Systematic Analysis of Structure-Function Relationships of Conserved Sequence Motifs in the NADH-Binding Lobe of Cytochrome \u3cem\u3eb\u3csub\u3e5\u3c/sub\u3e\u3c/em\u3e Reductase

NADH:Cytochrome b5 Reductase (cb5r) catalyzes the reduction of the ferric iron (Fe3+) atom of the heme cofactor found within cytochrome b5 (cb5) by the reduction of the FAD cofactor of cb5r from reducing equivalents of the physiological electron donor, reduced nicotinamide adenine dinucleotide (NADH). Cb5r is characterized by the presence of 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 correct 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 of residues located within the motifs to allow for the full characterizations. Second, naturally occurring recessive congenital methemoglobinemia (RCM) mutants found in proximity to these highly conserved motifs were analyzed utilizing site-directed mutagenesis. In addition, a canine variant of the cb5r soluble domain was cloned, generated and characterized and compared with the WT rat domain. The canine construct showed a high degree of sequence homology to that of the corresponding human and rat sequences. Characterization of the canine variant indicated that it possessed comparable functional characteristics to the rat variant. Investigation of the pyrophosphate-associating residues, Y112 and Q210, indicated that each played a role in the proper association and anchoring of NADH to the enzyme. The RCM type I mutants, T116S and E212K, caused a moderate decrease in efficiency of the enzyme. The presence of both mutations interact synergistically to generate a more substantially decreased function Analysis of the 180GtGitP185 NADH-binding motif and the preceding residue G179 revealed that these residues are vital in enabling proper NADH association. The residues of this motif were shown to be important in determining nucleotide specificity and properly positioning the NADH and flavin cofactor for efficient electron transfer. RCM variants A178T and A178V were shown to decrease catalytic efficiency or protein stability respectively, leading to disease phenotype. Analysis of the NADH-binding motif 273CGxxxM278 indicated that this motif facilitates electron transfer from substrate to cofactor and is important in release of NAD+ from the enzyme after electron transfer