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 H-2-production activity determined under standardized conditions (almost-equal-to 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 02-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 E(m) 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, 18 732 - 18 738] 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, E(m,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 H-2/H+ couple (E(m)' = - 330 mV) or by dithionite-titrating a recombinant protein that lacks the H-cluster active site (E(m,7.5) = - 340 mV). There is no significant redox interaction between the two F clusters (n almost-equal=to 1).