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Coupled Motions Direct Electrons along Human Microsomal P450 Chains

Protein domain motion is often implicated in biological electron transfer, but the general significance of motion is not clear. Motion has been implicated in the transfer of electrons from human cytochrome P450 reductase (CPR) to all microsomal cytochrome P450s (CYPs). Our hypothesis is that tight c...

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Autores principales: Pudney, Christopher R., Khara, Basile, Johannissen, Linus O., Scrutton, Nigel S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3243717/
https://www.ncbi.nlm.nih.gov/pubmed/22205878
http://dx.doi.org/10.1371/journal.pbio.1001222
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author Pudney, Christopher R.
Khara, Basile
Johannissen, Linus O.
Scrutton, Nigel S.
author_facet Pudney, Christopher R.
Khara, Basile
Johannissen, Linus O.
Scrutton, Nigel S.
author_sort Pudney, Christopher R.
collection PubMed
description Protein domain motion is often implicated in biological electron transfer, but the general significance of motion is not clear. Motion has been implicated in the transfer of electrons from human cytochrome P450 reductase (CPR) to all microsomal cytochrome P450s (CYPs). Our hypothesis is that tight coupling of motion with enzyme chemistry can signal “ready and waiting” states for electron transfer from CPR to downstream CYPs and support vectorial electron transfer across complex redox chains. We developed a novel approach to study the time-dependence of dynamical change during catalysis that reports on the changing conformational states of CPR. FRET was linked to stopped-flow studies of electron transfer in CPR that contains donor-acceptor fluorophores on the enzyme surface. Open and closed states of CPR were correlated with key steps in the catalytic cycle which demonstrated how redox chemistry and NADPH binding drive successive opening and closing of the enzyme. Specifically, we provide evidence that reduction of the flavin moieties in CPR induces CPR opening, whereas ligand binding induces CPR closing. A dynamic reaction cycle was created in which CPR optimizes internal electron transfer between flavin cofactors by adopting closed states and signals “ready and waiting” conformations to partner CYP enzymes by adopting more open states. This complex, temporal control of enzyme motion is used to catalyze directional electron transfer from NADPH→FAD→FMN→heme, thereby facilitating all microsomal P450-catalysed reactions. Motions critical to the broader biological functions of CPR are tightly coupled to enzyme chemistry in the human NADPH-CPR-CYP redox chain. That redox chemistry alone is sufficient to drive functionally necessary, large-scale conformational change is remarkable. Rather than relying on stochastic conformational sampling, our study highlights a need for tight coupling of motion to enzyme chemistry to give vectorial electron transfer along complex redox chains.
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spelling pubmed-32437172011-12-28 Coupled Motions Direct Electrons along Human Microsomal P450 Chains Pudney, Christopher R. Khara, Basile Johannissen, Linus O. Scrutton, Nigel S. PLoS Biol Research Article Protein domain motion is often implicated in biological electron transfer, but the general significance of motion is not clear. Motion has been implicated in the transfer of electrons from human cytochrome P450 reductase (CPR) to all microsomal cytochrome P450s (CYPs). Our hypothesis is that tight coupling of motion with enzyme chemistry can signal “ready and waiting” states for electron transfer from CPR to downstream CYPs and support vectorial electron transfer across complex redox chains. We developed a novel approach to study the time-dependence of dynamical change during catalysis that reports on the changing conformational states of CPR. FRET was linked to stopped-flow studies of electron transfer in CPR that contains donor-acceptor fluorophores on the enzyme surface. Open and closed states of CPR were correlated with key steps in the catalytic cycle which demonstrated how redox chemistry and NADPH binding drive successive opening and closing of the enzyme. Specifically, we provide evidence that reduction of the flavin moieties in CPR induces CPR opening, whereas ligand binding induces CPR closing. A dynamic reaction cycle was created in which CPR optimizes internal electron transfer between flavin cofactors by adopting closed states and signals “ready and waiting” conformations to partner CYP enzymes by adopting more open states. This complex, temporal control of enzyme motion is used to catalyze directional electron transfer from NADPH→FAD→FMN→heme, thereby facilitating all microsomal P450-catalysed reactions. Motions critical to the broader biological functions of CPR are tightly coupled to enzyme chemistry in the human NADPH-CPR-CYP redox chain. That redox chemistry alone is sufficient to drive functionally necessary, large-scale conformational change is remarkable. Rather than relying on stochastic conformational sampling, our study highlights a need for tight coupling of motion to enzyme chemistry to give vectorial electron transfer along complex redox chains. Public Library of Science 2011-12-20 /pmc/articles/PMC3243717/ /pubmed/22205878 http://dx.doi.org/10.1371/journal.pbio.1001222 Text en Pudney et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Pudney, Christopher R.
Khara, Basile
Johannissen, Linus O.
Scrutton, Nigel S.
Coupled Motions Direct Electrons along Human Microsomal P450 Chains
title Coupled Motions Direct Electrons along Human Microsomal P450 Chains
title_full Coupled Motions Direct Electrons along Human Microsomal P450 Chains
title_fullStr Coupled Motions Direct Electrons along Human Microsomal P450 Chains
title_full_unstemmed Coupled Motions Direct Electrons along Human Microsomal P450 Chains
title_short Coupled Motions Direct Electrons along Human Microsomal P450 Chains
title_sort coupled motions direct electrons along human microsomal p450 chains
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3243717/
https://www.ncbi.nlm.nih.gov/pubmed/22205878
http://dx.doi.org/10.1371/journal.pbio.1001222
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