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.

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.

Orientadores: Anibal Eugênio Vercesi, Helena Coutinho Franco de Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Aplicadas / Made available in DSpace on 2018-08-24T17:38:21Z (GMT). No. of bitstreams: 1 Moraes_Audreyde_M.pdf: 2287376 bytes, checksum: 667ac52246453deec2cd72f05541e70c (MD5) Previous issue date: 2014 / Resumo: Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital quando liberada / Abstract: Note: The complete abstract is available with the full electronic document / Mestrado / Fisiopatologia Médica / Mestra em Ciências