* To whom correspondence should be addressed.
Received February 26, 2008; Revision received March 28, 2008
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 b5 and truncated from the N-terminal sequence (Δ23) form of NADH-cytochrome b5 reductase. The kinetics and mechanism of electron transfer between NADH-cytochrome b5 reductase and cytochrome b5 in the frames of fusion protein consisting of cytochrome b5 (94 amino acids) and truncated form of NADH-cytochrome b5 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.
KEY WORDS: microsomal cytochrome b5, expression in Escherichia coli, affinity chromatography, purification