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.

S.A Ensign,P.W Ludden

doi:10.1016/s0021-9258(18)55283-2

S.A Ensign, P.W Ludden

Open Access

https://doi.org/10.1016/s0021-9258(18)55283-2

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Abstract

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.

Highlights

A diverse set of bacteria iscapable of oxidizing CO through monoxide dehydrogenase (CODH) from Rhodospiril- theaction of carbon monoxidedehydrogenases(CODH).’
The thawing has been shown previously to be associated with the chrobuffer, and all subsequent buffers used in handling of hydrogenase matophore membrane fraction of broken cells after centrifumembranes, contained dithionite,15% glycerol, 0.2 mM phenylmethylsulfonylfluoride, 0.5 pg/ml leupeptin,and 1 mM EDTA.The inclusion of these stabilizing agents was necessary to preventloss of gation (13)
Requirements for Reconstituting CO-dependent Hydrogen-Metal analysis of the isolated 22-kDa protein by plasma ase Activity withHydrogenase-containing Membranes and Puemission spectroscopy shows 4.0 Fe/mol, suggesting that it rified CODH-Since there is no obvious in uitro role for the may contain a single reducible low-potential 4Fe-4S cluster. 22-kDa protein in catalyzingCO oxidation, we considered the Noothertransitionmetals,including nickel,which is a n possibility that it functions tocouple the oxidation of CO to integral component of the active site of the 62-kDa subunit the reductionof protons by the R. rubrum CO-induced hydro

Summary

RESULTS

Solubilization Propertiesof CODH-CODH from R. rubrum and lysozyme, DNase, and RNase as described above. Effect of oxidant and reductant on dao.u\cholate-.solubilization of carbon monoxide deh.vdrogenase from anaerobic chromatophores. Membrane suspensions were prepared and incubations performed a s described under "Materials and Methods." The protein concentration in thedetergent/chromatophoresuspensions was 4.11 mg/ml. I' Membrane suspensions were oxidized by the addition of indigocarmine to 1.0 mM from a stock solution of 20 mM prepared in 100 mM MOPS, pH 8.0. Membrane suspensions were reduced by the addition of sodium dithionite to3.0 mM from a stock solutionof 100 mM prepared in 100 mM MOPS 8.0. Detergent treatmentof membrane suspensions and assaoyf CODH activity was performed as described under "Materials andMethods."

SupernaMtaenmt hranes

CODH form

DISCUSSION

Methanosarcina thermophila which was required for coupling