S. Junemann et al., Effects of mutation of the conserved glutamic acid-286 in subunit I of cytochrome c oxidase from Rhodobacter sphaeroides, BIOCHEM, 38(16), 1999, pp. 5248-5255
We have studied the effects of mutations, E286Q and E286D, of the conserved
glutamate in subunit I of cytochrome c oxidase from Rhodobacter sphaeroide
s 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 enz
yme properties and retained 50% of wild-type catalytic activity. For E286Q
a fi action of the binuclear center was trapped in an unreactive, spectrall
y distinct form which is most likely due to misfolded protein, but the majo
rity of E286Q reacted normally with formate and cyanide in the oxidized sta
te, and with carbon monoxide and cyanide in the dithionite-reduced form. Th
e mutation also had little effect on the pH-dependent redox properties of h
aem a in the reactive fraction. However, formation of the P state from oxid
ized enzyme with hydrogen peroxide or by aerobic incubation with carbon mon
oxide was inhibited. In particular, only an F-type product was obtained, at
less than 25% yield, in the reaction with hydrogen peroxide. The aerobic s
teady state in the presence of ferrous cytochrome c was characterized by es
sentially fully reduced haem a and ferric haem a(3), suggesting that the mu
tation hinders electron transfer from haem a to the binuclear center. Under
these conditions or after reoxidation, on a seconds time scale, of haem a(
3) following anaerobiosis, there was no indication of accumulation of signi
ficant amounts of P state. We propose that the glutamate is implicated in s
everal 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.