An unusual cytochrome o '-type cytochrome c oxidase in a Bacillus cereus cytochrome a(3) mutant has a very high affinity for oxygen

Bacillus cereus strain PYM1 is a mutant unable to synthesize haem A or spec trally detectable cytochromes aa(3) or caa(3). The nature of the remaining oxidase(s) catalysing oxygen uptake has been studied. Respiratory oxidase a ctivities and the levels of cytochromes b and c increased 2.6- to 4.2-fold on transition from exponential growth, in either of two media, to sporulati on stage ill, as previously observed for the parent wild-type strain. NADH oxidase activity at both stages of culture was several-fold higher than asc orbate plus tetramethylp-phenylenediamine (TMPD) oxidase activity, consiste nt with the TMPD- phenotype of strain PYM1. Oxidase activity with ascorbate as substrate was significant even in the absence of TMPD as electron media tor, suggesting that the terminal oxidase receives electrons from a cytochr ome c. Carbon monoxide (CID) difference spectra of membranes were obtained using various reductants (ascorbate+/-TMPD, NADH, dithionite) and revealed a haemoprotein resembling cytochrome o'. The CO complex of this cytochrome was photodissociable: the phatodissociation spectrum (photolysed minus CO-l igated) exhibited a trough at 416 nm and a peak at 436 nm, together with mi nor features in the odp region of the spectrum, consistent with the presenc e of a cytochrome o'-like pigment. Ca recombination occurred at -85 to -95 degrees C, No other haemoproteins showing photoreversible CO binding under these conditions were detected. Evidence that this pigment was the oxidase responsible for substrate oxidation was obtained by photodissociating the C O complex at subzero temperatures in the presence of oxygen; this resulted in faster ligand recombination, attributed to oxygen binding, and extensive oxidation of cytochromes c and b. The oxygen affinity of the oxidase was d etermined by using the deoxygenation of oxyleghaemoglobin as a sensitive re porter of dissociated oxygen concentration. A single oxidase was revealed w ith a K-m for oxygen of about 8 nM; this is one of the highest affinities y et reported for a terminal oxidase.