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Structure Function Studies of “CGxxxM” NADH Binding Motif of Cytochrome b5 Reductase
Author(s) -
Roma Glenn William,
Melichar Jamie,
Barber Michael
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.634.4
Subject(s) - flavoprotein , mutant , cytochrome b5 , reductase , ferredoxin—nadp(+) reductase , biochemistry , wild type , site directed mutagenesis , alanine , mutant protein , flavin group , nad+ kinase , chemistry , biology , mutagenesis , enzyme , gene , amino acid
Rat hepatic cytochrome b5 reductase (cb5r), a member of the ferredoxin:NADP+ reductase (FNR) family of flavoprotein transhydrogenases, catalyzes the NADH‐dependent reduction of cytochrome b5 (cb5r). Using site directed mutagenesis, the roles of three residues comprising a conserved pyridine nucleotide‐binding motif, “CGxxxM”, present in FNR family members, were probed. Alanine screening suggested residues C273 and G274 play a crucial role in conserving WT functionality. Thus, four more variants, C273S, C273M, G274P, and G274S, were analyzed. Additionally, two insertion mutants, CGPPPaM and CGPPPgM, were generated to further probe the role of M278. All cb5r variants were constructed, produced in E. coli and purified to homogeneity. Compared to the wild‐type (WT) cb5r, all mutants were efficiently expressed, showed the appropriate molecular masses. Absorption and CD spectra were comparable to the wild type rat cb5r diaphorase domain, suggesting similar protein folding and flavin incorporation. In contrast, all mutants exhibited decreased NADH:ferrricyanide reductase (NADH:FR) activities with the greatest impairment shown in the C273 and G274 variants and the two insertion mutants. These results confirm that the conserved C273, G274 and M278 residues play important roles in both modulating the protein's affinity for NADH and regulating its subsequent rate of oxidation.