Structural biology of redox partner interactions in P450cam monooxygenase: A fresh look at an old system

Sevrioukova, Irina F.; Poulos, T.L.

doi:10.1016/j.abb.2010.08.022

Cited by 50 publications

(45 citation statements)

References 66 publications

(106 reference statements)

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“…However, since CPR affinity is measured indirectly through substrate turnover, it is not possible to distinguish the effect of mutagenesis on protein activity from CPR-P450 complex formation by relying exclusively on steady-state measurements. Additionally, the significant biochemical and crystallographic work performed on the P450cam and P450BM3 redox complexes suggests the likelihood that ionic interactions do not exclusively dominate protein-protein contact regions in all P450s(18). The structure between the heme domain of P450BM3 and its FMN domain is the only available P450-redox partner complex structure and reveals that only a single intermolecular ionic bond mediates domain interaction(19).…”

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confidence: 99%

Uncovering the Role of Hydrophobic Residues in Cytochrome P450−Cytochrome P450 Reductase Interactions

et al. 2011

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Cytochrome P450 (CYP or P450)-mediated drug metabolism requires the interaction of P450s with their redox partner, cytochrome P450 reductase (CPR). In this work, we have investigated the role of P450 hydrophobic residues in complex formation with CPR and uncovered novel roles for the surface-exposed residues V267 and L270 of CYP2B4 in mediating CYP2B4-CPR interactions. Using a combination of fluorescence labeling and stopped-flow spectroscopy we have investigated the basis for these interactions. Specifically, in order to study P450-CPR interactions, a single reactive cysteine was introduced in to a genetically engineered variant of CYP2B4 (C79SC152S) at each of 7 strategically selected surface-exposed positions. Each of these cysteine residues was modified by reaction with fluorescein-5-maleimide (FM) and the CYP2B4-FM variants were then used to determine the Kd of the complex by monitoring fluorescence enhancement in the presence of CPR. Furthermore, the intrinsic Km values of the CYP2B4 variants for CPR were measured and stopped-flow spectroscopy was used to determine the intrinsic kinetics and the extent of reduction of the ferric P450 mutants to the ferrous P450-CO adduct by CPR. A comparison of the results from these three approaches reveals that the sites on P450 exhibiting the greatest changes in fluorescence intensity upon binding CPR are associated with the greatest increases in the Km values of the P450 variants for CPR and with the greatest decreases in the rates and extents of reduced P450-CO formation.

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confidence: 99%

Uncovering the Role of Hydrophobic Residues in Cytochrome P450−Cytochrome P450 Reductase Interactions

et al. 2011

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“…P450cam catalyzes the hydroxylation of D-camphor to 5-exohydroxycamphor using two electrons, two protons, and molecular oxygen. Putidaredoxin (Pdx) reductase oxidizes NADH and transfers the electrons to Pdx, which, in turn, shuttles electrons to P450cam in two consecutive steps (3)(4)(5). This complex reaction is controlled by the enzyme to ensure an efficient coupling between dioxygen reduction and substrate hydroxylation and to avoid side reactions.…”

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confidence: 99%

Delicate conformational balance of the redox enzyme cytochrome P450cam

Skinner

Liu

Hiruma

et al. 2015

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

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The energy landscapes of proteins are highly complex and can be influenced by changes in physical and chemical conditions under which the protein is studied. The redox enzyme cytochrome P450cam undergoes a multistep catalytic cycle wherein two electrons are transferred to the heme group and the enzyme visits several conformational states. Using paramagnetic NMR spectroscopy with a lanthanoid tag, we show that the enzyme bound to its redox partner, putidaredoxin, is in a closed state at ambient temperature in solution. This result contrasts with recent crystal structures of the complex, which suggest that the enzyme opens up when bound to its partner. The closed state supports a model of catalysis in which the substrate is locked in the active site pocket and the enzyme acts as an insulator for the reactive intermediates of the reaction.uring catalysis, an enzyme will traverse a conformational energy landscape that can be highly complex and easily changed by external factors, such as temperature, ionic strength, and crystallization. Cytochromes P450 (CYPs) are b-type hemecontaining monooxygenases found throughout the three domains of life. These enzymes catalyze the regiospecific and stereospecific hydroxylation of various aliphatic and aromatic compounds and are involved in a considerable number of metabolic processes, such as steroid biosynthesis and metabolism of xenobiotics in mammals. The CYP superfamily has been extensively studied over the past five decades, and an understanding of its mechanism is crucial for the development of pharmaceutical compounds that do not inhibit these enzymes. Under other circumstances, it is desirable to inhibit a CYP for therapeutic reasons. For example, compounds that inhibit CYP1B1 and CYP1A2 in humans have been proposed as promising anticancer agents (1). The archetypal member of this superfamily is CYP101A1 (more commonly known as P450cam) from Pseudomonas putida, which was the first CYP for which the 3D structure was determined by X-ray crystallography (2). P450cam catalyzes the hydroxylation of D-camphor to 5-exohydroxycamphor using two electrons, two protons, and molecular oxygen. Putidaredoxin (Pdx) reductase oxidizes NADH and transfers the electrons to Pdx, which, in turn, shuttles electrons to P450cam in two consecutive steps (3-5). This complex reaction is controlled by the enzyme to ensure an efficient coupling between dioxygen reduction and substrate hydroxylation and to avoid side reactions. P450cam must go through a cycle of several conformational and electronic states to perform its catalytic task (6).The first X-ray crystal structures of P450cam showed that the enzyme occupied a closed conformation, both in the presence and absence of substrate; therefore, it was unclear how the substrate was able to bind into the active site (2). Moreover, structures of the intermediates of the catalytic cycle were also in a closed conformation (7), leading to the idea that during the catalytic cycle, P450cam opens to bind camphor, closes to perform hydroxylation, and then re...

