Coupled Motions Direct Electrons along Human Microsomal P450 Chains

Pudney, Christopher R.; Khara, Basile; Johannissen, Linus O.; Scrutton, Nigel S.

doi:10.1371/journal.pbio.1001222

Cited by 50 publications

(100 citation statements)

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“…Because previous ensemble studies could only report on the averaged protein conformational distributions, and could not provide insight into the conformational dynamics (34,37,38,40), our study represents a significant advance and provides an interesting perspective on how CaM may regulate nNOSr electron transfer reactions through its control of protein conformational behavior.…”

Section: Discussionmentioning

confidence: 99%

“…two to four discreet conformational populations in NOS enzymes and in related flavoproteins, and in some cases, have also estimated the distances between the bound FAD and FMN cofactors in the different species (26,36,37,39,40), and furthermore, have confirmed that CaM shifts the NOS population distribution toward more open conformations (34,36,45). Although valuable, such ensemble-averaged results about conformational states cannot explain how electrons transfer through these enzymes, or how CaM increases the electron flux in NOS, because answering these questions requires a coordinate understanding of the dynamics of the conformational fluctuations.…”

Section: Significancementioning

confidence: 99%

“…In the electron-accepting closed conformation, the FMN domain interacts with the NADPH/FAD domain (FNR domain) to receive electrons, whereas in the electron donating open conformation the FMN domain has moved away to expose the bound FMN cofactor so that it may transfer electrons to a protein acceptor like the NOS oxygenase domain, or to a generic protein acceptor like cytochrome c. In this way, the reductase domain structure cycles between closed and open conformations to deliver electrons, according to a conformational equilibrium that determines the movements and thus the electron flux capacity of the FMN domain (25,28,32,34,35,37). A similar conformational switching mechanism is thought to enable electron transfer through the FMN domain in the related flavoproteins NADPH-cytochrome P450 reductase and methionine synthase reductase (38)(39)(40)(41)(42).NOS enzymes also contain a calmodulin (CaM) binding domain that is located just before the N terminus of the FMN domain (Fig. 1B), and this provides an important layer of regulation (25,27).…”

mentioning

confidence: 99%

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Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

Haque

Stuehr

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS.A lthough proteins adopt structures determined by their amino acid sequences, they are not static objects and fluctuate among ensembles of conformations (1). Transitions between these states can occur on a variety of length scales (Å to nm) and time scales (ps to s) and have been linked to functionally relevant phenomena such as allosteric signaling, enzyme catalysis, and protein-protein interactions (2-4). Indeed, protein conformational fluctuations and dynamics, often associated with static and dynamic inhomogeneity, are thought to play a crucial role in biomolecular functions (5-11). It is difficult to characterize such spatially and temporally inhomogeneous dynamics in bulk solution by an ensemble-averaged measurement, especially in proteins that undergo multiple-conformation transformations. In such cases, single-molecule spectroscopy is a powerful approach to analyze protein conformational states and dynamics under physiological conditions, and can provide a molecular-level perspective on how a protein's structural dynamics link to its functional mechanisms (12-21).A case in point is the nitric oxide synthase (NOS) enzymes (22-24), whose nitric oxide (NO) biosynthesis involves electron transfer reactions that are associated with relatively large-scale movement (tens of Å) of the enzyme domains (Fig. 1A). During catalysis, NADPH-derived electrons first transfer into an FAD domain and an FMN domain in NOS that together comprise the NOS reductase domain (NOSr), and then transfer from the FMN domain to a heme group that is bound in a separate attached "oxygenase" domain, which then enables NO synthesis to begin (22,(25)(26)(27). The electron transfers into and out of the FMN domain are the key steps for catalysis, and they appear to rely on the FMN domain cycling between electron-accepting and electron-donating conformational states (28, 29) (Fig. 1B). In this model, the FMN domain is suggested to be highly...

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Section: Discussionmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

mentioning

confidence: 99%

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Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

Haque

Stuehr

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…In the absence of 2Ј-AMP (or NADP(H)), this region of the polypeptide may be less ordered in A287P/P228L/A503V, thus rendering the two subdomains more easily separable and allowing the polypeptide to be more easily degraded by trypsin. Furthermore, it has been shown that the POR molecule adopts two conformations, open and closed forms (33,34), and it has been suggested that binding NADP(H)/2Ј-AMP favors the closed form (33)(34)(35). In the absence or diminished occupancy of the nucleotide, the POR molecule would more likely be in an open state, which would be more susceptible to trypsinolysis.…”

Section: Structural Characterization Of A287p Por In the Vicinity Of mentioning

confidence: 99%

Instability of the Human Cytochrome P450 Reductase A287P Variant Is the Major Contributor to Its Antley-Bixler Syndrome-like Phenotype

