Purification and characterization of an O2-utilizing cytochrome-c oxidase complex from Bradyrhizobium japonicum bacteroid membranes.

A cytochrome-c (cyt c) oxidase supercomplex consisting of 7-8 subunits and possessing a mass of 358-425 kDa was purified from Bradyrhizobium japonicum bacteroid membranes. At least two subunits possess c-type heme as a prosthetic group. One of the c-heme-containing components was detected in bacteroid membranes, but not in free-living cells. The complex also contains b-heme, and both b-type and c-type heme proteins were spectrophotometrically shown to form complexes with carbon monoxide. A CO difference spectrum showed an absorption minimum (trough) at 551.7 nm, possibly corresponding to a previously described cyt c-552 in bacteroid membranes. 1 mM quinacrine (Atebrin) had no effect on O2 uptake by the cytochrome-c oxidase complex, but 10 mM inhibited O2 uptake by 90%. Cytochromes b and c1 of the cytochrome bc1 respiratory complex were identified as two of the components of the bacteroid complex based upon immunoreaction with antibodies against these two proteins from B. japonicum. The oxidase complex oxidized exogenously added horse heart ferrocytochrome c concomitant with the uptake of oxygen. It could also oxidize the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine in the absence of added cytochrome c. Oxygen uptake activity was completely inhibited by 10 microM NaCN and 38% by 0.1 microM NaCN. The oxidase complex was not able to oxidize a ubiquinol homolog possessing a single isoprenoid unit side chain. Solubilization of bacteroid membranes in the presence of 1.0 mM EDTA resulted in complete loss of cytochrome-c oxidase activity. Leghemoglobin deoxygenation data indicated that the oxidase complex can efficiently function at free oxygen concentrations well below 1.0 microM, even though attempts to determine the oxidase's specific affinity oxygen were unsuccessful due to the formation of oxidized leghemoglobin derivatives.