Modulation of the electron-proton coupling at cytochrome a by the ligation of the oxidized catalytic center in bovine cytochrome c oxidase.

Cytochrome a was suggested as the key redox center in the proton pumping process of bovine cytochrome c oxidase (CcO). Recent studies showed that both the structure of heme a and its immediate vicinity are sensitive to the ligation and the redox state of the distant catalytic center composed of iron of cytochrome a3 (Fea3) and copper (CuB). Here, the influence of the ligation at the oxidized Fea33+-CuB2+ center on the electron-proton coupling at heme a was examined in the wide pH range (6.5-11). The strength of the coupling was evaluated by the determination of pH dependence of the midpoint potential of heme a (Em(a)) for the cyanide (the low-spin Fea33+) and the formate-ligated CcO (the high-spin Fea33+). The measurements were performed under experimental conditions when other three redox centers of CcO are oxidized. Two slightly differing linear pH dependencies of Em(a) were found for the CN- and the formate-ligated CcO with slopes of -13 mV/pH unit and -23 mV/pH unit, respectively. These linear dependencies indicate only a weak and unspecific electron-proton coupling at cytochrome a in both forms of CcO. The lack of the strong electron-proton coupling at the physiological pH values is also substantiated by the UV-Vis absorption and electron-paramagnetic resonance spectroscopy investigations of the cyanide-ligated oxidized CcO. It is shown that the ligand exchange at Fea3+ between His-Fea3+-His and His-Fea3+-OH- occurs only at pH above 9.5 with the estimated pK >11.0.

Response of Heme Symmetry to the Redox State of Bovine Cytochrome c Oxidase.

Second-derivative absorption spectroscopy was employed to monitor the response of effective symmetry of cytochromes a and a3 to the redox and ligation states of bovine cytochrome c oxidase (CcO). The Soret band π → π* electronic transitions were used to display the changes in symmetry of these chromophores induced by the reduction of CcO inhibited by the exogenous ligands and during catalytic turnover. The second derivative of the difference absorption spectra revealed only a single Soret band for the oxidized cytochromes a and a3 and cyanide-ligated oxidized cytochrome a3. In contrast, two absorption bands were resolved in ferrous cytochrome a and ferrous cytochrome a3 ligated with cyanide. A transition from one-band spectrum to two-band spectrum indicates the lowering of symmetry of these hemes due to the alteration of their immediate surroundings. It is suggested that the changes in polarity occurring in the vicinity of these cofactors are main reason for the split of the Soret band of both ferrous cytochrome a and cyanide-bound ferrous cytochrome a3.