McCammon

Panda

Xia

et al. 2016

Journal of Biological Chemistry

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Human NADPH-cytochrome P450 oxidoreductase (POR) gene mutations are associated with severe skeletal deformities and disordered steroidogenesis. The human POR mutation A287P presents with disordered sexual development and skeletal malformations. Difficult recombinant expression and purification of this POR mutant suggested that the protein was less stable than WT. The activities of cytochrome P450 17A1, 19A1, and 21A2, critical in steroidogenesis, were similar using our purified, full-length, unmodified A287P or WT POR, as were those of several xenobiotic-metabolizing cytochromes P450, indicating that the A287P protein is functionally competent in vitro, despite its functionally deficient phenotypic behavior in vivo. Differential scanning calorimetry and limited trypsinolysis studies revealed a relatively unstable A287P compared with WT protein, leading to the hypothesis that the syndrome observed in vivo results from altered POR protein stability. The crystal structures of the soluble domains of WT and A287P reveal only subtle differences between them, but these differences are consistent with the differential scanning calorimetry results as well as the differential susceptibility of A287P and WT observed with trypsinolysis. The relative in vivo stabilities of WT and A287P proteins were also examined in an osteoblast cell line by treatment with cycloheximide, a protein synthesis inhibitor, showing that the level of A287P protein post-inhibition is lower than WT and suggesting that A287P may be degraded at a higher rate. Current studies demonstrate that, unlike previously described mutations, A287P causes POR deficiency disorder due to conformational instability leading to proteolytic susceptibility in vivo, rather than through an inherent flavin-binding defect.

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“…It is not clear why the ΔN‐CPR should exhibit higher rates, but presumably loss of the MAD makes CPR more freely accessible to interact with cytochrome c , which, unlike the natural CYP redox partner of CPR, is small, not membrane‐bound and freely diffusible in aqueous medium. It is known that CPR adopts multiple conformations and that each will react differently with cytochrome c 54, 55. Truncation of the MAD in the ΔN‐CPR is expected to affect the conformational landscape of CPR.…”

Section: Discussionmentioning

confidence: 99%

Liver microsomal lipid enhances the activity and redox coupling of colocalized cytochrome P450 reductase‐cytochrome P450 3A4 in nanodiscs

et al. 2017

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The haem‐containing mono‐oxygenase cytochrome P450 3A4 (CYP3A4) and its redox partner NADPH‐dependent cytochrome P450 oxidoreductase (CPR) are among the most important enzymes in human liver for metabolizing drugs and xenobiotic compounds. They are membrane‐bound in the endoplasmic reticulum (ER). How ER colocalization and the complex ER phospholipid composition influence enzyme activity are not well understood. CPR and CYP3A4 were incorporated into phospholipid bilayer nanodiscs, both singly, and together in a 1 : 1 ratio, to investigate the significance of membrane insertion and the influence of varying membrane composition on steady‐state reaction kinetics. Reaction kinetics were analysed using a fluorimetric assay with 7‐benzyloxyquinoline as substrate for CYP3A4. Full activity of the mono‐oxygenase system, with electron transfer from NADPH via CPR, could only be reconstituted when CPR and CYP3A4 were colocalized within the same nanodiscs. No activity was observed when CPR and CYP3A4 were each incorporated separately into nanodiscs then mixed together, or when soluble forms of CPR were mixed with preassembled CYP3A4‐nanodiscs. Membrane integration and colocalization are therefore essential for electron transfer. Liver microsomal lipid had an enhancing effect compared with phosphatidylcholine on the activity of CPR alone in nanodiscs, and a greater enhancing effect on the activity of CPR‐CYP3A4 nanodisc complexes, which was not matched by a phospholipid mixture designed to mimic the ER composition. Furthermore, liver lipid enhanced redox coupling within the system. Thus, natural ER lipids possess properties or include components important for enhanced catalysis by CPR‐CYP3A4 nanodisc complexes. Our findings demonstrate the importance of using natural lipid preparations for the detailed analysis of membrane protein activity.

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Coupled Motions Direct Electrons along Human Microsomal P450 Chains

Abstract: Directional electron transfer through biological redox chains can be achieved by coupling reaction chemistry to conformational changes in individual redox enzymes.

Cited by 50 publications

References 36 publications

Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

Instability of the Human Cytochrome P450 Reductase A287P Variant Is the Major Contributor to Its Antley-Bixler Syndrome-like Phenotype

Liver microsomal lipid enhances the activity and redox coupling of colocalized cytochrome P450 reductase‐cytochrome P450 3A4 in nanodiscs

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