STRUCTURE/FUNCTION STUDIES OF THE GxGxxP NADH‐BINDING MOTIF IN CYTOCHROME b5 REDUCTASE

Cytochrome b 5 reductase (c b 5 r), a member of the FNR family of flavoprotein transhydrogenases, catalyzes the NADH‐dependent reduction of cytochrome b 5 . Multiple sequence alignments revealed a conserved “GxGxxP” motif in this family involved in binding reduced pyridine nucleotides. X‐ray crystallography shows that this motif is important in binding the adenosine moiety of NADH. To investigate the roles of these residues, a series of alanine variants were generated, recombinantly expressed in E. coli and purified to homogeneity. Each mutant protein incorporated FAD in a 1:1 cofactor/protein stoichiometry and exhibited absorption and CD spectra that were identical to those of wild‐type c b 5 r, indicating both correct protein folding and similar flavin environments. Oxidation/reduction potentials for the FAD/FADH2 couple ( n =2) were comparable to wild‐type protein ( E ° = −272 mV). All six mutants showed decreased NADH:FR activities, on the order of WT > I183A > P185A > T181A > G182A > G180A > T184A with T184A retaining only 18% of the wild‐type activity. The affinity for NADH was increased in the G180A and T184A variants, with all others showing decreased affinity on the order of G182A > T184A > WT > I183A > P185A > T181A > G180A with the K m NADH for G180A 11‐fold greater than that of the wild type. Both K s H 4 NAD and K s NAD+ values confirmed that the GxGxxP mutants compromised both NADH and NAD+ binding affinities.