Identification and characterization of the novel FAD-binding lobe G75S mutation in cytochrome b5 reductase: An aid to determine recessive congenital methemoglobinemia status in an infant

NADH–cytochrome b5 reductase deficiency results clinically in either type I or type II recessive congenital methemoglobinemia. The more severe type II form is associated with a global deficiency of cytochrome b5 reductase and is characterized by cyanosis with neurological dysfunction. In contrast, the only symptom for type I is cyanosis. We have identified a novel G to A mutation at position 15,635 in the DIAI gene of a 4-month-old baby that results in a glycine to serine substitution at codon 75 in the cytochrome b5 reductase protein. The G75S mutation, located in the FAD-binding lobe of cytochrome b5 reductase, was found in association with the previously described V252M variant. The V252M mutation is present in the NADH-binding domain and associated with both types I and II recessive congenital methemoglobinemia. Since the G75S and V252M mutations represent radical changes in differing regions of cytochrome b5 reductase, generating and characterizing these variants singly and in combination using a rat heterologous expression system would provide insight into the differences between types I and II disease at the molecular level. Although all three variants were found to retain stoichiometric levels of FAD with spectroscopic and thermodynamic properties comparable to those of native cytochrome b5 reductase, all exhibited decreased catalytic efficiency and reduced protein stability reflecting the position of the mutations in the primary structure. The G75S variant retained only 11% of the catalytic efficiency of the wild-type enzyme. Thus, cytochrome b5 reductase deficient patients who are heterozygous for either FAD- or NADH-binding lobe mutations can exhibit the clinically less severe type I phenotype.