A structure-based model for cytochrome P450cam-putidaredoxin interactions

Pochapsky, Thomas C.; Lyons, Teresa A.; Kazanis, Sophia; Arakaki, T.L.; Ratnaswamy, Gayathri

doi:10.1016/s0300-9084(97)82530-8

Cited by 103 publications

(147 citation statements)

References 36 publications

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“…All of these observations support our previous identification of the CYP101 structural features perturbed by binding of Pdx (22). In particular, the first three turns of the C helix (residues 107-118), previously proposed as a primary interaction site for Pdx (19), are now seen, along with the preceding B' helix, to be among the CYP101 structural features most uniformly affected by Pdx binding. Fig.…”

Section: Chemical Shift Perturbations In the Pdx R -Cyp-s-co Complexsupporting

confidence: 88%

Comparison of the Complexes Formed by Cytochrome P450_c_am with Cytochrome b₅ and Putidaredoxin, Two Effectors of Camphor Hydroxylase Activity

Rui

Pochapsky

2006

Biochemistry

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Structural perturbations in cytochrome P450 cam (CYP101) induced by the soluble fragment of rate cytochrome b 5 , a non-physiological effector of CYP101, were investigated by NMR spectroscopy and compared with the perturbations induced by the physiological reductant and effector, putidaredoxin (Pdx). Chemical shifts of perdeuterated [U-15 The results are in accord with our previous suggestion that the observed perturbations are related to effector activity and support the proposal that the primary role of the effector is to populate the active conformation of CYP101 to prevent uncoupling [Pochapsky et al. Biochemistry 42, 5649-5656 (2003)]. A titratable perturbation is observed at the 1 H resonance of the 8-CH 3 of CYP101-bound camphor upon addition of cytochrome b 5 , a phenomenon also associated with the formation of CYP101·Pdx complex albeit with larger perturbations [Wei et al., J. Am. Chem. Soc. 127, 6974-6 976 (2005)]. The effector activity of the particular rat cytochrome b 5 construct used for NMR studies was confirmed by monitoring the enzymatic turnover to yield 5-exo-hydroxy camphor using gas chromatography/mass spectrometry. Finally, the common features of the perturbations observed in the NMR spectra of the two complexes are discussed and their relevance to effector activity considered.

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Section: Chemical Shift Perturbations In the Pdx R -Cyp-s-co Complexsupporting

confidence: 88%

Comparison of the Complexes Formed by Cytochrome P450_c_am with Cytochrome b₅ and Putidaredoxin, Two Effectors of Camphor Hydroxylase Activity

Rui

Pochapsky

2006

Biochemistry

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“…In a primary interaction region, polar contacts are formed between residues of the NADP domain of AR and the Adx side chains belonging to the interaction domain (7) of the protein. Further polar interactions take place in a secondary interaction region where the core domain (7) of Adx contacts the FAD domain of AR and the covalent cross-link is formed linking Adx Asp 39 with AR Lys 27 . In a third interaction region, the C-terminal polypeptide stretch of Adx dips into a deep cleft between the two globular domains of AR.…”

Section: Resultsmentioning

confidence: 99%

Adrenodoxin Reductase-Adrenodoxin Complex Structure Suggests Electron Transfer Path in Steroid Biosynthesis

