Mutants of Cytochrome P450 Reductase Lacking Either Gly-141 or Gly-143 Destabilize Its FMN Semiquinone

Rwere, Freeborn; Xia, Chuanwu; Im, Sangchoul; Haque, Mohammad M.; Stuehr, Dennis J.; Waskell, Lucy; Kim, Jung-Ja P.

doi:10.1074/jbc.m116.724625

Title: Mutants of Cytochrome P450 Reductase Lacking Either Gly-141 or Gly-143 Destabilize Its FMN Semiquinone

Journal Article · Mon May 09 00:00:00 EDT 2016 · Journal of Biological Chemistry

DOI:https://doi.org/10.1074/jbc.m116.724625· OSTI ID:1328799

Rwere, Freeborn ^[1]; Xia, Chuanwu ^[2]; Im, Sangchoul ^[1]; Haque, Mohammad M. ^[3]; Stuehr, Dennis J. ^[3]; Waskell, Lucy ^[1]; Kim, Jung-Ja P. ^[2]

Univ. of Michigan and Veterans Affairs Medical Center, Ann Arbor, MI (United States)
Medical College of Wisconsin, Milwaukee, WI (United States)
Cleveland Clinic, OH (United States)

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. Further, the ΔGly-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 ΔGly-141 and ΔG141/E142N mutants were inactive with cytochrome P450 but fully active in reducing cytochrome c. In the ΔGly-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 ΔG141/E142N was slightly more stable than that of ΔGly-141, consistent with its crystallographically demonstrated more rigid loop. Nonetheless, both ΔGly-141 red semiquinones were less stable than those of the corresponding loop in cytochrome P450 BM3 and the neuronal NOS mutant (ΔGly-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.

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Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: National Institutes of Health (NIH); US Department of Veterans Affairs (VA)

Grant/Contract Number:: GM035533; GM094209; GM097031; GM51491

OSTI ID:: 1328799

Journal Information:: Journal of Biological Chemistry, Vol. 291, Issue 28; ISSN 0021-9258

Publisher:: American Society for Biochemistry and Molecular BiologyCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

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Cited by: 16 works

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59 BASIC BIOLOGICAL SCIENCES
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
cytochrome P450
electron transfer
flavoprotein
mutagenesis
oxidation-reduction (redox)
cytochrome P45 oxidoreductase
diflavin oxidoreductase
microsomal electron transport system
redox potential

Title: Mutants of Cytochrome P450 Reductase Lacking Either Gly-141 or Gly-143 Destabilize Its FMN Semiquinone

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