Catalytically-relevant electron transfer between two hemes b(L) in the hybrid cytochrome bc(1)-like complex containing a fusion of Rhodobacter sphaeroides and capsulatus cytochromes b

To address mechanistic questions about the functioning of dimeric cytochrome bc(1) 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 bc(1)-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 (B(S)–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 b(L) in the core of dimer. Kinetic data obtained with a series of B(S)–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 B(S)–B) consistently implicate that the heme-b(L)–b(L) bridge forms an electronic connection available for inter-monomer electron transfer in cytochrome bc(1).