Spectral components of the α-band of cytochrome oxidase

AbstractOxidative 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 (aH) or low (aL) midpoint potential (Em) 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 aL has a maximum at 605nm whereas the spectrum of aH has a maximum at 602nm. The ΔΨ-dependent shift in the Em of aH 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 aH 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 Em changes during redox titrations. The redox anti-cooperativity between heme a and heme a3, and proton binding, could be simulated with a model where the pump proton interacted with heme a and the substrate proton interacted with heme a3 with anti-cooperativity between proton binding sites, but not with a single proton binding site coupled to both hemes