title: Mechanism of electron transfer in fusion protein cytochrome b5-NADH-cytochrome b5 reductase.
abstract: In the present work we summarize results on construction of expression plasmid, heterologous expression in Escherichia coli, isolation and purification, as well as physicochemical characterization of chimeric protein consisting of hydrophilic domain of cytochrome b(5) and truncated from the N-terminal sequence (Delta(23)) form of NADH-cytochrome b(5) reductase. The kinetics and mechanism of electron transfer between NADH-cytochrome b(5) reductase and cytochrome b(5) in the frames of fusion protein consisting of cytochrome b(5) (94 amino acids) and truncated form of NADH-cytochrome b(5) reductase (277 amino acids) have been studied. It is shown that electron transfer takes place between redox partners belonging to two different molecules of the chimeric protein. Using computer modeling, we built the model of the tertiary structure of the fusion protein, which is in agreement with experimental data. By using Marcus theory of electron transfer in polar media, we demonstrate the inability of the hypothesis of electrostatic repulsions to explain the increase of electron transfer rate on increase of ion concentration in media due to elimination of the repulsion of similar charges. The real reason for the increase of the first order rate constant in some oxidation-reduction reactions between proteins, as shown in the present work, is a decrease of the media reorganization energy resulting in decrease of activation energy for oxidation-reduction reactions.
authors.affiliation: Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Minsk, 220141, Belarus.
authors.forename: A V
authors.initials: AV
authors.lastname: Yantsevich
authors.forename: A A
authors.initials: AA
authors.lastname: Gilep
authors.forename: S A
authors.initials: SA
authors.lastname: Usanov
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D042966:Cytochrome-B(5) Reductase / Q000235:genetics* / Q000378:metabolism*; D015786:Cytochromes b5 / Q000235:genetics* / Q000378:metabolism*; D004579:Electron Transport / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D011993:Recombinant Fusion Proteins; D015786:Cytochromes b5; D042966:Cytochrome-B(5) Reductase