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Accelerated Evolution of Cytochrome c in Higher Primates, and Regulation of the Reaction between Cytochrome c and Cytochrome Oxidase by Phosphorylation.

ABSTRACT: Cytochrome c (Cc) underwent accelerated evolution from the stem of the anthropoid primates to humans. Of the 11 amino acid changes that occurred from horse Cc to human Cc, five were at Cc residues near the binding site of the Cc:CcO complex. Single-point mutants of horse and human Cc were made at each of these positions. The Cc:CcO dissociation constant K_D of the horse mutants decreased in the order: T89E > native horse Cc > V11I Cc > Q12M > D50A > A83V > native human. The largest effect was observed for the mutants at residue 50, where the horse Cc D50A mutant decreased K_D from 28.4 to 11.8 μM, and the human Cc A50D increased K_D from 4.7 to 15.7 μM. To investigate the role of Cc phosphorylation in regulating the reaction with CcO, phosphomimetic human Cc mutants were prepared. The Cc T28E, S47E, and Y48E mutants increased the dissociation rate constant k_d, decreased the formation rate constant k_f, and increased the equilibrium dissociation constant K_D of the Cc:CcO complex. These studies indicate that phosphorylation of these residues plays an important role in regulating mitochondrial electron transport and membrane potential ΔΨ.

SUBMITTER: Brand SE

PROVIDER: S-EPMC9777161 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Accelerated Evolution of Cytochrome c in Higher Primates, and Regulation of the Reaction between Cytochrome c and Cytochrome Oxidase by Phosphorylation.

Brand Sue Ellen SE Scharlau Martha M Geren Lois L Hendrix Marissa M Parson Clayre C Elmendorf Tyler T Neel Earl E Pianalto Kaila K Silva-Nash Jennifer J Durham Bill B Millett Francis F

Cells 20221212 24

Cytochrome c (Cc) underwent accelerated evolution from the stem of the anthropoid primates to humans. Of the 11 amino acid changes that occurred from horse Cc to human Cc, five were at Cc residues near the binding site of the Cc:CcO complex. Single-point mutants of horse and human Cc were made at each of these positions. The Cc:CcO dissociation constant KD of the horse mutants decreased in the order: T89E > native horse Cc > V11I Cc > Q12M > D50A > A83V > native human. The larg ...[more]

PMID: 36552779

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Project description:In stopped-flow experiments in which oxidized cytochrome c oxidase was mixed with ferrocytochrome c in the presence of a range of oxygen concentrations and in the absence and presence of cyanide, a fast phase, reflecting a rapid approach to an equilibrium, was observed. Within this phase, one or two molecules of ferrocytochrome were oxidized per haem group of cytochrome a, depending on the concentration of ferrocytochrome c used. The reasons for this are discussed in terms of a mechanism in which all electrons enter through cytochrome a, which, in turn, is in rapid equilibrium with a second site, identified with 'visible' copper (830 nm-absorbing) Cud (Beinert et al., 1971). The value of the bimolecular rate constant for the reaction between cytochromes c2+ and a3+ was between 10(6) and 10(7) M(-1)-S(-1); some variability from preparation to preparation was observed. At high ferrocytochrome c concentrations, the initial reaction of cytochrome c2+ with cytochrome a3+ could be isolated from the reaction involving the 'visible' copper and the stoicheiometry was found to approach one molecule of cytochrome c2+ oxidized for each molecule of cytochrome a3+ reduced. At low ferrocytochrome c concentrations, however, both sites (i.e. cytochrome a and Cud) were reduced simultaneously and the stoicheiometry of the initial reaction was closer to two molecules of cytochrome c2+ oxidized per molecule of cytochrome a reduced. The bleaching of the 830 nm band lagged behind or was simultaneous with the formation of the 605 nm band and does not depend on the cytochrome c concentration, whereas the extinction at the steady-state does. The time-course of the return of the 830 nm-absorbing species is much faster than the bleaching of the 605 nm-absorbing component, and parallels that of the turnover phase of cytochrome c2+ oxidation. Additions of cyanide to the oxidase preparations had no effect on the observed stoicheiometry or kinetics of the reduction of cytochrome a and 'visible' copper, but inhibited electron transfer to the other two sites, cytochrome a3 and the undetectable copper, Cuu.

Dataset Information

Accelerated Evolution of Cytochrome c in Higher Primates, and Regulation of the Reaction between Cytochrome c and Cytochrome Oxidase by Phosphorylation.

Publications

Accelerated Evolution of Cytochrome <i>c</i> in Higher Primates, and Regulation of the Reaction between Cytochrome <i>c</i> and Cytochrome Oxidase by Phosphorylation.

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