Role of carboxyl residues surrounding heme of human cytochrome b5 in the electrostatic interaction with NADH-cytochrome b5 reductase.
To identify the cytochrome b5 residues responsible for the electrostatic interaction with NADH-cytochrome b5 reductase (b5R), we prepared and characterized the cytochrome b5 mutants in which Glu41, Glu42, Glu63, Asp70, and Glu73 were replaced by Ala, utilizing site-directed mutagenesis and the expression system for cytochrome b5 in Escherichia coli. Apparent Km values of the wild type b5R for Glu42Ala cytochrome b5 and Asp70Ala cytochrome b5 were approximately three-fold and six-fold higher than that for the wild type cytochrome b5, respectively, while the kcat values for those mutants were not remarkably affected. In contrast, Glu41Ala, Glu63Ala, and Glu73Ala cytochrome b5 showed almost the same kinetic properties as the wild type cytochrome b5. Furthermore, kinetic studies on combinations of the cytochrome b5 and b5R mutants suggested the interaction between Glu42 and Asp70 of cytochrome b5 and Lys125 and Lys41 of b5R, respectively, in the reaction.
Kawano
M
M
Department of Biochemistry, Oita Medical University, Japan.
Shirabe
K
K
Nagai
T
T
Takeshita
M
M
D019943:Amino Acid Substitution; D003579:Cytochrome Reductases / Q000737:chemistry / Q000378:metabolism*; D042966:Cytochrome-B(5) Reductase; D015786:Cytochromes b5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D055672:Static Electricity
D006418:Heme; D015786:Cytochromes b5; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase