Effects of flavin-binding motif amino acid mutations in the NADH-cytochrome b5 reductase catalytic domain on protein stability and catalysis

Porcine NADH-cytochrome b5 reductase catalytic domain (Pb5R) has the RXY(T/S)+(T/S) flavin-binding motif that is highly conserved among the structurally related family of flavoprotein reductases. Mutations were introduced that alter the Arg(63), Tyr(65), and Ser(99) residues within this motif. The mutation of Tyr(65) to either alanine or phenylalanine destabilized the protein, produced an accelerated release of FAD in the presence of 1.5 M guanidine hydrochloride, and decreased the k(cat) values of the enzyme. These results indicate that Tyr(65) contributes to the stability of the protein and is important in the electron transfer from NADH to FAD. The mutation of Ser(99) to either alanine or valine, and of Arg(63) to either alanine or glutamine increased both the K(m) values for NADH (K(m)(NADH)) and the dissociation constant for NAD(+) (K(d)(NAD+)). However, the mutation of Ser(99) to threonine and of Arg(63) to lysine had very little effect on the K(m)(NADH) and K(d)(NAD+) values, and resulted in small changes in the absorption and circular dichroism spectra. These results suggest that the hydroxyl group of Ser(99) and the positive charge of Arg(63) contribute to the maintenance of the properties of FAD and to the effective binding of Pb5R to both NADH and NAD(+). In addition, the mutation of Arg(63) to either alanine or glutamine increased the apparent K(m) values for porcine cytochrome b5 (Pb5), while changing Arg(63) to lysine did not. The positive charge of Arg(63) may regulate the electron transfer through the electrostatic interaction with Pb5. These results substantiate the important role of the flavin-binding motif in Pb5R.