1ib0
From Proteopedia
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CRYSTAL STRUCTURE OF RAT B5R IN COMPLEX WITH FAD AND NAD
Overview
Cytochrome b5 reductase (cb5r) (EC 1.6.6.2) catalyzes the reduction of two, molecules of cytochrome b5 using NADH as the physiological electron donor., The structure of pig cb5r at 2.4 A resolution was previously reported 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 A resolution and in a complex with, NAD+ at 2.3 A resolution. We found significant differences throughout the, rat structure compared to that of pig, including the locations of the, lysine and cysteine residues mentioned above. To test the structural, models, we made single amino acid substitutions of this lysine and showed, that all substitutions produced correctly folded proteins and exhibited, normal flavin behavior. However, the apparent kcat(NADH) decreased, and, the apparent K(m) for NADH increased; the K(m)'s for cytochrome b5 were, unchanged relative to that of the wild type. The largest effect was for, the glutamate-substituted protein, which was further characterized using a, charge transfer 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 stabilizing the NADH-bound form of cb5r. We have concluded that, the pig structure was mistraced in several regions and have reinterpreted, mutants in these regions that give rise to the hereditary disease, methemoglobinemia.
About this Structure
1IB0 is a Single protein structure of sequence from Rattus norvegicus with FAD and NAD as ligands. Active as Cytochrome-b5 reductase, with EC number 1.6.2.2 Full crystallographic information is available from OCA.
Reference
The structure and biochemistry of NADH-dependent cytochrome b5 reductase are now consistent., Bewley MC, Marohnic CC, Barber MJ, Biochemistry. 2001 Nov 13;40(45):13574-82. PMID:11695905
Page seeded by OCA on Tue Nov 20 17:13:51 2007
