Rotation of cytochrome P-450. I. Investigations of protein-protein interactions of cytochrome P-450 in phospholipid vesicles and liver microsomes.

Kawato, Suguru; Gut, Josef; Cherry, Richard J.; Winterhalter, Kaspar H.; Richter, Christoph

doi:10.1016/s0021-9258(18)34532-0

Cited by 94 publications

(36 citation statements)

References 39 publications

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“…The authors acknowledge that only one P450 isoform was used in the study and do not exclude that specific P450−P450 interactions are possible in other isoforms. Notwithstanding, we noticed that increasing the concentration of CYP2C9 caused only the formation of nonspecific protein aggregates (data not shown), which was already observed by Kawato et al, 45 and accounted as a possible source of immobile P450 in highly dense protein reconstitution systems.…”

Section: Discussionsupporting

confidence: 69%

“…At the high protein concentrations needed to carry out a FRAP experiment, the labeled CPR ox in solution strongly interferes with the FRAP signal emanating from the portion of proteins associated with the membrane. Second, it is well known that at high concentrations, cytochromes P450s will precipitate in the membrane creating large immobile aggregates. , For the single-molecule tracking experiments, there were on average 8.8 × 10 6 CYP2C9 molecules/cm 2 when a 500 pM solution of protein was used to make the sample and 1.6 × 10 7 CYP2C9 molecules/cm 2 of CYP2C9 when a 900 pM solution of the protein was used. At these densities, no aggregation was observed.…”

Section: Resultsmentioning

confidence: 99%

“…Second, it is well known that at high concentrations, cytochromes P450s will precipitate in the membrane creating large immobile aggregates. 45,46 For the single-molecule tracking experiments, there were on average 8.8 × 10 6 CYP2C9 molecules/cm 2 when a 500 pM solution of protein was used to make the sample and 1.6 × 10 7 CYP2C9 molecules/cm 2 of CYP2C9 when a 900 pM solution of the protein was used. At these densities, no aggregation was observed.…”

Section: Resultsmentioning

confidence: 99%

“…The non-stoichiometric imbalance between CPR and P450 has intrigued the scientific community since the beginning of P450 research . Although the stoichiometry of the electron-transfer complex has been resolved in the early 1980s, , the dynamics of these interactions are mostly unknown. Ensemble studies in reconstituted microsomal systems fail to achieve the spatial and temporal resolution to address these issues.…”

Section: Discussionmentioning

confidence: 99%

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Dissociation Constants of Cytochrome P450 2C9/Cytochrome P450 Reductase Complexes in a Lipid Bilayer Membrane Depend on NADPH: A Single-Protein Tracking Study

2017

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Cytochrome P450-reductase (CPR) is a versatile NADPH-dependent electron donor located in the cytoplasmic side of the endoplasmic reticulum. It is an electron transferase that is able to deliver electrons to a variety of membrane-bound oxidative partners, including the drug-metabolizing enzymes of the cytochrome P450s (P450). CPR is also stoichiometrically limited compared to its oxidative counterparts, and hypotheses have arisen about possible models that can overcome the stoichiometric imbalance, including quaternary organization of P450 and diffusion-limited models. Described here are results from a single-protein tracking study of fluorescently labeled CPR and cytochrome P450 2C9 (CYP2C9) molecules in which stochastic analysis was used to determine the dissociation constants of CPR/CYP2C9 complexes in a lipid bilayer membrane for the first time. Single-protein trajectories demonstrate the transient nature of these CPR-CYP2C9 interactions, and the measured K values are highly dependent on the redox state of CPR. It is shown that CPR/CYP2C9 complexes have a much higher dissociation constant than CPR/CYP2C9 or CPR/CYP2C9 complexes, and a model is presented to account for these results. An Arrhenius analysis of diffusion constants was also carried out, demonstrating that the reduced forms of CPR and CYP2C9 interact differently with the biomimetic ER and may, in addition to protein conformational changes, contribute to the observed NADPH-dependent shift in K. Finally, it is also shown that the CPR/CYP2C9 affinity depends on the nature of the ligand, being higher when a substrate is bound, compared to an inhibitor.