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“…However, depending on the identities of the surface residues of the individual P450s, non-charged attractions including hydrophobic and intermolecular hydrogen bonding interactions have also been suggested to be dominant factors in stabilizing the binding of the hemoprotein to the flavoprotein (16, 32-34). Thus, by comparing the differential effect of PN on P450 reduction to give the reduced P450-CO spectrum by NADPH/CPR to that by dithionite as well as the differential effect of PN on the BHP- and CPR-dependent catalytic activities, we concluded that nitrotyrosine formation on the proximal side of CYP3A4 impairs its interaction with CPR.…”

Section: Discussionmentioning

confidence: 99%

Reaction of Human Cytochrome P450 3A4 with Peroxynitrite: Nitrotyrosine Formation on the Proximal Side Impairs Its Interaction with NADPH-Cytochrome P450 Reductase

Lin

Kenaan

Zhang

et al. 2012

Chem. Res. Toxicol.

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The reaction of peroxynitrite (PN) with purified human cytochrome P450 3A4 (CYP3A4) resulted in the loss of the reduced-CO difference spectrum, but the absolute absorption spectrum of the heme was not significantly altered. The loss of 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC) O-debenzylation activity of CYP3A4 was concentration dependent with respect to PN and the loss of BFC activity supported by NADPH-cytochrome P450 reductase (CPR) was much greater than that supported by tert-butyl hydroperoxide. Moreover, the PN-treated CYP3A4 exhibited a reduced-CO spectrum when reduced by CPR that was much smaller than when it was reduced by dithionite. These results suggest that modification of CYP3A4 by PN may impair its interaction with CPR leading to the loss of catalytic activity. Tyrosine nitration, as measured by an increase in mass of 45 Da due to the addition of a nitro group, was used as a biomarker for protein modification by PN. PN-treated CYP3A4 was digested by trypsin and endoproteinase Glu C, and nitrotyrosine formation was then determined by using electrospray ionization-liquid chromatography-tandem mass spectrometry. Tyr residues 99, 307, 347, 430 and 432 were found to be nitrated. Using the GRAMM-X docking program, the structure for the CYP3A4-CPR complex shows that Tyr99, Tyr347 and Tyr430 are on the proximal side of CYP3A4 and are in close contact with three acidic residues in the FMN domain of CPR, suggesting that modification of one or more of these tyrosine residues by PN may influence CPR binding or the transfer of electrons to CYP3A4. Mutagenesis of Tyr430 to Phe or Val revealed that both the aromatic and hydroxyl groups of Tyr are required for CPR-dependent catalytic activity and thus support the idea that the proximal side Tyr participates in the 3A4-CPR interaction. In conclusion, modification of tyrosine residues by PN and their subsequent identification can be used to enhance our knowledge of the structure/function relationships of the P450s with respect to the electron transfer steps which are critical for P450 activity.

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Structural biology of redox partner interactions in P450cam monooxygenase: A fresh look at an old system

Cited by 50 publications

References 66 publications

Uncovering the Role of Hydrophobic Residues in Cytochrome P450−Cytochrome P450 Reductase Interactions

Uncovering the Role of Hydrophobic Residues in Cytochrome P450−Cytochrome P450 Reductase Interactions

Delicate conformational balance of the redox enzyme cytochrome P450cam

Reaction of Human Cytochrome P450 3A4 with Peroxynitrite: Nitrotyrosine Formation on the Proximal Side Impairs Its Interaction with NADPH-Cytochrome P450 Reductase

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