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The FMN “140s Loop” of Cytochrome P450 Reductase Controls Electron Transfer to Cytochrome P450

Cytochrome P450 reductase (CYPOR) provides electrons to all human microsomal cytochrome P450s (cyt P450s). The length and sequence of the “140s” FMN binding loop of CYPOR has been shown to be a key determinant of its redox potential and activity with cyt P450s. Shortening the “140s loop” by deleting...

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Autores principales: Rwere, Freeborn, Im, Sangchoul, Waskell, Lucy
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8508823/
https://www.ncbi.nlm.nih.gov/pubmed/34638963
http://dx.doi.org/10.3390/ijms221910625
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author Rwere, Freeborn
Im, Sangchoul
Waskell, Lucy
author_facet Rwere, Freeborn
Im, Sangchoul
Waskell, Lucy
author_sort Rwere, Freeborn
collection PubMed
description Cytochrome P450 reductase (CYPOR) provides electrons to all human microsomal cytochrome P450s (cyt P450s). The length and sequence of the “140s” FMN binding loop of CYPOR has been shown to be a key determinant of its redox potential and activity with cyt P450s. Shortening the “140s loop” by deleting glycine-141(ΔGly141) and by engineering a second mutant that mimics flavo-cytochrome P450 BM3 (ΔGly141/Glu142Asn) resulted in mutants that formed an unstable anionic semiquinone. In an attempt to understand the molecular basis of the inability of these mutants to support activity with cyt P450, we expressed, purified, and determined their ability to reduce ferric P450. Our results showed that the ΔGly141 mutant with a very mobile loop only reduced ~7% of cyt P450 with a rate similar to that of the wild type. On the other hand, the more stable loop in the ΔGly141/Glu142Asn mutant allowed for ~55% of the cyt P450 to be reduced ~60% faster than the wild type. Our results reveal that the poor activity of the ΔGly141 mutant is primarily accounted for by its markedly diminished ability to reduce ferric cyt P450. In contrast, the poor activity of the ΔGly141/Glu142Asn mutant is presumably a consequence of the altered structure and mobility of the “140s loop”.
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spelling pubmed-85088232021-10-13 The FMN “140s Loop” of Cytochrome P450 Reductase Controls Electron Transfer to Cytochrome P450 Rwere, Freeborn Im, Sangchoul Waskell, Lucy Int J Mol Sci Communication Cytochrome P450 reductase (CYPOR) provides electrons to all human microsomal cytochrome P450s (cyt P450s). The length and sequence of the “140s” FMN binding loop of CYPOR has been shown to be a key determinant of its redox potential and activity with cyt P450s. Shortening the “140s loop” by deleting glycine-141(ΔGly141) and by engineering a second mutant that mimics flavo-cytochrome P450 BM3 (ΔGly141/Glu142Asn) resulted in mutants that formed an unstable anionic semiquinone. In an attempt to understand the molecular basis of the inability of these mutants to support activity with cyt P450, we expressed, purified, and determined their ability to reduce ferric P450. Our results showed that the ΔGly141 mutant with a very mobile loop only reduced ~7% of cyt P450 with a rate similar to that of the wild type. On the other hand, the more stable loop in the ΔGly141/Glu142Asn mutant allowed for ~55% of the cyt P450 to be reduced ~60% faster than the wild type. Our results reveal that the poor activity of the ΔGly141 mutant is primarily accounted for by its markedly diminished ability to reduce ferric cyt P450. In contrast, the poor activity of the ΔGly141/Glu142Asn mutant is presumably a consequence of the altered structure and mobility of the “140s loop”. MDPI 2021-09-30 /pmc/articles/PMC8508823/ /pubmed/34638963 http://dx.doi.org/10.3390/ijms221910625 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Communication
Rwere, Freeborn
Im, Sangchoul
Waskell, Lucy
The FMN “140s Loop” of Cytochrome P450 Reductase Controls Electron Transfer to Cytochrome P450
title The FMN “140s Loop” of Cytochrome P450 Reductase Controls Electron Transfer to Cytochrome P450
title_full The FMN “140s Loop” of Cytochrome P450 Reductase Controls Electron Transfer to Cytochrome P450
title_fullStr The FMN “140s Loop” of Cytochrome P450 Reductase Controls Electron Transfer to Cytochrome P450
title_full_unstemmed The FMN “140s Loop” of Cytochrome P450 Reductase Controls Electron Transfer to Cytochrome P450
title_short The FMN “140s Loop” of Cytochrome P450 Reductase Controls Electron Transfer to Cytochrome P450
title_sort fmn “140s loop” of cytochrome p450 reductase controls electron transfer to cytochrome p450
topic Communication
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8508823/
https://www.ncbi.nlm.nih.gov/pubmed/34638963
http://dx.doi.org/10.3390/ijms221910625
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