title: An unusual cytochrome o'-type cytochrome c oxidase in a Bacillus cereus cytochrome a3 mutant has a very high affinity for oxygen.
abstract: Bacillus cereus strain PYM1 is a mutant unable to synthesize haem A or spectrally detectable cytochromes aa3 or caa3. 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 III, as previously observed for the parent wild-type strain. NADH oxidase activity at both stages of culture was several-fold higher than ascorbate plus tetramethyl-p-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 (CO) 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 photodissociation spectrum (photolysed minus CO-ligated) exhibited a trough at 416 nm and a peak at 436 nm, together with minor features in the alpha/beta region of the spectrum, consistent with the presence of a cytochrome o'-like pigment. CO 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.
authors.affiliation: Instituto de Fisiología Celular y Depto de Bioquimica, Facultad de Medicina, Universidad Nacional Autónoma de México, Apartado postal 70-242, Mexico 04510, D.F., Mexico.
authors.forename: Martha L
authors.initials: ML
authors.lastname: Contreras
authors.affiliation: Instituto de Fisiología Celular y Depto de Bioquimica, Facultad de Medicina, Universidad Nacional Autónoma de México, Apartado postal 70-242, Mexico 04510, D.F., Mexico.
authors.forename: J Edgardo
authors.initials: JE
authors.lastname: Escamilla
authors.affiliation: Instituto de Fisiología Celular y Depto de Bioquimica, Facultad de Medicina, Universidad Nacional Autónoma de México, Apartado postal 70-242, Mexico 04510, D.F., Mexico.
authors.forename: I Patricia
authors.initials: IP
authors.lastname: Del Arenal
authors.affiliation: Escuela de Biología, Universidad Autónoma de Puebla, Puebla, Mexico.
authors.forename: J R
authors.initials: JR
authors.lastname: Dávila
authors.affiliation: Department of Paediatrics, Imperial College School of Medicine at St Mary's, London W2 1PG, UK.
authors.forename: Rita
authors.initials: R
authors.lastname: D'mello
authors.affiliation: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, UK.
authors.forename: Robert K
authors.initials: RK
authors.lastname: Poole
mesh_terms: D001409:Bacillus cereus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D002248:Carbon Monoxide / Q000378:metabolism; D003573:Cytochrome b Group; D003580:Cytochromes / Q000235:genetics / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008024:Ligands; D009097:Multienzyme Complexes / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D010782:Photolysis; D013053:Spectrophotometry; D013057:Spectrum Analysis; D013171:Spores, Bacterial / Q000201:enzymology / Q000254:growth & development; D013696:Temperature
chemical_list: D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D008024:Ligands; D009097:Multienzyme Complexes; D002248:Carbon Monoxide; C017168:cytochrome bo, E coli; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D003576:Electron Transport Complex IV; D010100:Oxygen