spectrum from a slower-relaxing species at g = 2.34, 2.12.     

Study of the respiratory chain in Micrococcus luteus (lysodeikticus) by electron-spin-resonance spectroscopy

Low-temperature electron spin resonance spectroscopy was used to investigate the redox centres of Micrococcus luteus membranes. Three different types of iron-sulphur centres were distinguished. Two of these, a [4Fe-4S]3+-type cluster giving rise to a signal at g = 2.01 in the oxidized state and a [2Fe-2S] cluster with a spectrum at g = 2.03 and 1.93 in the reduced state, were attributable to succinate dehydrogenase. Another, generating signals in the reduced state at g = 2.027, 1.90 and 1.78 was identified as a 'Rieske' iron-sulphur centre. This latter cluster had a mid-point potential (pH 7.0) of +130 mV. In addition, signals characteristic of high-spin ferric haem (g = 6.20), low-spin ferric haem (g = 3.67, 3.36 and 3.01) and Cu2+ (g = 2.18 and 2.02) were also detected. The ferric-haem features, together with the Cu2+ and 'Rieske' centres, were enriched in membrane residues insoluble in Triton X-100, which are known from difference spectroscopy to contain cytochromes b-560, c-550 and a-601 (aa3 oxidase). The signals demonstrated by electron spin resonance for M. luteus membranes showed marked similarities to those documented for the complexes II, III, and IV of mitochondria. However, signals analogous to complex I (NADH-ubiquinone reductase) could not be demonstrated for M. luteus membranes.     

Endonuclease III is an iron-sulfur protein

Elemental analyses, M?ssbauer, and EPR data are reported to show that endonuclease III of Escherichia coli is an iron-sulfur protein. M?ssbauer spectra of protein freshly prepared from E. coli grown on 57Fe-enriched medium demonstrate that the native enzyme contains a single 4Fe-4S cluster in the 2+ oxidation state, with a net spin of zero. Upon treatment with ferricyanide, a fraction (less than 25%) of the clusters is oxidized into a state which yields an EPR spectrum near g = 2.01 typical of a 3Fe-4S cluster. The magnetic field dependence of the linear electric field effect verifies this assignment. Electron spin echo modulation on the g = 2.01 form of the protein in deuterated solvent indicates the presence of exchangeable protons in the vicinity of the 3Fe-4S cluster. The data obtained show that the [4Fe-4S]2+ cluster of the native enzyme is resistant to either oxidation or reduction, although photoreduction elicited a g = 1.94 type EPR signal characteristic of a [4Fe-4S]1+ cluster. These studies show that endonuclease III is unique in being both a DNA repair enzyme and an iron-sulfur protein. The function of the 4Fe-4S cluster remains to be established.     

Redox intermediates of Desulfovibrio gigas [NiFe] hydrogenase generated under hydrogen. M?ssbauer and EPR characterization of the metal centers

The hydrogenase (EC 1.2.2.1) of Desulfovibrio gigas is a complex enzyme containing one nickel center, one [3Fe-4S] and two [4Fe-4S] clusters. Redox intermediates of this enzyme were generated under hydrogen (the natural substrate) using a redox-titration technique and were studied by EPR and M?ssbauer spectroscopy. In the oxidized states, the two [4Fe-4S]2+ clusters exhibit a broad quadrupole doublet with parameters (apparent delta EQ = 1.10 mm/s and delta = 0.35 mm/s) typical for this type of cluster. Upon reduction, the two [4Fe-4S]1+ clusters are spectroscopically distinguishable, allowing the determination of their midpoint redox potentials. The cluster with higher midpoint potential (-290 +/- 20 mV) was labeled Fe-S center I and the other with lower potential (-340 +/- 20 mV), Fe-S center II. Both reduced clusters show atypical magnetic hyperfine coupling constants, suggesting structural differences from the clusters of bacterial ferredoxins. Also, an unusually broad EPR signal, labeled Fe-S signal B', extending from approximately 150 to approximately 450 mT was observed concomitantly with the reduction of the [4Fe-4S] clusters. The following two EPR signals observed at the weak-field region were tentatively attributed to the reduced [3Fe-4S] cluster: (i) a signal with crossover point at g approximately 12, labeled the g = 12 signal, and (ii) a broad signal at the very weak-field region (approximately 3 mT), labeled the Fe-S signal B. The midpoint redox potential associated with the appearance of the g = 12 signal was determined to be -70 +/- 10 mV. At potentials below -250 mV, the g = 12 signal began to decrease in intensity, and simultaneously, the Fe-S signal B appeared. The transformation of the g = 12 signal into the Fe-S signal B was found to parallel the reduction of the two [4Fe-4S] clusters indicating that the [3Fe-4S]o cluster is sensitive to the redox state of the [4Fe-4S] clusters. Detailed redox profiles for the previously reported Ni-signal C and the g = 2.21 signal were obtained in this study, and evidence was found to indicate that these two signals represent two different oxidation states of the enzyme. Finally, the mechanistic implications of our results are discussed.     

