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The effect of a single site mutation of Arg-54 to methionine in Paracoccus denitrificans cytochrome c oxidase was studied using a combination of optical spectroscopy, electrochemical and rapid kinetics techniques, and time-resolved measurements of electrical membrane potential. The mutation resulted in a blue-shift of the heme a alpha-band by 15 nm and partial occupation of the low-spin heme site by heme O. Additionally, there was a marked decrease in the midpoint potential of the low-spin heme, resulting in slow reduction of this heme species. A stopped-flow investigation of the reaction with ferrocytochrome c yielded a kinetic difference spectrum resembling that of heme a(3). This observation, and the absence of transient absorbance changes at the corresponding wavelength of the low-spin heme, suggests that, in the mutant enzyme, electron transfer from Cu(A) to the binuclear center may not occur via heme a but that instead direct electron transfer to the high-spin heme is the dominating process. This was supported by charge translocation measurements where Deltapsi generation was completely inhibited in the presence of KCN. Our results thus provide an example for how the interplay between protein and cofactors can modulate the functional properties of the enzyme complex.
The cytochrome bc1 complex is a dimeric enzyme of the inner mitochondrial membrane that links electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which ubiquinol is oxidized at one center in the enzyme, referred to as center P, and ubiquinone is rereduced at a second center, referred to as center N. To better understand the mechanism of ubiquinol oxidation, we have examined catalytic activities and pre-steady-state reduction kinetics of yeast cytochrome bc1 complexes with mutations in cytochrome b that we expected would affect oxidation of ubiquinol. We mutated two residues thought to be involved in proton conduction linked to ubiquinol oxidation, Tyr132 and Glu272, and two residues proposed to be involved in docking ubiquinol into the center P pocket, Phe129 and Tyr279. Substitution of Phe129 by lysine or arginine yielded a respiration-deficient phenotype and lipid-dependent catalytic activity. Increased bypass reactions were detectable for both variants, with F129K showing the more severe effects. Substitution with lysine leads to a disturbed coordination of a b heme as deduced from changes in the midpoint potential and the EPR signature. Removal of the aromatic side chain in position Tyr279 lowers the catalytic activity accompanied by a low level of bypass reactions. Pre-steady-state kinetics of the enzymes modified at Glu272 and Tyr132 confirmed the importance of their functional groups for electron transfer. Altered center N kinetics and activation of ubiquinol oxidation by binding of cytochrome c in the Y132F and E272D enzymes indicate long range effects of these mutations.
The proton-pumping NADH:ubiquinone oxidoreductase (complex I) couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Electron transfer is accomplished by FMN and a series of iron-sulfur clusters. Its coupling with proton translocation is not yet understood. Here, we report that the redox reaction of the FeS cluster N2 located on subunit NuoB of the Escherichia coli complex I induces a protonation/deprotonation of tyrosine side chains. Electrochemically induced FT-IR difference spectra revealed characteristic tyrosine signals at 1,515 and 1,498 cm−1 for the protonated and deprotonated form, respectively. Mutants of three conserved tyrosines on NuoB were generated by complementing a chromosomal in-frame deletion strain with nuoB on a plasmid. Though the single mutations did not alter the electron transport activity of complex I, the EPR signal of cluster N2 was slightly shifted. The tyrosine signals detected by FT-IR spectroscopy were roughly halved in the mutants Y114C and Y139C while only minor changes were detected in the Y154H mutant. The enzymatic activity of the Y114C/Y139F double mutant was 80% reduced, and FT-IR difference spectra of the double mutant revealed a complete loss the modes characteristic for protonation reactions of tyrosines. Therefore, we propose that tyrosines 114 and 139 on NuoB were protonated upon reduction of cluster N2 and were thus involved in the proton-transfer reaction coupled with its redox reaction.