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Section: Discussionsupporting

confidence: 69%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Dissociation Constants of Cytochrome P450 2C9/Cytochrome P450 Reductase Complexes in a Lipid Bilayer Membrane Depend on NADPH: A Single-Protein Tracking Study

2017

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“…Preparation of Proteoliposomes. Reconstitution of P-450 in phosphatidylcholine/phosphatidylserine/phosphatidylethanolamine (PC/PE/PS) (10:5:1) (w/w) vesicles was done by cholate dialysis (Kawato et al, 1982). The buffer was 50 mM potassium phosphate, pH 7.4, 0.1 mM ethylenediaminetetraacetic acid, and 20% glycerol (standard buffer).…”

Section: Methodsmentioning

confidence: 99%

Identification of the membrane anchor of microsomal rat liver cytochrome P-450

Vergères

Winterhalter²,

Richter³

1989

Biochemistry

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Cytochrome P450IIB1 isolated from rat liver microsomes was incorporated into phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine (10:5:1 w/w) liposomes. Trypsinolysis of proteoliposomes and sequencing of the membrane-bound domains revealed that only one peptide, comprising amino acid residues 1-21, spans the membrane. Modification of the N-terminal methionine by membrane-impermeable fluorescein isothiocyanate occurred with the protein in solution but not in proteoliposomes. We conclude that in proteoliposomes cytochrome P-450 spans the membrane only with amino acid residues 1-21, the N-terminal methionine facing the lumen.

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Reconstitution of the Cytb₅–CytP450 Complex in Nanodiscs for Structural Studies using NMR Spectroscopy

et al. 2016

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Cytochrome P450s (P450s) are as uperfamily of enzymes responsible for the catalysis of aw ide range of substrates.D ynamic interactions between full-length membrane-bound P450 and its redox partner cytochrome b 5 (cytb 5 ) have been found to be important for the enzymatic activity of P450. However,t he stability of the circa 70 kDa membranebound complex in model membranes renders high-resolution structural NMR studies particularly difficult. To overcome these challenges,r econstitution of the P450-cytb 5 complex in peptide-based nanodiscs,c ontaining no detergents,h as been demonstrated, whicha re characterized by size exclusion chromatography and NMR spectroscopy. In addition, NMR experiments are used to identify the binding interface of the P450-cytb 5 complex in the nanodisc. This is the first successful demonstration of ap rotein-protein complex in an anodisc using NMR structural studies and should be useful to obtain valuable structural information on membrane-bound protein complexes.

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Rotation of cytochrome P-450. I. Investigations of protein-protein interactions of cytochrome P-450 in phospholipid vesicles and liver microsomes.

Cited by 94 publications

References 39 publications

Dissociation Constants of Cytochrome P450 2C9/Cytochrome P450 Reductase Complexes in a Lipid Bilayer Membrane Depend on NADPH: A Single-Protein Tracking Study

Dissociation Constants of Cytochrome P450 2C9/Cytochrome P450 Reductase Complexes in a Lipid Bilayer Membrane Depend on NADPH: A Single-Protein Tracking Study

Identification of the membrane anchor of microsomal rat liver cytochrome P-450

Reconstitution of the Cytb₅–CytP450 Complex in Nanodiscs for Structural Studies using NMR Spectroscopy

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Rotation of cytochrome P-450. I. Investigations of protein-protein interactions of cytochrome P-450 in phospholipid vesicles and liver microsomes.

Cited by 94 publications

References 39 publications

Dissociation Constants of Cytochrome P450 2C9/Cytochrome P450 Reductase Complexes in a Lipid Bilayer Membrane Depend on NADPH: A Single-Protein Tracking Study

Dissociation Constants of Cytochrome P450 2C9/Cytochrome P450 Reductase Complexes in a Lipid Bilayer Membrane Depend on NADPH: A Single-Protein Tracking Study

Identification of the membrane anchor of microsomal rat liver cytochrome P-450

Reconstitution of the Cytb5–CytP450 Complex in Nanodiscs for Structural Studies using NMR Spectroscopy

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Reconstitution of the Cytb₅–CytP450 Complex in Nanodiscs for Structural Studies using NMR Spectroscopy