Redox properties of the iron‐sulfur clusters in activated Fe‐hydrogenase from Desulfovibrio vulgaris (Hildenborough)

The periplasmic Fe‐hydrogenase from Desulfovibrio vulgaris (Hildenborough) contains three iron‐sulfur prosthetic groups: two putative electron transferring [4Fe‐4S] ferredoxin‐like cubanes (two F‐clusters), and one putative Fe/S supercluster redox catalyst (one H‐cluster). Combined elemental analysis by proton‐induced X‐ray emission, inductively coupled plasma mass spectrometry, instrumental neutron activation analysis, atomic absorption spectroscopy and colorimetry establishes that elements with Z > 21 (except for 12–15 Fe) are present in 0.001–0.1 mol/mol quantities, not correlating with activity. Isoelectric focussing reveals the existence of multiple charge conformers with pI in the range 5.7–6.4. Repeated re‐chromatography results in small amounts of enzyme of very high H2‐production activity determined under standardized conditions (∼ 7000 U/mg). The enzyme exists in two different catalytic forms: as isolated the protein is ‘resting’ and O2‐insensitive; upon reduction the protein becomes active and O2‐sensitive. EPR‐monitored redox titrations have been carried out of both the resting and the activated enzyme. In the course of a reductive titration, the resting protein becomes activated and begins to produce molecular hydrogen at the expense of reduced titrant. Therefore, equilibrium potentials are undefined, and previously reported apparent Em and n values [Patil, D. S., Moura, J. J. G., He, S. H., Teixeira, M, Prickril, B. C., DerVartanian, D. V., Peck, H. D. Jr, LeGall, J. & Huynh, B.‐H. (1988) J. Biol. Chem. 263, 18732–18738] are not thermodynamic quantities. In the activated enzyme an S= 1/2 signal (g= 2.11, 2.05, 2.00; 0.4 spin/ protein molecule), attributed to the oxidized H cluster, exhibits a single reduction potential, Em, 7=–307 mV, just above the onset potential of H2 production. The midpoint potential of the two F clusters (2.0 spins/protein molecule) has been determined either by titrating active enzyme with the H2/H+ couple (E'm=–330 mV) or by dithionite‐titrating a recombinant protein that lacks the Hcluster active site (Em7.5=–340 mV). There is no significant redox interaction between the two F clusters (n ∼ 1).