Mi. Verkhovsky et al., CONTROL OF ELECTRON DELIVERY TO THE OXYGEN REDUCTION SITE OF CYTOCHROME-C-OXIDASE - A ROLE FOR PROTONS, Biochemistry, 34(22), 1995, pp. 7483-7491
We have studied the reaction of oxidized ''pulsed'' cytochrome c oxida
se with reduced cytochrome c and with ruthenium(II) hexaammine using s
topped-flow mixing. The rate of reduction of Fe-alpha 3 (the oxygen-bi
nding heme) is not a linear function of the population of reduced Fe-a
lpha (the low-spin heme), as would be expected if electron transfer be
tween these sites is rate-limiting. Instead, the rate can be increased
significantly by increasing the driving force of the reductant (lower
ing of E(h)) even after Fe-alpha is almost completely reduced. Reducti
on of Fe-alpha 3 becomes slower as the pH is raised, and consumption o
f protons can be seen simultaneously with electron entry into Fe-alpha
3. Both the reduction of Fe-alpha 3 and the proton uptake are biphasi
c. To explain these findings, we propose a model in which (1) intramol
ecular heme-heme electron transfer is fast, and has an essentially con
stant rate; (2) when reduction begins, the midpoint potentials of Fe-a
lpha 3 and Cu-B are initially low, and only a small fraction of these
centers become reduced; and (3) this reduced population is then stabil
ized by the uptake of protons. Thus, net reduction of Fe-alpha 3 and C
u-B is controlled by the amount of the low-potential population which
becomes reduced together with the rate of proton uptake by this reduce
d low-potential species. Important consequences of this mechanism for
the function of the enzyme and for the respiratory chain as a whole ar
e discussed.