Spectral components of the α-band of cytochrome oxidase.

Oxidative redox titrations of the mitochondrial cytochromes were performed in near-anoxic RAW 264.7 cells by inhibiting complex I. Cytochrome oxidation changes were measured with multi-wavelength spectroscopy and the ambient redox potential was calculated from the oxidation state of endogenous cytochrome c. Two spectral components were separated in the α-band range of cytochrome oxidase and they were identified as the difference spectrum of heme a when it has a high (a(H)) or low (a(L)) midpoint potential (E(m)) by comparing their occupancy during redox titrations carried out when the membrane potential (ΔΨ) was dissipated with a protonophore to that predicted by the neoclassical model of redox cooperativity. The difference spectrum of a(L) has a maximum at 605nm whereas the spectrum of a(H) has a maximum at 602nm. The ΔΨ-dependent shift in the E(m) of a(H) was too great to be accounted for by electron transfer from cytochrome c to heme a against ΔΨ but was consistent with a model in which a(H) is formed after proton uptake against ΔΨ suggesting that the spectral changes are the result of protonation. A stochastic simulation was implemented to model oxidation states, proton uptake and E(m) changes during redox titrations. The redox anti-cooperativity between heme a and heme a(3), and proton binding, could be simulated with a model where the pump proton interacted with heme a and the substrate proton interacted with heme a(3) with anti-cooperativity between proton binding sites, but not with a single proton binding site coupled to both hemes.