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 (approximately 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, 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, 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 H-cluster active site (Em,7.5 = -340 mV). There is no significant redox interaction between the two F clusters (n approximately 1).
Pierik
A J
AJ
Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
Hagen
W R
WR
Redeker
J S
JS
Wolbert
R B
RB
Boersma
M
M
Verhagen
M F
MF
Grande
H J
HJ
Veeger
C
C
Mutsaers
P H
PH
Sands
R H
RH
D003300:Copper / Q000032:analysis; D016969:Desulfovibrio vulgaris / Q000201:enzymology*; D004789:Enzyme Activation; D006859:Hydrogen / Q000378:metabolism; D006864:Hydrogenase / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D007501:Iron / Q000032:analysis; D007506:Iron-Sulfur Proteins / Q000032:analysis / Q000378:metabolism*; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D009532:Nickel / Q000032:analysis; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000378:metabolism; D015032:Zinc / Q000032:analysis
D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D003300:Copper; D009532:Nickel; D006859:Hydrogen; D007501:Iron; D006864:Hydrogenase; D015032:Zinc