title: 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 (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.
authors.affiliation: Department of Radiology, Dartmouth Medical School, Hanover, NH, USA.
authors.forename: N
authors.initials: N
authors.lastname: Kim
authors.forename: M O
authors.initials: MO
authors.lastname: Ripple
authors.forename: R
authors.initials: R
authors.lastname: Springett
mesh_terms: D000818:Animals; D002460:Cell Line; D003576:Electron Transport Complex IV / Q000737:chemistry*; D006418:Heme / Q000031:analogs & derivatives / Q000737:chemistry; D008264:Macrophages / Q000378:metabolism; D008564:Membrane Potentials; D051379:Mice; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D013057:Spectrum Analysis / Q000379:methods; D013269:Stochastic Processes
chemical_list: C027728:heme a; D006418:Heme; D003576:Electron Transport Complex IV; D010100:Oxygen