Isolated cytochrome-c oxidase ligated with cyanide was titrated by Flash-Induced chemical photoREduction (FIRE) (Moody, A.J. and Rich, P.R. (1988) EBEC Short Rep. 5, 69) using cytochrome c as a redox indicator. Haem a is found to titrate in a complex manner consistent with its interacting anticooperatively with at least two other components. We assign CuB as the major interactant at neutral pH, and CuA as the minor interactant. In the pH range 7.0–8.1 the strength of the interaction with CuB is found to decrease with increasing pH, while the interaction with CuA remains essentially constant.
The decrease in the interaction with CuB appears to continue above pH 8.1 such that at pH 9.2 the titration curve for haem a is only slightly distorted from an ‘n = 1’ shape, although it is not possible from the titration data to assess the relative contributions of CuB and CuA to the total interaction observed at pH values greater than 8.1.
Haem a and CuB show similar pH-dependence and, to account for this, we present a model in which the oxidoreductions of both haem a and CuB are linked to the (de)protonation of a common acid/base group. The model predicts a pH-dependent indirect cooperative interaction between haem a and CuB in addition to the direct anticooperative interaction, thereby explaining the observed pH-dependence of the redox interaction between haem a and CuB
Moody, A.J. and Rich, P.R., 1990. The effect of pH on redox titrations of haem a in cyanide-liganded cytochrome-c oxidase: experimental and modelling studies. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 1015(2), pp.205-215.
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