RHODOBACTER-CAPSULATUS CONTAINS A NOVEL CB-TYPE CYTOCHROME-C-OXIDASE WITHOUT A CU-A CENTER

The facultative phototrophic bacterium Rhodobacter capsulatus is capab le of growth in a wide range of environmental conditions using a highl y branched electron-transfer chain. During respiratory growth of this organism reducing equivalents are conveyed to oxygen via two terminal oxidases, previously called ''cyt b(410)'' (cytochrome c oxidase) and ''cyt b(260)'' (quinol oxidase). The cytochrome c oxidase was purified to homogeneity from a semiaerobically grown R. capsulatus strain. The purified enzyme consumes oxygen at a rate of 600 s(-1), oxidizes redu ced equine cyt c and R. capsulatus cyt c(2), and has high sensitivity to cyanide. The complex is composed of three major polypeptides of app arent molecular masses 45, 32, and 28 kDa on SDS-PAGE. The 32- and 28- kDa proteins also stain with tetramethylbenzidine, indicating that the y are c-type cytochromes. Partial amino acid sequences obtained from e ach of the subunits reveal significant homology to the fixN, fixO, and fixP gene products of Bradyrhizobium japonicum and Rhizobium meliloti . The reduced enzyme has an optical absorption spectrum with distinct features near 550 and 560 nm and an asymmetric Soret band centered at 418 nm, indicating the presence of both c- and b-type cytochromes. Two electrochemically distinct cyt c are apparent, with redox midpoint po tentials (E(m7)) of 265 and 320 mV, while the low-spin cyt b has an E( m7) value of 385 mV. The enzyme binds carbon monoxide, and the CO diff erence spectrum indicates that CO binds to a high-spin cyt b. Pyridine hemochrome and HPLC analyses suggest that the complex contains 1 mol of heme C to 1 mol of protoheme and that neither heme O nor heme A is present. Further, the R. capsulatus mutant M7 and its derivatives know n to lack ''cyt b(410)'' also lack the 32-kDa cyt c subunit, and redox titrations of the mutant membranes indicate that the 320-mV cyt c cor relates with the remaining 28-kDa cyt c. Electron paramagnetic resonan ce spectroscopy of the purified enzyme performed at 10 and 77 K shows the presence of both low-spin and high-spin ferricytochromes, but in c ontrast to all other previously characterized cyt c oxidases, no indic ation for a Cu-A-like signal was found at either temperature. The over all data therefore indicate that R. capsulatus contains a novel cb-typ e cyt c oxidase without a Cu-A center.