Desulfovibrio Gigas Hydrogenase: Redox Properties of the Nickel and Iron‐Sulfur Centers

Abstract

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...

A pulsed EPR study of redox‐dependent hyperfine interactions for the nickel centre of Desulfovibrio gigas hydrogenase

“…They also catalyze hydrogen-isotope exchange between H2 and H20. The hydrogenase from Desulfovibrio gigas contains a nickel centre [1,2], and in addition, one [3Fe-4S] cluster and two [4Fe-4S] clusters per molecule [3]. Abbreviations: ESEEM, electron spin echo envelope modulation; EPR, electron paramagnetic resonance; shf, superhyperfine; nqi, nuclear quadrupolar interaction EPR signals dependent upon the redox state of the protein.…”

“…The oxidized enzyme, as isolated, is in an 'unready state', and is unreactive towards H2 [4]. It is EPR active, having a signal arising from nickel, designated as Ni-A (g = 2.32, 2.23, 2.01) [5]. After prolonged reductive activation, the EPR spectrum changes and gives the Ni-C signal (g = 2.19, 2.16, 2.01) [6].…”

“…After prolonged reductive activation, the EPR spectrum changes and gives the Ni-C signal (g = 2.19, 2.16, 2.01) [6]. Ni-C has been proposed as an intermediate in the reaction cycle [5,7]. The pH dependence of the midpoint potentials for the protein suggests that reduction of the Ni-A to the Ni-C state involves the addition of two electrons and 2-3 protons to the enzyme [8].…”

Infrared-Detectable Group Senses Changes in Charge Density on the Nickel Center in Hydrogenase from Chromatium vinosum

“…The well-studied enzyme from Desulfovibrio gigas (Fernandez et al, 1986;Teixeira et al, 1989) belongs to this category. Although the amino acid sequence of the Chromatium vinosum enzyme is not yet known, its properties are very similar to those of the D. gigas' enzyme.…”

Infrared Studies on the Interaction of Carbon Monoxide with Divalent Nickel in Hydrogenase from Chromatium vinosum

“…Redox titrations of nickel hydrogenases in the presence of mediating dyes have made it clear that in enzymes from a variety of sources [D. gigas (Teixeira et al, 1983(Teixeira et al, , 1989, C. vinosum (Coremans et al, 1992a,b) and Desulfovibrio vulgaris, strain Miyazaki (Asso et al, 1992)] at least four different redox states with respect to the EPR properties of nickel can be observed. In both the inactive forms of hydrogenase from C. vinosum, Ni,(III) and Niu(III), a n = 1 transition, as evidenced by the disappearance of the S = '/z EPR signals associated with the Ni, could be observed in the presence of mediating dyes at pH = 8 and 30 OC at exactly the same midpoint potential (Eo' = -1 15 mV) (Coremans et al, 1992a).…”