DECOMPOSITION OF CARBON-MONOXIDE DEHYDROGENASE INTO ALPHA-METALLOSUBUNITS AND A CATALYTICALLY-ACTIVE FORM CONSISTING PRIMARILY OF BETA-METALLOSUBUNITS

Carbon monoxide dehydrogenase from Clostridium thermoaceticum, with an alpha(3) beta(3) quaternary structure, was incubated with dithiothrei tol and 740-78 000 equiv/alpha(3) beta(3) of sodium dodecyl sulfate (S DS), followed by electrophoresis under anaerobic native conditions. Th ree catalytically-active bands and four inactive bands were obtained, in proportions dependent on the amount of SDS added. The catalytically -active bands, called SM-CODH, NM-CODH, and FM-CODH, migrated slower t han, similar to, and faster than native enzyme, respectively. Two-dime nsional electrophoresis revealed that SM-CODH and NM-CODH contained ap proximately equal proportions of the alpha and beta subunits, while th e beta/alpha ratio for FM-CODH was about 2.7. The four inactive bands were identified as the beta subunit, two forms of the alpha subunit (c alled alpha and alpha'), and a form that migrated similarly to native enzyme. Overloaded gels revealed that alpha and each active band had b rown color, indicating intact Fe-S clusters in these species. Higher c oncentrations of SDS (>1600 equiv/alpha(3) beta(3)) and/or incubation at temperatures >15 degrees C yielded more alpha and beta subunits at the expense of the catalytically-active bands. When incubated at 70 de grees C in 78 000 equiv/alpha(3) beta(3) of SDS, alpha quantitatively converted to alpha', suggesting that alpha' is a denatured form. FM-CO DH appears to be an intermediate in the decomposition of CODH by SDS a nd to have either an alpha(1) beta(3) or alpha(1) beta(2) quaternary s tructure. FM-CODH catalyzed CO oxidation at essentially the same turno ver frequency as NM-CODH, indicating that the dissociable subunits are not involved in the oxidation of CO. The beta subunits appear to cont ain the clusters required for CO oxidation, while alpha subunits may f unction in the synthesis of acetyl-CoA.