PROTON INTERACTIONS IN THE RESTING FORM OF CYTOCHROME-OXIDASE

The effect of pH on the near-UV absorption spectrum of cytochrome oxidase has been examined. Several lines of evidence implicate a proton binding site that can modulate the optical properties of cytochrome alpha-3 in the resting enzyme. Changing the pH within the range 6.5-10.5 was found to reversibly shift the position of the Soret band over an 11-nm range. The lower pH values caused a progressive blue shift in the Soret band, whereas the high-pH range promoted a gradual red shift. Limiting band positions were approximately 416 and 427 nm. The incubation time required to reach a stable band position varied somewhat as did the actual extent of the shift. In most cases, the shift was associated with an isosbestic point. A pH titration profile for the apparent equilibrium position of the Soret band was obtained. Nonlinear least-squares fitting to a scatter plot, assuming a single acid/base group, showed an apparent pK(a) of 7.8. Magnetic circular dichroism (MCD) spectra of the low-pH form at 416 nm, the high-pH form at 427 nm, and the cyanide derivative at 428 nm were compared. No evidence of a high-pH-dependent low-spin transition or a change in the redox state of cytochrome alpha-3 was found, confirming earlier work [Baker, G. M., Noguchi, M., & Palmer, G. (1987) J. Biol. Chem. 262, 595-604]. Subtraction of ferricytochrome alpha [spectrum taken from Vanneste, W. H. (1966) Biochemistry 5, 838-848] from a series of blue-shifting spectra showed a band at 414 nm that progressively gained amplitude and a band at 430 nm that correspondingly lost amplitude. A series of red-shifting spectra showed the opposite behavior with a clear isosbestic point being evident in both cases. The difference extinction change at 414 and 430 nm depended linearly on the position of the Soret band, both showing a reversible dependence on pH. The 430-nm band is noted to be unusually red-shifted for high-spin ferric heme alpha. An additional, pH-insensitive band was observed at 408-410 nm which was eliminated by treatment with cyanide. The kinetics of the pH-induced blue shift and red shift were obtained at 416 nm by using dual-wavelength method and found to be biphasic, despite the occurrence of an isosbestic point. Two-exponential fitting gave observed rate constants of 2 x 10(-2) and 3 x 10(-4) s-1 for both the low- and high-pH-induced kinetics, with the rapid phase accounting for 45-55% of the total absorbance change. Incubation times of 2-3 h were generally required to achieve apparent equilibrium following a 1.6-2.0 unit change in pH. The low-pH-induced shift in the Soret band was not kinetically correlated with the fast to slow conversion in cyanide binding behavior that is also known to occur at low pH (Baker et al., 1987). Various explanations for the pH-induced shift in the absorption profile of cytochrome alpha-3 are considered. A model based on external point charge interaction with the pi-pi* Soret resonance is consistent with the available data.