Kinetic analysis of electron flux in cytochrome P450 reductases reveals differences in rate-determining steps in plant and mammalian enzymes

Whitelaw, Douglas A.; Tonkin, Rochelle; Meints, Carla E.; Wolthers, Kirsten R.

doi:10.1016/j.abb.2015.09.002

Cited by 13 publications

(24 citation statements)

References 36 publications

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“…4F,G,J, and K). This speculation is consistent with the kinetic assays [29]. In plant CPRs, interflavin electron transfer is slower than NADPH hydride transfer; in hCPR, transfer of a hydride ion from NADPH to FAD is tightly coupled with subsequent FAD to FMN electron transfer.…”

Section: Rate-limiting Stepsupporting

confidence: 87%

“…In the ATR2 structure, the flavin ring of FAD is stacked by W711. Previous kinetics study has revealed that electron transferring from NADPH to FAD is much faster in plant CPR than that in hCPR , which could be a reason that NADPH cannot be ‘captured’ by ATR2 in this study.…”

Section: Discussionmentioning

confidence: 74%

“…In plant CPRs, interflavin electron transfer is slower than NADPH hydride transfer; in hCPR, transfer of a hydride ion from NADPH to FAD is tightly coupled with subsequent FAD to FMN electron transfer. The rate of the transfer of electrons from FMN to cytochrome c in all CPRs is rapid . All these results suggest that the rate‐limiting step for plant CPR is the interflavin electron transfer, and for hCPR/rCPR/yCPR is the electron transfer from NADPH to FAD.…”

Section: Discussionmentioning

confidence: 89%

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Structure of the Arabidopsis thaliana NADPH‐cytochrome P450 reductase 2 (ATR2) provides insight into its function

et al. 2017

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Section: Rate-limiting Stepsupporting

confidence: 87%

Section: Discussionmentioning

confidence: 74%

Section: Discussionmentioning

confidence: 89%

See 1 more Smart Citation

Structure of the Arabidopsis thaliana NADPH‐cytochrome P450 reductase 2 (ATR2) provides insight into its function

et al. 2017

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“…SM fluorescence studies of a nanodisc‐bound CPR cysteine knock‐in variant from Sorghum bicolor have been informative , and extended through the use of SM Förster resonance energy transfer (FRET). Plant CPRs may be different to their mammalian counterparts as they crystallize in ‘open’ states and display different catalytic turnover kinetics . Notwithstanding, SM fluorescence studies have demonstrated that S. bicolor CPR populates at least two states in solution, one with high activity and another with low activity .…”

Section: Binary Solution Structural Modelsmentioning

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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“…POR is a highly dynamic protein oscillating between compact and extended conformations to execute electron transfer to CYPs. This has been verified by ensemble techniques including electron paramagnetic resonance (EPR) 10 , nuclear magnetic resonance (NMR) 6,9 , small-angle X-ray scattering (SAXS) 6 , small-angle neutron scattering (SANS) 5 , fluorescence 24 , and stopped-flow ultraviolet-visible (UV-VIS) spectroscopy 27 , providing insights into POR conformational sampling recently confirmed by smFRET burst analysis 18,28 . Mutations known to control POR specificity and to cause metabolic disorders are often found in the hinge region of POR that controls conformational dynamics 4,12,13 .…”

Section: Direct Observation Of Por Conformational Sampling and Its Rementioning

confidence: 85%

Across kingdom biased CYP-mediated metabolism via small-molecule ligands docking on P450 oxidoreductase

Jensen

Thodberg

Parween

et al. 2020

Preprint

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Metabolic control is mediated by the dynamic assemblies and function of multiple redox enzymes. A key element in these assemblies, the P450 oxidoreductase (POR), donates electrons and selectively activates numerous (>50 in humans and >300 in plants) cytochromes P450 (CYPs) controlling metabolism of drugs, steroids and xenobiotics in humans and natural product biosynthesis in plants. The mechanisms underlying POR-mediated CYP metabolism remain poorly understood and to date no ligand binding has been described to regulate the specificity of POR. Here, using a combination of computational modeling and functional assays, we identified ligands that dock on POR and bias its specificity towards CYP redox partners. Single molecule FRET studies revealed ligand docking to alter POR conformational sampling, which resulted in biased activation of metabolic cascades in whole cell assays. We propose the model of biased metabolism, a mechanism akin to biased signaling of GPCRs, where ligand docking on POR stabilizes different conformational states that are linked to distinct metabolic outcomes. Biased metabolism may allow designing pathway-specific therapeutics or personalized food suppressing undesired, disease related, metabolic pathways.

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Kinetic analysis of electron flux in cytochrome P450 reductases reveals differences in rate-determining steps in plant and mammalian enzymes

Cited by 13 publications

References 36 publications

Structure of the Arabidopsis thaliana NADPH‐cytochrome P450 reductase 2 (ATR2) provides insight into its function

Structure of the Arabidopsis thaliana NADPH‐cytochrome P450 reductase 2 (ATR2) provides insight into its function

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

Across kingdom biased CYP-mediated metabolism via small-molecule ligands docking on P450 oxidoreductase

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