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Below 30 K, oxidized Desulfovibrio gigas hydrogenase presents an intense electron paramagnetic resonance (EPR) signal centered at g=2.02, typical of an iron-sulfur center. In addition a rhombic EPR signal, attributed to Ni(1II) species, is also observed [LeGall, J., Ljungdahl, P., Moura, I., Peck, H. D., Jr, Xavier, A. V., Moura, J. J. G., Teixeira, M., Huynh, B. H., and DerVartanian, D. V. (1982) Biochem. Biop!?j>s. Rcs. Commun. 106,[610][611][612][613][614][615][616] and Cammack, R., Patil, D., Aguirre, R., and Hatchikian, E. C., (1982) FEBS Lett. 142, 289-2921, At higher temperatures (77 K) the iron-sulfur EPR signal is broader and all the EPR features of the rhombic nickel signal can easily be observed. We have now obtained additional information concerning the redox properties of these EPR active centers, using an EPR redox titration method in the presence of dye mediators at pH = 8.5, The mid-point potential was determined to be -70 mV for the Fe,S cluster and -220 mV for the Ni center. Intermediate oxidation states were obtained upon partial reduction with either dithionite or hydrogen. Although upon dithionite reduction the centers are reduced in the order of decreasing mid-point reduction potentials, under a hydrogen atmosphere the nickel center reduces preferentially. This suggests a catalytic involvement of the nickel redox center in the binding of hydrogen. Preliminary Mossbauer studies on Desulfovibrio gigas hydrogenase reveal the presence of a paramagnetic 3 Fe center and two 4 Fe centers. The 3 Fe center is responsible for the g= 2.02 EPR signal but the two 4 Fe centers have been so far undetectable by EPR.Iron-sulfur centers have been implicated in the simplest known oxidation-reduction process which is mediated by hydrogenase, i.e. the reduction of the proton [I].Flavins have also been indicated as prosthetic groups in a few cases [2]. Nickel has been shown to be required for the biosynthesis of hydrogenase [3] and found in the hydrogenase of Methanobacterium thermoautotrophicum [4].Recently the presence of Ni in several hydrogenases was firmly established by EPR studies of 61Ni-enriched samples. A rhombic EPR signal with g values at 2.30, 2.23 and 2.02 was previously observed in oxidized membranes of Methanobacterium bryantii [ 5 ] . This rhombic signal was later proven to originate from Ni through the observation of hyperfine structure on the EPR spectrum of samples prepared from cells grown in 61Ni-enriched culture medium [6]. The same type of Ni signal was then observed in hydrogenases from Mb. thermoautotrophicum [7], Desulfovibrio gigas [8] and Desulfovibrio desulfuricans (strain 27774) [9].Desulfovibrio gigas hydrogenase was recently purified by a new improved method and a nickel content of approximately 0.7 mol Nii89 kDa was found IS]. In the oxidized state (native form) superimposed with an intense narrow EPR signal centered around g = 2.02, a rhombic signal was also detected with g values of 2.31,2.23, and around 2.00. The g values, line shape and saturation characteristics were si...

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Three types of hydrogenases have been isolated from the sulfate‐reducing bacteria of the genus Desulfobibrio. They differ in their subunit and metal compositions, physico‐chemical characteristics, amino acid sequences, immunological ractivities, gene structures and their catalytic properties. Broadly, the hydrogenases can be considered as ‘iron only’ hydrogenases and nickel‐containing hydrogenases. The iron‐sulfur‐containg hydrogenase ([Fe] hydrogenase) contains two ferredoxin‐type (4Fe‐4S) clusters and an atypical iron‐sulfur center belived to be involved in the activation of H2. The [Fe] hydrogenase has the highest specific activity in the evolution and consumption of hydrogen and in the proton‐deuterium exchange reaction and this enzyme is the most sensitive to CO and NO2−. It is not present in all species of Desulfovibrio The nickel‐(iron‐sulfur)‐containing hydrogenases ([NiFe] hydrogenase) posses two (4Fe‐4S) centers and one (3Fe‐xS) cluster in addition to nickel and have been found in all species of Desulfovibrio so far investigated. The redox active nickel is ligated by at least two cysteinyl thiolate residues and the [NiFe] hydrogenases are particularly resistant to inhibitors such as CO and NO2−. The genes encoding the large and small subunits of a periplasmic and a membrane‐bound species of the [NiFe] hydrogenase have been cloned in Eschierichia (E.) coli and sequenced. Their derived amino acid sequences exhibit a high degree of homology (70%); however, they show no obvious metal‐binding sites or homology with the derived amino acid sequence of the [Fe] hydrogenase. The third class is represented by the nickel‐iron‐sulfur)‐selenium‐containing hydrogenases ([NiFe‐Se] hydrohenases) which contain nickel and selenium in equimoleular amounts plus (4Fe‐4S) centers and are only found in some species of Desulfovibrio. The genes encoding the large and small subunits of the periplasmic hydrogenase from Desulfrovibio (D) baculatus (DSM 1743) (for abbrviations see appendix) have been cloned