4yau | pdb_00004yau
Reduced CYPOR mutant - G141del/E142N
Structural highlights
FunctionNCPR_RAT This enzyme is required for electron transfer from NADP to cytochrome P450 in microsomes. It can also provide electron transfer to heme oxygenase and cytochrome B5. Publication Abstract from PubMedNADPH-Cytochrome P450 oxidoreductase (CYPOR) transfers electrons from NADPH to cytochromes P450 via its FAD and FMN. To understand the biochemical and structural basis of electron transfer from FMN-hydroquinone to its partners, three deletion-mutants in a conserved loop near the FMN were characterized. Comparison of oxidized and reduced wild type and mutant structures reveals that the basis for the air-stability of the neutral-blue semiquinone is protonation of the flavin N5 and strong H-bond formation with the Gly-141 carbonyl. The Gly-143 protein had moderately decreased activity with cytochrome P450 and cytochrome c. It formed a flexible loop, which transiently interacts with the flavin N5, resulting in the generation of both an unstable neutral-blue semiquinone and hydroquinone. The Gly-141 and Gly-141/Glu-142Asn mutants were inactive with cytochrome P450, but fully active with reduced cytochrome c . In the Gly-141 mutants, the backbone amide of Glu/Asn 142 forms an H-bond to the N5 of the oxidized flavin, which leads to formation of an unstable red-anionic semiquinone with a more negative potential than the hydroquinone. The semiquinone of G141/E142N was slightly more stable than that of G141, consistent with its crystallographically demonstrated more rigid loop. Nonetheless, both Gly-141 red semiquinones were less stable than those of the corresponding loop in cytochrome P450 BM3 and the nNOS mutant (DeltaGly-810). Our results indicate that the catalytic activity of CYPOR is a function of the length, sequence, and flexibility of the 140s loop and illustrate the sophisticated variety of biochemical mechanisms employed in fine-tuning its redox properties and function. Mutants of Cytochrome P450 Reductase Lacking Either Gly-141 or Gly-143 Destabilize Its FMN Semiquinone.,Rwere F, Xia C, Im S, Haque MM, Stuehr DJ, Waskell L, Kim JP J Biol Chem. 2016 May 9. pii: jbc.M116.724625. PMID:27189945[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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