Systematic mutations of highly conserved His49 and carboxyl-terminal of recombinant porcine liver NADH-cytochrome b5 reductase solubilized domain.

The cDNA encoding solubilized porcine liver NADH-cytochrome b5 reductase catalytic domain (Pb5R) was cloned and overexpressed in Escherichia coli. A highly conserved His49 and a C-terminal Phe272 of Pb5R, which are located near the isoalloxazine moiety of the FAD, were systematically modulated by site-directed mutagenesis. Large structural change was not detected on the absorption and circular dichroism spectra of mutant proteins. Drastic changes in enzymatic properties were not observed, but the apparent Km value for soluble form of porcine liver cytochrome b5 (Pb5) was affected by the substitutions of His49 with glutamic acid and with lysine, deletion of C-terminal Phe272, and addition of Gly273. The values of the catalytic constant (kcat) were obviously decreased by the substitution of His49 with glutamic acid or the addition of Gly273. In these two mutants, the rate for reduction of FAD was decreased, and the rate for autoxidation of reduced FAD was increased. These results showed that His49 and C-terminal carboxyl group in Pb5R are not critical for the electron transfer to Pb5, but the electrostatic environmental changes at these positions could affect the recognition of Pb5 and modulate the catalytic function of the enzyme by changing the stability of reduced FAD.