Abstract
To identify the cytochrome b5residues responsible for the electrostatic interaction with NADH-cytochrome b5reductase (b5R), we prepared and characterized the cytochrome b5mutants in which Glu41, Glu42, Glu63, Asp70, and Glu73 were replaced by Ala, utilizing site-directed mutagenesis and the expression system for cytochrome b5inEscherichia coli.Apparent Km values of the wild type b5R for Glu42Ala cytochrome b5and Asp70Ala cytochrome b5were 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 b5showed almost the same kinetic properties as the wild type cytochrome b5. Furthermore, kinetic studies on combinations of the cytochrome b5and b5R mutants suggested the interaction between Glu42 and Asp70 of cytochrome b5and Lys125 and Lys41 of b5R, respectively, in the reaction.
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