The resonance Raman spectra of ferredoxins (Fd) I and II from Desulfovibrio gigas are reported using 4579 A Ar+ laser excitation. The (3Fe-3S) center in Fd II has a characteristic resonance Raman spectrum, readily distinguishable from those of (2Fe-2S) or (4Fe-4S) clusters. Reduction of Fd II produces a marked alteration in the resonance Raman spectrum. Fd I is shown to contain both (3Fe-3S) and (4Fe-4S) Fd-type clusters. The results illustrate the potential of resonance Raman spectroscopy in Fe-S cluster identification, even in cases where more than one cluster type is present.

Ferredoxin II from Desulphovibrio gigas is a tetrameric protein containing a novel iron-sulphur cluster consisting of three iron atoms. The low-temperature magnetic circular dichroism (MCD) spectra of the oxidized and dithionite-reduced forms of ferredoxin II have been measured over the wavelength range approx. 300-800 nm. Both oxidation levels of the cluster are shown to be paramagnetic, although only the oxidized form gives an EPR signal. MCD magnetization curves have been constructed over the temperature range approx. 1.5-150 K and at fields between 0 and 5.1 Tesla. The curve for the oxidized protein can be fitted to a ground state of spin S = 1/2 with an isotropic g factor of 2.01. There is evidence for the thermal population of a low-lying electronic state above 50 K. The reduced protein gives a distinctive set of magnetization curves that are tentatively assigned to a ground state of S = 2, with a predominantly axial zero-field distortion that leaves the doublet Ms = +/-2 lowest in energy. The zero-field components have a maximum energy spread of approx. 15 cm-1. which places an upper limit of 4 cm-1 on the axial zero-field parameter D. The MCD spectra of the oxidized and reduced forms of the cluster are quite distinctive from one another. The spectra of the oxidized state are also different from those of oxidized high-potential iron protein from Chromatium and should provide a useful criterion for distinguishing between four- and three-iron clusters in their highest oxidation levels.

A flavodoxin and a rubredoxin have been isolated from the sulfate-reducing bacterium Desulphovibrio salexigens (strain British Guiana, NICB 8403). Their amino acid composition and spectral characteristics did not differ markedly from the homologous proteins presented in other Desulphovibrio spp. Flavodoxin was shown to be active in the electron transport of the sulfite reductase system.

The tetrameric form of a Desulfovibrio gigas ferredoxin, named Fd II, mediates electron transfer between cytochrome c3 and sulfite reductase. We have studied two stable oxidation states of this protein with Mossbauer spectroscopy and electron paramagnetic resonance. We found 3 iron atoms/monomer and a spin concentration of 0.9 spins/monomer for the oxidized protein. Taken together, the EPR and Mossbauer data demonstrate conclusively the presence of a spin-coupled structure containing 3 iron atoms and labile sulfur. The Mossbauer data show also that this metal center is structurally similar, if not identical, with the low potential center of a ferredoxin from Azotobacter vinelandii, a novel cluster described recently (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).

1. Ferricytochrome c3 from D. gigas exhibits two low-spin ferric heme EPR resonances with gz-values at 2.959 and 2.853. Ferrocytochrome c3 is diamagnetic based on the absence of any EPR signals. 2. EPR potentiometric titrations result in the resolution of the two low-spin ferric heme resonances into two additional heme components representing in total the four hemes of the cytochrome, with EM values of -235 mV and -315 mV at heme resonance I and EM values of -235 mV and -306 mV at heme resonance II. 3. EPR spectroscopy has detected a significant diminution of intensity (approx. 60 p. 100) in the gx amplitude of ferricytochrome c3 in the presence of D. gigas ferredoxin II. The presence of ferredoxin II also causes a more negative shift in the EM of the second components of the signals at heme resonances I and II of cytochrome C3. Both observations suggest that an interaction has occurred between cytochrome C3 and ferredoxin II. 4. The results presented suggest that the heme ligand environment of ferricytochrome c3 from D. gigas is less perturbed and/or less asymmetric than environment for ferricytochrome c3 from D. vulgaris whose EPR behavior indicates the non-equivalence of all four hemes.

