Structural role of serine 127 in the NADH-binding site of human NADH-cytochrome b5 reductase

Serine 127 of human NADH-cytochrome b5 reductase was replaced by proline and alanine by site-directed mutagenesis. The former mutation has been found in the genes of patients with hereditary deficiency of the Enzyme. Both the mutant Enzymes (Ser-127----Pro mutant and Ser-127----Ala mutant) were overproduced in Escherichia coli and purified to homogeneity. The two purified mutant Enzymes showed indistinguishable spectral properties which differed from those of the wild-type Enzyme. The mutant Enzymes showed higher molecular extinction coefficients at 462 nm than that of the wild-type Enzyme. Quenching of FAD fluorescence in these mutant Enzymes was significantly less than that in the wild-type Enzyme. Furthermore, circular dichroism spectra of the mutant Enzymes were different, in both the visible and ultraviolet regions, from that of the wild-type Enzyme. The spectra of the mutant Enzymes in the visible region were restored to almost the same spectrum as the wild type upon reduction with NADH. Ser-127----Pro mutant and Ser-127----Ala mutant showed very low Kcat/Km (NADH) values (5 x 10(7) and 3.5 x 10(7) s-1 M-1, respectively) with cytochrome b5 as an electron acceptor, than that of the wild-type Enzyme (Kcat/Km (NADH) = 179 x 10(7) s-1 M-1), while the Kcat/Km (cytochrome b5) value for each Enzyme was similar. The mutant Enzymes were less thermostable than the wild-type Enzyme. These results indicate that serine 127 plays an important role to maintain the structure of the NADH-binding site in the Enzyme.