title: Electrostatic interaction between NADH-cytochrome b5 reductase and cytochrome b5 studied by site-directed mutagenesis.
abstract: Electrostatic interaction between NADH-cytochrome b5 reductase and cytochrome b5 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 b5. Mutant reductases that lost one of the above-listed Lys residues showed higher K(m) values for cytochrome b5 and lower kcat values than those of the wild type, suggesting that all of the examined Lys residues participate in binding with cytochrome b5 as reported previously. In contrast, a removal of one of (or even all of) the group A residues from cytochrome b5 by mutagenesis caused no significant effect on the catalytic properties of cytochrome b5. 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 b5 which participate in binding with NADH-cytochrome b5 reductase.
authors.affiliation: Department of Biochemistry, Oita Medical University, Japan. shirabe@oita-med.ac.jp
authors.forename: K
authors.initials: K
authors.lastname: Shirabe
authors.forename: T
authors.initials: T
authors.lastname: Nagai
authors.forename: T
authors.initials: T
authors.lastname: Yubisui
authors.forename: M
authors.initials: M
authors.lastname: Takeshita
mesh_terms: D001665:Binding Sites / Q000235:genetics; D003579:Cytochrome Reductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D042966:Cytochrome-B(5) Reductase; D015786:Cytochromes b5 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004579:Electron Transport; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D055672:Static Electricity; D013379:Substrate Specificity
chemical_list: D015786:Cytochromes b5; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase