Kinetic and Structural Characterization of the Interaction between the FMN Binding Domain of Cytochrome P450 Reductase and Cytochrome c

Huang, Rui; Zhang, Meng; Rwere, Freeborn; Waskell, Lucy; Ramamoorthy, Ayyalusamy

doi:10.1074/jbc.m114.582700

Cited by 22 publications

(22 citation statements)

References 56 publications

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“…This suggests that cyt c does not bind to oxidized CPR and/ or that the interactions between these two proteins upon binding do not cause significant structural rearrangement of CPR. A recent NMR study of the interaction between the FMN domain of CPR and cyt c has demonstrated that transient dynamic complexes are formed between the two proteins [39]. Taken together, these data are consistent with a model whereby ET from CPR to cyt c occurs in a diffusion-controlled manner through formation of a collisional complex that does not require domain re-organization in CPR.…”

Section: Discussionsupporting

confidence: 81%

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

2015

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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.

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Section: Discussionsupporting

confidence: 81%

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

2015

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“…Cyt c is a component of the electron transfer chain in mitochondria required for the production of ATP. The heme group of cyt c performs electron transfer with membrane proteins bc1 complex and complex 4 47Fig. 5A.…”

Section: Resultsmentioning

confidence: 99%

Designed multi-stranded heme binding β-sheet peptides in membrane

D’Souza¹,

Mahajan²,

Bhattacharjya³

2016

Chem. Sci.

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“…Only a few reports have appeared of structures of complexes [45, 107, 116] (Figure 6). Some NMR studies have been done to identify parts of proteins involved in complexation [104, 117], but many questions still remain, as discussed under the description of P450 17A1.…”

Section: Future Needs In P450 Structural Workmentioning

confidence: 99%

Recent Structural Insights into Cytochrome P450 Function

Guengerich

Waterman

Egli

2016

Trends in Pharmacological Sciences

279

178

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Cytochrome P450 (P450) enzymes are important in the metabolism of drugs, steroids, fat-soluble vitamins, carcinogens, pesticides, and many other types of chemicals. Their catalytic activities are important issues in areas such as drug-drug interactions and endocrine function. During the past 30 years, structures of P450s have been very helpful in understanding function, particularly the mammalian P450 structures available in the past 15 years. We review recent activity in this area, focusing on the past two years (2014–2015). Structural work with microbial P450s includes studies related to the biosynthesis of natural products and the use of parasitic and fungal P450 structures as targets for drug discovery. Studies on mammalian P450s include the utilization of information about ‘drug-metabolizing’ P450s to improve drug development and also to understand the molecular bases of endocrine dysfunction.

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Kinetic and Structural Characterization of the Interaction between the FMN Binding Domain of Cytochrome P450 Reductase and Cytochrome c

Cited by 22 publications

References 56 publications

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

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

Designed multi-stranded heme binding β-sheet peptides in membrane

Recent Structural Insights into Cytochrome P450 Function

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