The structure and biochemistry of NADH-dependent cytochrome b(5) reductaseare now consistent

Cytochrome b(5) reductase (cb5r) (EC 1.6.6.2) catalyzes the reduction of tw o molecules of cytochrome b(5) using NADH as the physiological electron don or. The structure of pig cb5r at 2.4 Angstrom resolution was previously rep orted in the literature, but it was inconsistent with the biochemistry; for example, K83 and C245 were both implicated in the mechanism, but were not located at the active site. To address this problem, we have determined the structures of cb5r from rat at 2.0 Angstrom resolution and in a complex wi th NAD(+) at 2.3 Angstrom resolution. We found significant differences thro ughout the rat structure compared to that of pig, including the locations o f the lysine and cysteine residues mentioned above. To test the structural models, we made single amino acid substitutions of this lysine and showed t hat all substitutions produced correctly folded proteins and exhibited norm al flavin behavior. However, the apparent k(cat)(NADH) decreased, and the a pparent Km for NADH increased; the K-m's for cytochrome b(5) were unchanged relative to that of the wild type. The largest effect was for the glutamat e-substituted protein, which was further characterized using a charge trans fer assay and found to be less efficient at NADH utilization than the wild type. These results are consistent with a role for this lysine in stabilizi ng the NADH-bound form of cb5r. We have concluded that the pig structure wa s mistraced in several regions and have reinterpreted mutants in these regi ons that give rise to the hereditary disease methemoglobinemia.