Potentiometric titration followed by e.p.r. measurements were used to determine the midpoint reduction potentials of the redox centres of a molybdenum-containing iron-sulphur protein previously isolated from Desulfovibrio gigas, a sulphate-reducing bacterium (Moura, Xavier, Bruschi, Le Gall, Hall & Cammack (1976) Biochem. Biophys. Res. Commun. 728 782-789; Moura, Xavier, Bruschi, Le Gall & Cabral (1977) J. Less Common Metals 54, 555-562). The iron-sulphur centres could readily be distinguished into three types by means of g values, temperature effect, oxidation-reduction potential values and reduction rates. The type-I Fe-S centres are observed at 77 K. They show mid-point potential values of -260mV (Fe-S type IA) and -440 mV (Fe-S type IB). Centres of types IA and IB appear to have similar spectra at 77 K and 24 K. The Fe-S type-II centres are only observed below 65 K and have a midpoint potential of -28mV. Long equilibration times (30 min) with dye mediators under reducing conditions were necessary to observe the very slow equilibrating molybdenum signals. The potential values associated with this signal were estimated to be approx. -415 mV for Mo(VI)/Mo(V) and-530mV for Mo(V)/Mo(IV).

A two cluster (4Fe-4S) ferredoxin and a rubredoxin have been isolated from the sulfur-reducing bacterium Desulfuromonas acetoxidans. Their amino acid compositions are reported and compared to those of other iron-sulfur proteins. The ferredoxin contains 8 cysteine residues, 8 atoms of iron and 8 atoms of labile sulfur per molecule; its minimum molecular weight is 6163. The protein exhibits an abosrbance ratio of A385/A283 = 0.74. Storage results in a bleaching of the chromophore; the denatured ferredoxin is reconstitutable with iron and sulfide. The instability temperature is 52 degrees C. The rubredoxin does not differ markedly from rubredoxins from other anaerobic bacteria.

The sulphate-reducing bacteria have a complex electron transfer system which leads to the reduction of sulphate by oxidation of either organic substrates or molecular hydrogen. These bacteria can either produce or consume molecular hydrogen. The central part of this electron pathway for Desulovibrio gigas is constituted by hydrogenase (3 X (4Fe-4S)). cytochrome c3 (4 haems with different redox potentials) and a one (4Fe-4S) cluster ferredoxin. This ferredoxin is isolated in different oligomeric forms, which stabilize different oxidation states and have different physiological roles; the trimer FdI being involved in the production of H2 and the tetramer FdII being more efficient for the consumption of H2. The presence of intrinsic probes (the iron ions) in these proteins is particularly helpful for structural studies using NMR spectroscopy. These studies allowed a characterization of the oxidation states used by the different oligomers of the ferredoxin and obtaintion of structural information on multi-haem cytochromes (c3 and c7). NMR is also suitable to study protein-protein interaction. The study of the complex formed between FdII and cytochrome c3 has shown that there is an alteration of the kinetics of electron transfer upon complexation.

A NMR and magnetic susceptibility study of the oxidized and reduced states of three different oligomers (forms) of a [4Fe-4S] ferrodoxin protein from Desulphovibrio gigas, FdI, FdI', and FdII was carried out. FdI and FdI' are different trimers and FdII a tetramer of the same basic subunit. A probable assignment of the contact shifted resonances is indicated. Since the temperature dependences of the contact shifted responances associated with each [4Fe-4S] are not all similar a delocalized model for the spin densities on the 4Fe does not apply. The exchange rate between oxidized and reduced states is slow on the NMR time scale. The three oligomers are not magnetically equivalent. Using the "three state hypothesis" terminology it is shown that FdIox is predominantly in the C2- state and changes upon reduction into the C3- state, while FdIIox is in the C- state and changes into the C2- state. FdI' does not easily fit into this classification. This study shows a similarity of magnetic behaviour between FdI and bacterial ferredoxins (e.g. Bacillus polymyxa) and between FdII and HiPIP from Chromatium sp. The influence of the quaternary structure on the stabilization of the different oxidation states of ferredoxins as well as on their redox potentials is discussed.

Electron paramagnetic resonance spectra were recorded of three forms of Desulphovibrio gigas ferredoxin, FdI, FdI' and FdII. The g = 1.94 signal seen in dithionite-reduced samples is strong in FdI, weaker in FdI' and very small in FdII. The g = 2.02 signal in the oxidized proteins is weak in FdI and strongest in FdII. It is concluded that most of the 4Fe-4S centres in FdI change between states C- and C2-; FdI' contain both types of centre. There is no evidence that any particular centre can change reversibly between all three oxidation states. Circular dichroism spectra show differences between FdI and FdII even in the diamagnetic C2- state. The redox potentials of the iron-sulphur centres of the three oligomers (forms) are different. After formation of the apo-protein of FdII and reconstitution with iron and sulphide, the protein behaves more like FdI, showing a strong g = 1.94 signal in the reduced states.