Preparation and characterization of some surface negatively charged residue mutants of cytochrome b5

Abstract

Site-directed mutagenesis was used to obtain seven variants of tryptic fragment of bovine liver cytochrome bm5 (cyt b5), in which the negatively charged residues around the heme exposed edge of cyt b5 were replaced by hydrophobic amino acid alanine. Double-site mutants, triple-site mutants and even quadruple-site mutants were obtained. DNA sequencing and molecular weight measurements of the mutant proteins both confirmed that these site-directed mutagenesises were successfully performed. Spectroelectrochemistry of these mutant proteins revealed that the apparent redox potentials of these mutant proteins caused a positive shift of 2–10 mV. The global structure of these mutant proteins did not show much difference from that of the wild type cyt b5, providing a solid base for the further study on the roles of the proteins’ surface charges.