Putidaredoxin-to-Cytochrome P450cam Electron Transfer:  Differences between the Two Reductive Steps Required for Catalysis

Kuznetsov, Vadim Yu.; Poulos, T.L.; Sevrioukova, Irina F.

doi:10.1021/bi0611154

Cited by 60 publications

(116 citation statements)

References 75 publications

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“…We speculate, however, that P450cam in this state also remains closed upon Pdx binding. It has been established that Pdx is specifically required as a donor for the second electron transfer step, somehow signaling the start of the catalytic reaction in the active site (43). This signal is called the effector activity of Pdx.…”

Section: Resultsmentioning

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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Section: Resultsmentioning

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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“…Structural models of PdR-Pdx and Pdx-CYP101A1 complexes have been proposed from the structures of the individual components and spectro-scopic data (52,65,69,70). The structures of cross-linked AdR and Adx, and a related ONFR-ferredoxin (BphA4-BphA3) complex from the biphenyl dioxygenase system of Acidocorax sp.…”

Section: Discussionmentioning

confidence: 99%

“…The Arx surface potential in the vicinity of the [2Fe-2S] cluster is mainly negative, with a neutral area immediately surrounding the cluster binding loop ( Extensive studies suggested that the interaction between CYP101A1 and Pdx is dominated by contacts between Trp 106 (C-terminal residue) and Asp 38 (before the cluster binding loop) on Pdx with residues on CYP101A1 (65,69,70 Fig. S14c) (27).…”

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confidence: 99%

Molecular Characterization of a Class I P450 Electron Transfer System from Novosphingobium aromaticivorans DSM12444

Yang

Bell

Wang

et al. 2010

Journal of Biological Chemistry

135

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Cytochrome P450 (CYP) 4 enzymes constitute a superfamily of heme-containing monooxygenases (1, 2) that participate in a variety of biological processes such as carbon source assimilation, biosynthesis and biodegradation, xenobiotic detoxification, and metabolism of medicines (1, 2). The most common activity of P450 enzymes is the insertion of an oxygen atom from dioxygen into chemically inert carbon-hydrogen bonds, but other reaction types including dealkylation, desaturation, heteroatom oxidation, epoxidation, phenol coupling, and reductive dehalogenation are also known (3-6). The various activities of P450 enzymes are of great interest due to their potential applications in, for example, synthesis of fine chemicals and drug metabolites under mild conditions with high specificity.P450 enzymatic activity requires two electrons that are usually derived from NAD(P)H and delivered to the P450s by electron transfer proteins which are broadly divided into two classes (7,8). Class I systems are diverse, usually consisting of an oxygenase-coupled NAD(P)H-dependent ferredoxin reductase (ONFR) and an iron-sulfur ferredoxin. Such systems are the predominant forms in prokaryotes but are also found in eukaryotic mitochondrial membranes. ONFRs typically contain an FAD cofactor. Ferredoxin cluster types include [2Fe-2S], [3Fe-4S], [4Fe-4S], and combinations of these (7, 9). Non-ferredoxin FMN proteins have also been identified (10). Class II P450 enzymes are most common in eukaryotes and utilize an NADPH-cytochrome P450 reductase (CPR) containing prosthetic groups FAD and FMN (11). Recently other more diverse electron transfer systems for P450 enzymes have been discovered and these have been defined into several new classes (7,8).An important difference between the two main classes is that, whereas a single CPR supports the activity of all 57 human P450s and yeast CPRs support the activity of numerous P450s heterologously expressed in the organism, most class I systems show redox partner specificity. Putidaredoxin (Pdx) is well known to have an effector role in CYP101A1 activity (12, 13). The activity of CYP199A2 from Rhodopseudomonas palustris CGA009 has been reconstituted with palustrisredoxin (Pux), a [2Fe-2S] ferredoxin genomically associated with CYP199A2, and an ONFR, palustrisredoxin reductase (PuR) (14). The high demethylation activity of this system is severely compromised in the hybrid PdR/Pdx/CYP199A2 system (14) due to weak ferredoxin/P450 binding (14,15). Numerous P450 enzymes with potentially interesting and desirable activities are

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“…[7][8][9][10][11] The electron transfer proteins can also have additional roles, for example, putidaredoxin (Pdx) has an effector role in CYP101A1 (P450cam) activity. [12][13][14] Therefore, the identification and characterisation of functional CYP electron transfer chains are imperative in order to optimise the catalytic activity.…”

Section: Introductionmentioning

confidence: 99%

Structural Analysis of CYP101C1 from Novosphingobium aromaticivorans DSM12444

Bell

Yang

et al. 2010

ChemBioChem

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CYP101C1 from Novosphingobium aromaticivorans DSM12444 is a homologue of CYP101D1 and CYP101D2 enzymes from the same bacterium and CYP101A1 from Pseudomonas putida. CYP101C1 does not bind camphor but is capable of binding and hydroxylating ionone derivatives including α- and β-ionone and β-damascone. The activity of CYP101C1 was highest with β-damascone (k(cat)=86 s(-1)) but α-ionone oxidation was the most regioselective (98 % at C3). The crystal structures of hexane-2,5-diol- and β-ionone-bound CYP101C1 have been solved; both have open conformations and the hexanediol-bound form has a clear access channel from the heme to the bulk solvent. The entrance of this channel is blocked when β-ionone binds to the enzyme. The heme moiety of CYP101C1 is in a significantly different environment compared to the other structurally characterised CYP101 enzymes. The likely ferredoxin binding site on the proximal face of CYP101C1 has a different topology but a similar overall positive charge compared to CYP101D1 and CYP101D2, all of which accept electrons from the ArR/Arx class I electron transfer system.

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Putidaredoxin-to-Cytochrome P450cam Electron Transfer: Differences between the Two Reductive Steps Required for Catalysis

Cited by 60 publications

References 75 publications

Delicate conformational balance of the redox enzyme cytochrome P450cam

Delicate conformational balance of the redox enzyme cytochrome P450cam

Molecular Characterization of a Class I P450 Electron Transfer System from Novosphingobium aromaticivorans DSM12444

Structural Analysis of CYP101C1 from Novosphingobium aromaticivorans DSM12444

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