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Real-time analysis of conformational control in electron transfer reactions of human cytochrome P450 reductase with cytochrome c.

ABSTRACT: Protein domain dynamics and electron transfer chemistry are often associated, but real-time analysis of domain motion in enzyme-catalysed reactions and the elucidation of mechanistic schemes that relate these motions to the reaction chemistry are major challenges for biological catalysis research. Previously we suggested that reduction of human cytochrome P450 reductase with the reducing coenzyme NADPH is accompanied by major structural re-orientation of the FMN- and FAD-binding domains through an inferred dynamic cycle of 'open' and 'closed' conformations of the enzyme (PLoS Biol, 2011, e1001222). However, these studies were restricted to stopped-flow/FRET analysis of the reductive half-reaction, and were compromised by fluorescence quenching of the acceptor by the flavin cofactors. Here we have improved the design of the FRET system, by using dye pairs with near-IR fluorescence, and extended studies on human cytochrome P450 reductase to the oxidative half-reaction using a double-mixing stopped-flow assay, thereby analysing in real-time conformational dynamics throughout the complete catalytic cycle. We correlate redox changes accompanying the reaction chemistry with protein dynamic changes observed by FRET, and show that redox chemistry drives a major re-orientation of the protein domains in both the reductive and oxidative half-reactions. Our studies using the tractable (soluble) surrogate electron acceptor cytochrome c provide a framework for analysing mechanisms of electron transfer in the endoplasmic reticulum between cytochrome P450 reductase and cognate P450 enzymes. More generally, our work emphasizes the importance of protein dynamics in intra- and inter-protein electron transfer, and establishes methodology for real-time analysis of structural changes throughout the catalytic cycle of complex redox proteins.

SUBMITTER: Hedison TM

PROVIDER: S-EPMC4973710 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Real-time analysis of conformational control in electron transfer reactions of human cytochrome P450 reductase with cytochrome c.

Hedison Tobias M TM Hay Sam S Scrutton Nigel S NS

The FEBS journal 20150916 22

Protein domain dynamics and electron transfer chemistry are often associated, but real-time analysis of domain motion in enzyme-catalysed reactions and the elucidation of mechanistic schemes that relate these motions to the reaction chemistry are major challenges for biological catalysis research. Previously we suggested that reduction of human cytochrome P450 reductase with the reducing coenzyme NADPH is accompanied by major structural re-orientation of the FMN- and FAD-binding domains through ...[more]

PMID: 26307151

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Project description:Tyrosyl radicals play essential roles in biological proton-coupled electron transfer (PCET) reactions. Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides and is vital in DNA replication in all organisms. Class Ia RNRs consist of ?2 and ?2 homodimeric subunits. In class Ia RNR, such as the E. coli enzyme, an essential tyrosyl radical (Y122O(•))-diferric cofactor is located in ?2. Although Y122O(•) is extremely stable in free ?2, Y122O(•) is highly reactive in the quaternary substrate-?2?2 complex and serves as a radical initiator in catalytic PCET between ?2 and ?2. In this report, we investigate the structural interactions that control the reactivity of Y122O(•) in a model system, isolated E. coli ?2. Y122O(•) was reduced with hydroxyurea (HU), a radical scavenger that quenches the radical in a clinically relevant reaction. In the difference FT-IR spectrum, associated with this PCET reaction, amide I (CO) and amide II (CN/NH) bands were observed. Specific (13)C-labeling of the tyrosine C1 carbon assigned a component of these bands to the Y122-T123 amide bond. Comparison to density functional calculations on a model dipeptide, tyrosine-threonine, and structural modeling demonstrated that PCET is associated with a Y122 rotation and a 7.2 Å translation of the Y122 phenolic oxygen. To test for the functional consequences of this structural change, a proton inventory defined the origin of the large solvent isotope effect (SIE = 16.7 ± 1.0 at 25 °C) on this reaction. These data suggest that the one-electron, HU-mediated reduction of Y122O(•) is associated with two, rate-limiting (full or partial) proton transfer reactions. One is attributable to HU oxidation (SIE = 11.9, net H atom transfer), and the other is attributable to coupled, hydrogen-bonding changes in the Y122O(•)-diferric cofactor (SIE = 1.4). These results illustrate the importance of redox-linked changes to backbone and ring dihedral angles in high potential PCET and provide evidence for rate-limiting, redox-linked hydrogen-bonding interactions between Y122O(•) and the iron cluster.

Dataset Information

Real-time analysis of conformational control in electron transfer reactions of human cytochrome P450 reductase with cytochrome c.

Publications

Real-time analysis of conformational control in electron transfer reactions of human cytochrome P450 reductase with cytochrome c.

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