Dataset Information

Enzymatic activities of isolated cytochrome bc?-like complexes containing fused cytochrome b subunits with asymmetrically inactivated segments of electron transfer chains.

ABSTRACT: Homodimeric structure of cytochrome bc?, a common component of biological energy conversion systems, builds in four catalytic quinone oxidation/reduction sites and four chains of cofactors (branches) that, connected by a centrally located bridge, form a symmetric H-shaped electron transfer system. The mechanism of operation of this complex system is under constant debate. Here, we report on isolation and enzymatic examination of cytochrome bc?-like complexes containing fused cytochrome b subunits in which asymmetrically introduced mutations inactivated individual branches in various combinations. The structural asymmetry of those forms was confirmed spectroscopically. All the asymmetric forms corresponding to cytochrome bc? with partial or full inactivation of one monomer retain high enzymatic activity but at the same time show a decrease in the maximum turnover rate by a factor close to 2. This strongly supports the model assuming independent operation of monomers. The cross-inactivated form corresponding to cytochrome bc? with disabled complementary parts of each monomer retains the enzymatic activity at the level that, for the first time on isolated from membranes and purified to homogeneity preparations, demonstrates that intermonomer electron transfer through the bridge effectively sustains the enzymatic turnover. The results fully support the concept that electrons freely distribute between the four catalytic sites of a dimer and that any path connecting the catalytic sites on the opposite sides of the membrane is enzymatically competent. The possibility to examine enzymatic properties of isolated forms of asymmetric complexes constructed using the cytochrome b fusion system extends the array of tools available for investigating the engineering of dimeric cytochrome bc? from the mechanistic and physiological perspectives.

SUBMITTER: Czapla M

PROVIDER: S-EPMC3269193 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Enzymatic activities of isolated cytochrome bc₁-like complexes containing fused cytochrome b subunits with asymmetrically inactivated segments of electron transfer chains.

Czapla Monika M Borek Arkadiusz A Sarewicz Marcin M Osyczka Artur A

Biochemistry 20120117 4

Homodimeric structure of cytochrome bc₁, a common component of biological energy conversion systems, builds in four catalytic quinone oxidation/reduction sites and four chains of cofactors (branches) that, connected by a centrally located bridge, form a symmetric H-shaped electron transfer system. The mechanism of operation of this complex system is under constant debate. Here, we report on isolation and enzymatic examination of cytochrome bc₁-like complexes containing fused cytochrome b subunit ...[more]

PMID: 22233445

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Project description:Cytochrome c(1) of Rhodobacter (Rba.) species provides a series of mutants which change barriers for electron transfer through the cofactor chains of cytochrome bc(1) by modifying heme c(1) redox midpoint potential. Analysis of post-flash electron distribution in such systems can provide useful information about the contribution of individual reactions to the overall electron flow. In Rba. capsulatus, the non-functional low-potential forms of cytochrome c(1) which are devoid of the disulfide bond naturally present in this protein revert spontaneously by introducing a second-site suppression (mutation A181T) that brings the potential of heme c(1) back to the functionally high levels, yet maintains it some 100 mV lower from the native value. Here we report that the disulfide and the mutation A181T can coexist in one protein but the mutation exerts a dominant effect on the redox properties of heme c(1) and the potential remains at the same lower value as in the disulfide-free form. This establishes effective means to modify a barrier for electron transfer between the FeS cluster and heme c(1) without breaking disulfide. A comparison of the flash-induced electron transfers in native and mutated cytochrome bc(1) revealed significant differences in the post-flash equilibrium distribution of electrons only when the connection of the chains with the quinone pool was interrupted at the level of either of the catalytic sites by the use of specific inhibitors, antimycin or myxothiazol. In the non-inhibited system no such differences were observed. We explain the results using a kinetic model in which a shift in the equilibrium of one reaction influences the equilibrium of all remaining reactions in the cofactor chains. It follows a rather simple description in which the direction of electron flow through the coupled chains of cytochrome bc(1) exclusively depends on the rates of all reversible partial reactions, including the Q/QH2 exchange rate to/from the catalytic sites.

Dataset Information

Enzymatic activities of isolated cytochrome bc?-like complexes containing fused cytochrome b subunits with asymmetrically inactivated segments of electron transfer chains.

Publications

Enzymatic activities of isolated cytochrome bc₁-like complexes containing fused cytochrome b subunits with asymmetrically inactivated segments of electron transfer chains.

Similar Datasets

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