Characterization of the CO oxidation/H2 evolution system of Rhodospirillum rubrum. Role of a 22-kDa iron-sulfur protein in mediating electron transfer between carbon monoxide dehydrogenase and hydrogenase.
The response of the membrane-associated carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum to solubilization by detergents and organic solvents, the properties of solubilized CODH, and the mechanism for coupling CO oxidation to hydrogen evolution via a CO-induced hydrogenase activity have been investigated. The release of CODH by a variety of ionic and nonionic detergents occurs in a redox-dependent fashion: CODH is solubilized in the presence of low-potential reductants (dithionite, CO, and H2) but is resistant to solubilization from membranes prepared in the absence of reductant or membranes prepared in the presence of reductant and subsequently dye-oxidized. This redox-dependent response to detergent solubilization has been exploited to release CODH from the membranes in a purified state. CODH can also be solubilized from deoxycholate-washed membranes in a redox-independent manner with 20% ethanol. CODH solubilized by deoxycholate or ethanol, when purified to homogeneity by the protocol previously described for heat-solubilized CODH (Bonam, D., and Ludden, P. W. (1987) J. Biol. Chem. 262, 2980-2987), is associated with a previously unobserved 22-kDa protein. The 22-kDa protein can be dissociated from CODH with acetonitrile and can be reconstituted with CODH, after removal of acetonitrile, in a stoichiometric (1:1) fashion. The isolated 22-kDa protein contained 4.0 iron atoms, a reducible Fe-S center, and was O2- and heat-labile. The 22-kDa protein did not alter the catalytic properties of CODH as assayed in vitro with methyl viologen as the electron acceptor for CO oxidation, but was required for reconstituting CO oxidation to hydrogen evolution via the CO-induced membrane-bound hydrogenase. Other electron carrier proteins (ferredoxins and flavodoxin) were ineffective at coupling CO oxidation and hydrogen evolution. We conclude that the 22-kDa protein is a reversibly dissociable subunit of CODH tha mediates electron transfer to hydrogenase.
Ensign
S A
SA
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706.
Ludden
P W
PW
D000445:Aldehyde Oxidoreductases / Q000378:metabolism*; D002248:Carbon Monoxide / Q000737:chemistry*; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002793:Cholic Acids; D002850:Chromatography, Gel; D003902:Detergents; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D006859:Hydrogen / Q000737:chemistry*; D006864:Hydrogenase / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000502:physiology*; D009097:Multienzyme Complexes; D010084:Oxidation-Reduction; D012247:Rhodospirillum rubrum / Q000201:enzymology / Q000378:metabolism*; D013057:Spectrum Analysis
D002793:Cholic Acids; D003902:Detergents; D007506:Iron-Sulfur Proteins; D009097:Multienzyme Complexes; D002248:Carbon Monoxide; D006859:Hydrogen; D006864:Hydrogenase; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate