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Characterization of the free energy dependence of an interprotein electron transfer reaction by variation of pH and site-directed mutagenesis.

ABSTRACT: The interprotein electron transfer (ET) reactions of the cupredoxin amicyanin, which mediates ET from the tryptophan tryptophylquinone (TTQ) cofactor of methylamine dehydrogenase to cytochrome c-551i have been extensively studied. However, it was not possible to perform certain key experiments in that native system. This study examines the ET reaction from reduced amicyanin to an alternative electron acceptor, the diheme protein MauG. It was possible to vary the ?G° for this ET reaction by simply changing pH to determine the dependence of kET on ?G°. A P94A mutation of amicyanin significantly altered its oxidation-reduction midpoint potential value. It was not possible to study the ET from reduced P94A amicyanin to cytochrome c-551i in the native system because that reaction was kinetically coupled. However, the reaction from reduced P94A amicyanin to MauG was a true ET reaction and it was possible to determine values of reorganization energy (?) and electronic coupling for the reactions of this variant as well as native amicyanin. Comparison of the ? values associated with the ET reactions between amicyanin and the TTQ of methylamine dehydrogenase, the diheme center of MauG and the single heme of cytochrome c-551i, provides insight into the factors that dictate the ? values for the respective reactions. These results demonstrate how study of ET reactions with alternative redox partner proteins can complement and enhance our understanding of the reactions with the natural redox partners, and further our understanding of mechanisms of protein ET reactions.

SUBMITTER: Dow BA

PROVIDER: S-EPMC4843787 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Characterization of the free energy dependence of an interprotein electron transfer reaction by variation of pH and site-directed mutagenesis.

Dow Brian A BA Davidson Victor L VL

Biochimica et biophysica acta 20150615 10

The interprotein electron transfer (ET) reactions of the cupredoxin amicyanin, which mediates ET from the tryptophan tryptophylquinone (TTQ) cofactor of methylamine dehydrogenase to cytochrome c-551i have been extensively studied. However, it was not possible to perform certain key experiments in that native system. This study examines the ET reaction from reduced amicyanin to an alternative electron acceptor, the diheme protein MauG. It was possible to vary the ΔG° for this ET reaction by simpl ...[more]

PMID: 26087387

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Project description:Secondary electron transfer in photosystem II (PSII), which occurs when water oxidation is inhibited, involves redox-active carotenoids (Car), as well as chlorophylls (Chl), and cytochrome b 559 (Cyt b 559), and is believed to play a role in photoprotection. CarD2 may be the initial point of secondary electron transfer because it is the closest cofactor to both P680, the initial oxidant, and to Cyt b 559, the terminal secondary electron donor within PSII. In order to characterize the role of CarD2 and to determine the effects of perturbing CarD2 on both the electron-transfer events and on the identity of the redox-active cofactors, it is necessary to vary the properties of CarD2 selectively without affecting the ten other Car per PSII. To this end, site-directed mutations around the binding pocket of CarD2 (D2-G47W, D2-G47F, and D2-T50F) have been generated in Synechocystis sp. PCC 6803. Characterization by near-IR and EPR spectroscopy provides the first experimental evidence that CarD2 is one of the redox-active carotenoids in PSII. There is a specific perturbation of the Car(∙+) near-IR spectrum in all three mutated PSII samples, allowing the assignment of the spectral signature of Car D2 (∙+) ; Car D2 (∙+) exhibits a near-IR peak at 980 nm and is the predominant secondary donor oxidized in a charge separation at low temperature in ferricyanide-treated wild-type PSII. The yield of secondary donor radicals is substantially decreased in PSII complexes isolated from each mutant. In addition, the kinetics of radical formation are altered in the mutated PSII samples. These results are consistent with oxidation of CarD2 being the initial step in secondary electron transfer. Furthermore, normal light levels during mutant cell growth perturb the shape of the Chl(∙+) near-IR absorption peak and generate a dark-stable radical observable in the EPR spectra, indicating a higher susceptibility to photodamage further linking the secondary electron-transfer pathway to photoprotection.

Dataset Information

Characterization of the free energy dependence of an interprotein electron transfer reaction by variation of pH and site-directed mutagenesis.

Publications

Characterization of the free energy dependence of an interprotein electron transfer reaction by variation of pH and site-directed mutagenesis.

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