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 1.5M 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 elec-tron 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
Km values for NADH (
KmNADH) and the dissociation constant for NAD
+(K
dNAD+). However, the mutation of Ser
99 to threonine and of Arg
63 to lysine had very little effect on the K
mNADH and K
dNAD+ 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
Km 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.
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