Electron spin relaxation of iron-sulphur proteins studied by microwave power saturation.

The electron-spin relaxation of iron-sulphur centres in a range of simple proteins (ferredoxin, high-potential iron-sulphur protein and rubredoxin) was investigated by means of the temperature dependence and microwave power saturation of the EPR signal. The proteins containing [2Fe-2S] centres all showed temperature optima higher than those for [4Fe-4S] centres, but the difference between the slowest-relaxing [4Fe-4S] protein (Chromatium high-potential iron-sulphur protein) and the fastest-relaxing [2Fe-2S] protein (Halobacterium halobium ferredoxin) was small. A greater distinction was seen in the power saturation behaviour at low temperature (10--20 K). The behaviour of the signal intensity as a function of microwave power was analyzed in terms of the power for half saturation P 1/2 and the degree of homogeneous/inhomogeneous broadening. The effect of distorting the protein structure by salts, organic solvents and urea was to decrease the electron-spin relaxation rate as shown by a decreased value of P 1/2. The addition of Ni2+ as a paramagnetic perturbing agent caused an increase in the electron-spin relaxation rate of all the proteins, with the exception of adrenal ferredoxin, as shown by an increased P 1/2 and, in a few cases, broadening of the linewidth. Ferricyanide, a commonly used oxidizing agent, has similar effects. These results are discussed in relation to the use of paramagnetic probes to determine whether iron-sulphur centres are near to a membrane surface. Spin-spin interactions between two paramagnetic centres in a protein molecule such as a 2[4Fe-4S] ferredoxin, lead to more rapid electron-spin relaxation. This method was used to detect a spin-spin interaction between molybdenum V and centre Fe-SI in xanthine oxidase.     

Characterization of the nickel-iron periplasmic hydrogenase from Desulfovibrio fructosovorans

The periplasmic hydrogenase from Desulfovibrio fructosovorans grown on fructose/sulfate medium was purified to homogeneity. It exhibits a molecular mass of 88 kDa and is composed of two different subunits of 60 kDa and 28.5 kDa. The absorption spectrum of the enzyme is characteristic of an iron-sulfur protein and its absorption coefficients at 400 and 280 nm are 50 and 180 mM-1 cm-1, respectively. D. fructosovorans hydrogenase contains 11 +/- 1 iron atoms, 0.9 +/- 0.15 nickel atom and 12 +/- 1 acid-labile sulfur atoms/molecule but does not contain selenium. The amino acid composition of the protein and of its subunits, as well as the N-terminal sequences of the small and large subunits, have been determined. The cysteine residues of the protein are distributed between the large (9 residues) and the small subunits (11 residues). Electron spin resonance (ESR) properties of the enzyme are consistent with the presence of nickel(III), [3Fe-4S] and [4Fe-4S] clusters. The hydrogenase of D. fructosovorans isolated under aerobic conditions required an incubation with hydrogen or other reductants in order to express its full catalytic activity. H2 uptake and H2 evolution activities doubled after a 3-h incubation under reducing conditions. Comparison with the (NiFe) hydrogenase from D. gigas shows great structural similarities between the two proteins. However, there are significant differences between the catalytic properties of the two enzymes which can be related to the respective state of their nickel atom. ESR showed a higher proportion of the Ni-B species (g = 2.33, 2.16, 2.01) which can be related to a more facile conversion to the ready state. The periplasmic location of the enzyme and the presence of hydrogenase activity in other cellular compartments are discussed in relation to the ability of D. fructosovorans to participate actively in interspecies hydrogen transfer.     

An investigation of the iron-sulphur proteins of benzene dioxygenase from Pseudomonas putida by electron-spin-resonance spectroscopy.

Benzene dioxygenase from Pseudomonas putida comprises three components, namely a flavoprotein (NADH:ferredoxin oxidoreductase; Mr 81000), an intermediate electron-transfer protein, or ferredoxin (Mr 12000) with a [2Fe-2S] cluster, and a terminal dioxygenase containing two [2Fe-2S] iron-sulphur clusters (Mr 215000), which requires two additional Fe2+ atoms/molecule for oxygenase activity. The ferredoxin and the dioxygenase give e.s.r. signals in the reduced state with rhombic symmetry and average g values of 1.92 and 1.896 respectively. The mid-point redox potentials were determined by e.s.r. titration at pH 7.0 to be -155 mV and -112 mV respectively. The signal from the dioxygenase shows pronounced g anisotropy and most closely resembles those of 4-methoxybenzoate mono-oxygenase from Pseudomonas putida and the [2Fe-2S] 'Rieske' proteins of the quinone-cytochrome c region of electron-transport chains of respiration and photosynthesis.