Conformational States of Cytochrome P450 Oxidoreductase Evaluated by Förster Resonance Energy Transfer Using Ultrafast Transient Absorption Spectroscopy

Kovrigina, Elizaveta A.; Pattengale, Brian; Xia, Chuanwu; Galiakhmetov, Azamat R.; Huang, Jier; Kim, Jung‐Ja P.; Kovrigin, Evgenii L.

doi:10.1021/acs.biochem.6b00623

Cited by 11 publications

(21 citation statements)

References 21 publications

(42 reference statements)

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“…). The mechanism by which CPR transfers electrons to partner proteins is complex and evidence suggests this is achieved through conformational sampling of CPR by relative reorganization of cofactor‐binding protein domains .…”

Section: Introductionmentioning

confidence: 99%

Tripping the light fantastic in membrane redox biology: linking dynamic structures to function in ER electron transfer chains

Hedison

Scrutton

2019

The FEBS Journal

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How the dynamics of proteins assist catalysis is a contemporary issue in enzymology. In particular, this holds true for membrane‐bound enzymes, where multiple structural, spectroscopic and biochemical approaches are needed to build up a comprehensive picture of how dynamics influence enzyme reaction cycles. Of note are the recent studies of cytochrome P450 reductases (CPR)–P450 (CYP) endoplasmic reticulum redox chains, showing the relationship between dynamics and electron flow through flavin and haem redox centres and the impact this has on monooxygenation chemistry. These studies have led to deeper understanding of mechanisms of electron flow, including the timing and control of electron delivery to protein‐bound cofactors needed to facilitate CYP‐catalysed reactions. Individual and multiple component systems have been used to capture biochemical behaviour and these have led to the emergence of more integrated models of catalysis. Crucially, the effects of membrane environment and composition on reaction cycle chemistry have also been probed, including effects on coenzyme binding/release, thermodynamic control of electron transfer, conformational coupling between partner proteins and vectorial versus ‘off pathway’ electron flow. Here, we review these studies and discuss evidence for the emergence of dynamic structural models of electron flow along human microsomal CPR–P450 redox chains.

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Section: Introductionmentioning

confidence: 99%

Tripping the light fantastic in membrane redox biology: linking dynamic structures to function in ER electron transfer chains

Hedison

Scrutton

2019

The FEBS Journal

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“…The sCPR concentration was determined by absorption spectroscopy based on the known absorbance maximum of the oxidized flavin cofactors (e 454 nm = 22 000 cm À1 m À1 ). Protein integrity was verified by SDS-PAGE, western blot (first antibody:H is 6 Ta gm onoclonal antibody (HIS.H8), MA1-21315, Thermo Fisher,France;second antibody:g oat anti-mouse IgG (H + L) secondary antibody,H RP,# 31430, Thermo Fisher;d etection:S IGMAFAST BCIP/NBT,S igma). Protein functionality was verified by determination of the cytochrome c reduction efficiency k obs (see below).…”

Section: Methodsmentioning

confidence: 99%

“…Large structural reorientations occurring below the millisecond timescale have been observed in the case, for example, of the human NADPH-cytochrome P450 reductase (CPR) by avariety of different biophysical methods. [2][3][4][5][6] CPR is ap aradigmatic diflavin reductase, localized att he endoplasmic reticulum, where it transfers electrons from NADPH to av ariety of acceptors including P450 enzymes and playsamajor role in essential physiological processes such as drug, steroid, andf atty acid metabolism. [7] CPR alternates between al ocked, compact state, competent for electron transfer between its two flavin cofactors, bound to distinct flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD)/NADPH domains, and an unlocked, open state, allowing electron transfer to acceptors (Figure 1).…”

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confidence: 99%

Accurate Determination of Human CPR Conformational Equilibrium by smFRET using Dual Orthogonal Non-Canonical Amino Acid Labeling

Quast

Fatemi

Kranendonk

et al. 2018

Preprint

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Conjugation of fluorescent dyes to proteins-a prerequisite for the study of conformational dynamics by single-molecule (sm) FRET-can lead to substantial changes in adye's photophysical properties, ultimately biasingt he determination of inter-dye distances. In particular,c yanine dyes andt heir derivatives, the most commonly used dyes in smFRET experiments,e xhibit such behavior.T oo vercome this, we developed ag eneral strategy to equip proteins site-specifically with FRET pairs through chemoselective reactions with two distinct noncanonical amino acids simultaneouslyi ncorporated throughg enetic code expansion in Escherichia coli. Application of this technique to human NADPH-cytochromeP 450 reductase (CPR) demonstrated the importance of homogenously labeled samples for accurate determination of FRET efficiencies and unveiled the effect of NADP + on the ionic-strength-dependent modulation of the conformational equilibrium of CPR. Thanks to itsg enerality and accuracy, the presented methodology establishes an ew benchmark for deciphering of complex molecular dynamics in single molecules.

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“…The cytosolic cysless POR constructs were prepared as described in our earlier report 12 . Size-exclusion chromatography was carried out using Superose 6 Increase 10/300 column on the Shimadzu HPLC system.…”

Section: Methodsmentioning

confidence: 99%

Detection of domain motion in NADPH-cytochrome P450 oxidoreductase through polarization anisotropy measurements

Kovrigina

Xia

Kim

et al. 2017

Preprint

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ABSTRACT:Conformational transitions between closed and open states in the NADPH-cytochrome P450 oxidoreductase (POR) play a critical role in its electron-transport function. In this study, we determined rotational diffusion coefficients of the EDANS fluorophore attached to the cytosolic POR construct lacking the N-terminal transmembrane region. We identified two dynamic modes, slow and fast, which are interpreted as the rotational diffusion of POR as a whole and the local domain motion, respectively. Timescale of the local rotational diffusion component suggests that it may correspond to the transient opening of the fully oxidized POR structure.NADPH-cytochrome P450 oxidoreductase (POR) transfers electrons from NADPH to multiple cytochromes P450 and other proteins in the endoplasmic reticulum of liver cells and other tissues 1 . Conformational changes in POR molecule are required for productive interaction with cytochromes P450, and the "open" and "closed" structures of POR were identified by X-ray crystallography 2-4 . Because the complex of POR with cytochrome P450 has never been structurally resolved, how these conformational states of POR relate to the steps in the functional cycle-remains debated. For example, oxidized POR has been demonstrated to assume closed conformation (blue ribbon in Figure 1) in solution by NMR spectroscopy and small-angle X-ray scattering 5-7 , while other reports indicated it is in an open form in the oxidized state 8,9 . In this study, we probed conformational states of the oxidized cytosolic fragment of POR, residues 57-678, (in the following text-POR) from viewpoint of the local conformational freedom measured by rotational diffusion constants. We used a single cysteine mutant of POR with otherwise cysteine-less background to introduce a longlived EDANS fluorophore in the FMN domain of POR. Two distinct time constants observed in fluorescence anisotropy decays allowed us to propose that FMN domain might have some degree of local mobility within the oxidized POR structure.

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Conformational States of Cytochrome P450 Oxidoreductase Evaluated by Förster Resonance Energy Transfer Using Ultrafast Transient Absorption Spectroscopy

Cited by 11 publications

References 21 publications

Tripping the light fantastic in membrane redox biology: linking dynamic structures to function in ER electron transfer chains

Tripping the light fantastic in membrane redox biology: linking dynamic structures to function in ER electron transfer chains

Accurate Determination of Human CPR Conformational Equilibrium by smFRET using Dual Orthogonal Non-Canonical Amino Acid Labeling

Detection of domain motion in NADPH-cytochrome P450 oxidoreductase through polarization anisotropy measurements

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