Electrostatic interaction between NADH-cytochrome b5 reductase and cytochrome b5 studied by site-directed mutagenesis

Electrostatic interaction between NADH-cytochrome b(5) reductase and cytochrome b(5) was studied by site-directed mutagenesis. The target residues for mutagenesis were selected on the basis of the previously reported chemical cross-linking study of these two proteins, which implicated possible charge-pair interactions between Lys-41, Lys-125, Lys-162, and Lys-163 of the enzyme, and Glu-47, Glu-48, Glu-52, Glu-60, Asp-64 (group A), and heme propionate of cytochrome b(5). Mutant reductases that lost one of the above-listed Lys residues showed higher K-m, values for cytochrome b(5) and lower k(cat) values than those of the wild type, suggesting that all of the examined Lys residues participate in binding with cytochrome b(5) as reported previously. In contrast, a removal of one of (or even all of) the group A residues from cytochrome b(5) by mutagenesis caused no significant effect on the catalytic properties of cytochrome b(5). Additional elimination of another set of negative residues (Glu-41, Glu-42, Asp-57, and Glu-63 (group B)), which are also located close to heme, elevated the K-m value by more than five folds. These results suggest that there should be other acidic residue(s) than group A in cytochrome b(5) which participate in binding with NADH-cytochrome b(5) reductase. (C) 199S Elsevier Science B.V.