Open AccessRedox properties of the iron‐sulfur clusters in activated Fe‐hydrogenase from Desulfovibrio vulgaris (Hildenborough)
Author(s) -
PIERIK Antonio J.,
HAGEN Wilfred R.,
REDEKER Jan S.,
WOLBERT Ronnie B. G.,
BOERSMA Marelle,
VERHAGEN Marc F. J. M.,
GRANDE Hans J.,
VEEGER Cees,
MUTSAERS Peter H. A.,
SANDS Richard H.,
DUNHAM W. Richard
Publication year - 1992
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1992.tb17261.x
Subject(s) - chemistry , redox titration , desulfovibrio vulgaris , titration , ferredoxin , hydrogenase , redox , periplasmic space , iron–sulfur cluster , crystallography , electron paramagnetic resonance , inorganic chemistry , isoelectric point , active site , catalysis , nuclear chemistry , enzyme , biochemistry , nuclear magnetic resonance , physics , escherichia coli , biology , bacteria , gene , genetics
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 (∼ 7000 U/mg). The enzyme exists in two different catalytic forms: as isolated the protein is ‘resting’ and O 2 ‐insensitive; upon reduction the protein becomes active and O 2 ‐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 , 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, E m, 7 =–307 mV, just above the onset potential of H 2 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 Hcluster active site ( E m7.5 =–340 mV). There is no significant redox interaction between the two F clusters (n ∼ 1).