4YAO: Reduced CYPOR mutant - G143del

Citation:
Abstract
NADPH-cytochrome P450 oxidoreductase 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 DeltaGly-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 DeltaGly-141 and DeltaG141/E142N mutants were inactive with cytochrome P450 but fully active in reducing cytochrome c In the DeltaGly-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 DeltaG141/E142N was slightly more stable than that of DeltaGly-141, consistent with its crystallographically demonstrated more rigid loop. Nonetheless, both DeltaGly-141 red semiquinones were less stable than those of the corresponding loop in cytochrome P450 BM3 and the neuronal NOS mutant (DeltaGly-810). Our results indicate that the catalytic activity of cytochrome P450 oxidoreductase 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.
PDB ID: 4YAODownload
MMDB ID: 136877
PDB Deposition Date: 2015/2/17
Updated in MMDB: 2017/10
Experimental Method:
x-ray diffraction
Resolution: 2.5  Å
Source Organism:
Similar Structures:
Biological Unit for 4YAO: monomeric; determined by author and by software (PISA)
Molecular Components in 4YAO
Label Count Molecule
Protein (1 molecule)
1
Nadph--cytochrome P450 Reductase(Gene symbol: Por)
Molecule annotation
Chemicals (5 molecules)
1
1
2
1
3
1
4
2
* Click molecule labels to explore molecular sequence information.

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