Proton Interactions with Hemesaanda3in Bovine Heart CytochromecOxidase

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Abstract
Three forms of cytochrome c oxidase, fully oxidized CcO (CcO−O), oxidized CcO complexed with cyanide (CcO·CN), and mixed valence CcO, in which both heme a3 and CuB are reduced and stabilized by carbon monoxide (MV·CO), were investigated by optical spectroscopy, MCD, and stopped-flow for the pH sensitivity of spectral features. In the pH range between pH 5.7 and 9.0, both heme a and heme a3 in CcO−O interact with a single protolytic group. From the variation of the position of the Soret peak with changes in pH, a pKa of 6.6 ± 0.2 was determined for this group. The pH sensitivity of heme a3 is lost in the CcO·CN complex, and only heme a responds to pH changes. In MV·CO the spectra of both hemes are almost independent of pH between 5.7 and 11.0. The stoichiometry of proton uptake in the conversion of CcO−O both to MV·CO and to fully reduced CcO was determined between pH 5.8 and pH 8.2. Formation of MV·CO from CcO−O was accompanied by the uptake of approximately two protons, and this value was almost independent of pH. Full reduction of oxidized CcO was associated with the uptake of approximately 2 H+ at basic pH, and this value increases with decreasing pH. On the basis of these proton uptake measurements, it is concluded that the pKa of the group is independent of the redox state of CcO. It is suggested that Glu60 of subunit II, located at the entrance of the proton conducting K-channel, is the protolytic residue that interacts with both hemes through a hydrogen-bonding network.

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