Structure-Function Studies of Conserved Sequence Motifs of Cytochrome \u3cem\u3eb\u3c/em\u3e\u3csub\u3e5\u3c/sub\u3e Reductase:

NADH:Cytochrome b5 Reductase (cb5r) catalyzes the two electron reduction of the iron center of the heme cofactor found within cytochrome b5 (cb5) utilizing reducing equivalents of the nicotinamide adenine dinucleotide (NADH) coenzyme. Cb5r is characterized by two domains necessary for proper enzyme function: a flavin-binding domain and a pyridine nucleotide-binding domain. Within these domains are highly conserved motifs necessary for the proper binding and orientation of both the NADH coenzyme and the FAD cofactor. To address the importance of these conserved motifs site-directed mutagenesis was utilized to generate a series of variants upon residues found within the motifs to allow for the full characterizations. Second, naturally occurring recessive congenital methemoglobinemia (RCM) mutants that are found within or in close proximity to these highly conserved motifs were analyzed utilizing site-directed mutagenesis. The flavin-binding motif 91RxYSTxxSN97 was characterized by the generation of variants T94H, T94G, T94P, P95I, V96S, and S97N. In addition to this, the naturally occurring double mutant P92H/E255- was fully characterized to establish a role of the P92 residue giving rise to RCM. The role of the 124GRxxST127 was determined by the introduction of a positive charge, charge reversal, and conserved amino acid mutations through site-directed mutagenesis of the G124, K125, and M126 residues. Based on the data presented here, each of the residues of the GRxxST motif are directly involved in maintaining the proper binding and orientation of the cb5r flavin prosthetic group. Analysis of the NADH-binding motif 273CGxxx-M278 was accomplished through the characterization of the type II RCM variant M272- and the type I RCM variant P275L. This demonstrates that the deletion of the M272 residue causes a frame shift leading to the inability of the NADH substrate to bind. The introduction of the P275L variant showed that substrate affinity was diminished, yet turnover was comparable to wild-type cytochrome b5 reductase, indicating that although P275 is required for proper substrate binding it is not essential for overall catalytic function. Finally, analysis of the naturally occurring double mutant G75S/V252M provided the first insight into a methemoglobinemia variant that possessed mutations in both the FAD-binding and NADH-binding domains