Arginine 391 in subunit I of the cytochrome bd quinol oxidase from Escherichia coli stabilizes the reduced form of the hemes and is essential for quinol oxidase activity.
The cytochrome bd quinol oxidase is one of two respiratory oxidases in Escherichia coli. It oxidizes dihydroubiquinol or dihydromenaquinol while reducing dioxygen to water. The bd-type oxidases have only been found in prokaryotes and have been implicated in the survival of some bacteria, including pathogens, under conditions of low aeration. With a high affinity for dioxygen, cytochrome bd not only couples respiration to the generation of a proton motive force but also scavenges O(2). In the current work, the role of a highly conserved arginine residue is explored by site-directed mutagenesis. Four mutations were made: R391A, R391K, R391M, and R391Q. All of the mutations except R391K result in enzyme lacking ubiquinol oxidase activity. Oxidase activity using the artificial reductant N,N,N',N'-tetramethyl-p-phenylenediamine in place of ubiquinol was, however, unimpaired by the mutations, indicating that the catalytic center where O(2) is reduced is intact. UV-visible spectra of each of the mutant oxidases show no perturbations to any of the three heme components (heme b(558), heme b(595), and heme d). However, spectroelectrochemical titrations of the R391A mutant reveal that the midpoint potentials of all of the heme components are substantially lower compared with the wild type enzyme. Since Arg(391) is close to Met(393), one of the axial ligands to heme b(558), it is to be expected that the R391A mutation might destabilize the reduced form of heme b(558). The fact that the midpoint potentials of heme d and heme b(595) are also significantly lowered in the R391A mutant is consistent with these hemes being physically close together on the periplasmic side of the membrane.
Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, USA.
Jie
J
Zhang
Petra
P
Hellwig
Jeffrey P
JP
Osborne
Robert B
RB
Gennis
D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics / Q000502:physiology*; D001665:Binding Sites; D002248:Carbon Monoxide; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003573:Cytochrome b Group / Q000378:metabolism; D003580:Cytochromes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D045222:Electron Transport Chain Complex Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006418:Heme / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019255:NADPH Oxidases / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011199:Potentiometry; D013053:Spectrophotometry; D017550:Spectroscopy, Fourier Transform Infrared; D013329:Structure-Activity Relationship; D013772:Tetramethylphenylenediamine / Q000378:metabolism; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000378:metabolism
D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; C435883:heme b(595); D014451:Ubiquinone; D006418:Heme; C080647:heme d; D002248:Carbon Monoxide; C036888:cytochrome b558; D001120:Arginine; D010088:Oxidoreductases; D019255:NADPH Oxidases; C040077:cytochrome bd terminal oxidase complex, E coli; C003741:ubiquinol; D013772:Tetramethylphenylenediamine; D010100:Oxygen