Effects of mutation of the conserved glutamic acid-286 in subunit I of cytochrome c oxidase from Rhodobacter sphaeroides.
We have studied the effects of mutations, E286Q and E286D, of the conserved glutamate in subunit I of cytochrome c oxidase from Rhodobacter sphaeroides with a view to evaluating the role of this residue in redox-linked proton translocation. The mutation E286D did not have any dramatic effects on enzyme properties and retained 50% of wild-type catalytic activity. For E286Q a fraction of the binuclear center was trapped in an unreactive, spectrally distinct form which is most likely due to misfolded protein, but the majority of E286Q reacted normally with formate and cyanide in the oxidized state, and with carbon monoxide and cyanide in the dithionite-reduced form. The mutation also had little effect on the pH-dependent redox properties of haem a in the reactive fraction. However, formation of the P state from oxidized enzyme with hydrogen peroxide or by aerobic incubation with carbon monoxide was inhibited. In particular, only an F-type product was obtained, at less than 25% yield, in the reaction with hydrogen peroxide. The aerobic steady state in the presence of ferrous cytochrome c was characterized by essentially fully reduced haem a and ferric haem a3, suggesting that the mutation hinders electron transfer from haem a to the binuclear center. Under these conditions or after reoxidation, on a seconds time scale, of haem a3 following anaerobiosis, there was no indication of accumulation of significant amounts of P state. We propose that the glutamate is implicated in several steps in the catalytic cycle, O --> R, P --> F, and, possibly, F --> O. The results are discussed in relation to the "glutamate trap" model for proton translocation.
Jünemann
S
S
Glynn Laboratory of Bioenergetics, Department of Biology, University College, London, U.K. s.junemann@ucl.ac.uk
Meunier
B
B
Fisher
N
N
Rich
P R
PR
D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D017124:Conserved Sequence / Q000235:genetics*; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005290:Ferric Compounds / Q000737:chemistry; D005561:Formates / Q000737:chemistry; D018698:Glutamic Acid / Q000235:genetics*; D005973:Glutamine / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics
D005290:Ferric Compounds; D005561:Formates; D008024:Ligands; D005973:Glutamine; C030544:formic acid; D001224:Aspartic Acid; D018698:Glutamic Acid; D003576:Electron Transport Complex IV