Bacillus cereus strain PYM1 is a mutant unable to synthesize haem A or spectrally detectable cytochromes aa(3) or caa(3). The nature of the remaining oxidase(s) catalysing oxygen uptake has been studied. Respiratory oxidase activities 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 sporulation stage ill, as previously observed for the parent wild-type strain. NADH oxidase activity at both stages of culture was several-fold higher than ascorbate plus tetramethylp-phenylenediamine (TMPD) oxidase activity, consistent with the TMPD- phenotype of strain PYM1. Oxidase activity with ascorbate as substrate was significant even in the absence of TMPD as electron mediator, suggesting that the terminal oxidase receives electrons from a cytochrome 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-ligated) exhibited a trough at 416 nm and a peak at 436 nm, together with minor features in the odp region of the spectrum, consistent with the presence 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 CO 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 determined by using the deoxygenation of oxyleghaemoglobin as a sensitive reporter of dissociated oxygen concentration. A single oxidase was revealed with a K-m for oxygen of about 8 nM; this is one of the highest affinities yet reported for a terminal oxidase.
Última actualización: 15/12/2017