ROLE OF CARBOXYL RESIDUES SURROUNDING HEME OF HUMAN CYTOCHROME B(5) IN THE ELECTROSTATIC INTERACTION WITH NADH-CYTOCHROME B(5) REDUCTASE

To identify the cytochrome b(5) residues responsible for the electrost atic interaction with NADH-cytochrome bg reductase (b(5)R), we prepare d and characterized the cytochrome b(5) mutants in which Glu41, Glu42, Glu63, Asp70, and Glu73 were replaced by Ala, utilizing site-directed mutagenesis and the expression system for cytochrome b(5), in Escheri chia coli. Apparent Km values of the wild type b(5)R for Glu42Ala cyto chrome b(5) and Asp70Ala cytochrome b(5) were approximately three-fold and six-fold higher than that for the wild type cytochrome b(5), resp ectively, while the kcat values for those mutants were not remarkably affected. In contrast, Glu41Ala, Glu63Ala, and Glu73Ala cytochrome b(5 ) showed almost the same kinetic properties as the wild type cytochrom e b(5). Furthermore, kinetic studies on combinations of the cytochrome b(5) and b(5)R mutants suggested the interaction between Glu42 and As p70 of cytochrome b(5) and Lys125 and Lys41 of b(5)R, respectively, in the reaction. (C) 1998 Academic Press.