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

Site-directed mutagenesis was used to obtain seven variants of tryptic fragment of bovine liver cytochrome b_m5 (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.