Abstract
To address mechanistic questions about the functioning of dimeric cytochrome bc1 new genetic approaches have recently been developed. They were specifically designed to enable construction of asymmetrically-mutated variants suitable for functional studies. One approach exploited a fusion of two cytochromes b that replaced the separate subunits in the dimer. The fusion protein, built from two copies of the same cytochrome b of purple bacterium Rhodobacter capsulatus, served as a template to create a series of asymmetrically-mutated cytochrome bc1-like complexes (B–B) which, through kinetic studies, disclosed several important principles of dimer engineering. Here, we report on construction of another fusion protein complex that adds a new tool to investigate dimeric function of the enzyme through the asymmetrically mutated forms of the protein. This complex (BS–B) contains a hybrid protein that combines two different cytochromes b: one coming from R. capsulatus and the other — from a closely related species, R. sphaeroides. With this new fusion we addressed a still controversial issue of electron transfer between the two hemes bL in the core of dimer. Kinetic data obtained with a series of BS–B variants provided new evidence confirming the previously reported observations that electron transfer between those two hemes occurs on a millisecond timescale, thus is a catalytically-relevant event. Both types of the fusion complexes (B–B and BS–B) consistently implicate that the heme-bL–bL bridge forms an electronic connection available for inter-monomer electron transfer in cytochrome bc1.
Highlights
Cytochrome bc1 is an integral component of many biological energy conversion systems
As prerequisite for experiments of fusing two different cytochromes b described we have tested the effect of replacing native cytochrome b of R. capsulatus cytochrome bc1 with that coming from the closely related strain, R. sphaeroides
To this end we constructed an expression vector pMTS1-BS, which in the place of native R. capsulatus petB gene in petABC operon contained the gene fbcB coding for R. sphaeroides cytochrome b (Fig. 1)
Summary
Cytochrome bc is an integral component of many biological energy conversion systems. The distances between the cofactors in different monomers, as revealed by X-ray crystallography, are large enough to exclude possibility of inter-monomer electron transfer except for one point: a bridge formed by two hemes bL which in the center of the dimer are at 14 Å edge to edge. This distance appears to be just at the limit of distances between the centers that exchange electrons within micro- to milliseconds, a timescale generally considered to be catalytically-relevant [4]. The revelation about the close distance between two hemes bL inspired an intense discussion about possible electron transfer between the monomers and its role in a catalytic cycle. The possibility of electron transfer between the hemes bL was so appealing that it became an integral part of several
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