Spectral components of the α-band of cytochrome oxidase

Abstract

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 (Δ Ψ) 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 605 nm whereas the spectrum of a H has a maximum at 602 nm. The Δ Ψ-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 Δ Ψ but was consistent with a model in which a H is formed after proton uptake against Δ Ψ 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.