title: Catalytically-relevant electron transfer between two hemes bL in the hybrid cytochrome bc1-like complex containing a fusion of Rhodobacter sphaeroides and capsulatus cytochromes b.
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.
authors: Department of Molecular Biophysics, Biophysics and Biotechnology, Jagiellonian University, 30-387 Kraków, Poland.
authors: Monika
authors: M
authors: Czapla
authors: Ewelina
authors: E
authors: Cieluch
authors: Arkadiusz
authors: A
authors: Borek
authors: Marcin
authors: M
authors: Sarewicz
authors: Artur
authors: A
authors: Osyczka
mesh_terms: D002384:Catalysis; D045303:Cytochromes b / Q000737:chemistry*; D004579:Electron Transport; D014450:Electron Transport Complex III / Q000737:chemistry*; D006418:Heme / Q000737:chemistry*; D008027:Light; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D016354:Rhodobacter capsulatus / Q000201:enzymology*; D012242:Rhodobacter sphaeroides / Q000201:enzymology*
chemical_list: D011993:Recombinant Fusion Proteins; D006418:Heme; D045303:Cytochromes b; D014450:Electron Transport Complex III