MUTAGENIC, ELECTROCHEMICAL, AND CRYSTALLOGRAPHIC INVESTIGATION OF THE CYTOCHROME-B5 OXIDATION REDUCTION EQUILIBRIUM - INVOLVEMENT OF ASPARAGINE-57, SERINE-64, AND HEME PROPIONATE-7

A gene coding for lipase-solubilized bovine liver microsomal cytochrome b5 has been synthesized, expressed in Escherichia coli, and mutated at functionally critical residues. Characterization of the recombinant protein revealed that it has a reduction potential that is approximately 17 mV lower than that of authentic wild-type protein at pH 7 (25°C). Structural studies determined that the recombinant protein differed in sequence from authentic wild-type cytochrome b5 owing to three errors in amidation status in the published sequence for the protein on which the gene synthesis was based. The structural origin of the lower reduction potential exhibited by the triple mutant has been investigated through X-ray crystallographic determination of the three-dimensional structure of this protein and is attributed to the presence of Asp-57 within 3.3 Å of heme vinyl-4 in the mutant. In addition, the model developed by Argos and Mathews [Argos, P., & Mathews, F. S. (1975) J. Biol. Chem. 250, 747] for the change in cytochrome b5 oxidation state has been studied through mutation of Ser-64 to Ala. In this model, Ser-64 is postulated to stabilize the oxidized protein through H-bonding interactions with heme propionate-7 that orients this propionate group 6.2 Å from the heme iron. Spectroelectrochemical studies of a mutant in which Ser-64 has been changed to an alanyl residue demonstrate that this protein has a reduction potential that is 7 mV lower than that of the wild-type protein; moreover, conversion of the heme propionate groups to the corresponding methyl esters increases the potential by 67 mV. These results establish that elimination of one of the hydrogen bonds formed between Ser-64 and heme propionate-7 in ferricytochrome b5 is not sufficient to destabilize this form of the protein. We conclude that the increase in reduction potential produced by heme propionate esterification results from elimination of a carboxylate inductive effect or simply from removal of two negatively charged functional groups that are in proximity to the heme group. © 1990, American Chemical Society.