Müller¹,

Лапко²,

Bourenkov³

et al. 2001

Journal of Biological Chemistry

155

122

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The steroid hydroxylating system of adrenal cortex mitochondria consists of the membrane-attached NADPH-dependent adrenodoxin reductase (AR), the soluble one-electron transport protein adrenodoxin (Adx), and a membrane-integrated cytochrome P450 of the CYP11 family. In the 2.3-Å resolution crystal structure of the Adx⅐AR complex, 580 In mitochondria of the adrenal cortex, the cytochrome P450 enzymes of the CYP11 family catalyze the side chain cleavage of cholesterol to form pregnenolone (P450scc, 1 CYP11A1) and are involved in the formation of cortisol (P45011␤, CYP11B1) and aldosterone (P450aldo, CYP11B2) (1). The enzymatic activity of the cytochrome P450-dependent steroid hydroxylases is based on their ability to activate molecular oxygen by reductive splitting of dioxygen. This multistep reaction requires the transfer of electrons from the flavoprotein adrenodoxin reductase (AR) via adrenodoxin (Adx) to the terminal cytochromes P450 as electron acceptors in dependence on the specific hydroxylation substrate (1-3). Several models for electron transfer have been discussed, including a shuttle model in which Adx forms consecutive 1:1 complexes (4) with AR and cytochrome P450scc and models requiring the formation of an organized 1:1:1 ternary complex (5) or a 1:2:1 quaternary complex (6) between AR, Adx, and cytochrome P450scc. Common to these models is a complex between AR and Adx during the first steps of electron transfer from the reductase to the cytochrome P450.Recently, the crystal structures of two forms of bovine adrenodoxin (7, 8) and of adrenodoxin reductase (9) were determined. These structures revealed the general topology of the two proteins and the molecular environments of the [2Fe-2S] cluster of Adx and the FAD moiety of AR. Here, we report the 2.3-Å resolution crystal structure of a cross-linked 1:1 complex of full-length Adx and AR. This structure shows the geometry of an electron transfer complex of soluble, freely dissociable proteins from a higher eukaryote for the first time, highlights structural adaptations that accompany the binding of AR to Adx, and permits us to predict electron transport paths in their complex. EXPERIMENTAL PROCEDURESSample Preparation-Recombinant bovine Adx and AR were purified and crystallized as described (10). The synthesized Adx differs from the wild-type protein by the exchange of Ser 1 for glycine and is composed of 128 amino acids, including the N-and C-terminal residues missing in the truncated adrenodoxin, Adx-(4 -108), studied earlier (7). Crosslinking of AR to Adx has also been described (11,12). The native complex is formed at low ionic strength between the two proteins, and the cross-linking was carried out with the water-soluble coupling reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, purchased from Sigma). The mixture of Adx (1.2 mol) and AR (300 nmol) was dialyzed for 18 -20 h against 20 mM potassium phosphate, pH 7.2, followed by addition of an equal volume of fresh 8 mM EDC solution in distilled water and incubation at 4°C in the dark wi...

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“…It should be stressed that the tilt of the heme plane upon the binding of Pdx would be mediated by Arg-112. Because Arg-112 in P450cam, which is crucial for the interaction with Asp-38 in Pdx (12,15,16), is hydrogen-bonded to 6-heme propionate (3,54), it is likely that the binding of Pdx to P450cam shifts the side chain of Arg-112, leading to the positional change of one of the propionate groups and the tilting of the heme plane. The positional change of Arg-112 is evident from the significant perturbation of the N⑀H resonance of Arg-112 upon the Pdx binding (28).…”

Section: Nmr Spectra In the Presencementioning

confidence: 99%

“…With the availability of the P450cam (3) and Pdx (4, 5) structures, many investigators have performed the kinetic (6 -9), mutational (10 -14), and theoretical (15, 16) studies to clarify the molecular mechanism for the ET reaction in the P450cam⅐Pdx system. These studies demonstrated that Pdx interacts with the proximal surface of P450cam through the electrostatic interaction (12,15,17) and suggested that Arg-112 at the putative Pdx binding site forms the ET pathway in the P450cam⅐Pdx complex (12, 16).It was also revealed that Pdx acts as the specific electron donor for the turnover reaction of P450cam (18). Lipscomb et al (18) carried out the mixing of oxy-P450cam with several electron donors, including the non-physiological electron donors as well as the physiological redox partner, Pdx, and examined the formation of the hydroxylation product.…”

mentioning

confidence: 99%

“…The ET from NADH to P450cam is sequentially mediated by a flavin group of putidaredoxin reductase and a [2Fe-2S] center of putidaredoxin (Pdx). With the availability of the P450cam (3) and Pdx (4,5) structures, many investigators have performed the kinetic (6 -9), mutational (10 -14), and theoretical (15,16) studies to clarify the molecular mechanism for the ET reaction in the P450cam⅐Pdx system. These studies demonstrated that Pdx interacts with the proximal surface of P450cam through the electrostatic interaction (12,15,17) and suggested that Arg-112 at the putative Pdx binding site forms the ET pathway in the P450cam⅐Pdx complex (12,16).…”

mentioning

confidence: 99%

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NMR Study on the Structural Changes of Cytochrome P450cam upon the Complex Formation with Putidaredoxin

Tosha

Shiro

Takahashi

et al. 2003

Journal of Biological Chemistry

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We investigated putidaredoxin-induced structural changes in carbonmonoxy P450cam by using NMR spectroscopy. The resonance from the ␤-proton of the axial cysteine was upfield shifted by 0.12 ppm upon the putidaredoxin binding, indicating that the axial cysteine approaches to the heme-iron by about 0.1 Å. The approach of the axial cysteine to the heme-iron would enhance the electronic donation from the axial thiolate to the heme-iron, resulting in the enhanced heterolysis of the dioxygen bond. In addition to the structural perturbation on the axial ligand, the structural changes in the substrate and ligand binding site were observed. The resonances from the 5-exo-and 9-methyl-protons of d-camphor, which were newly identified in this study, were upfield shifted by 1.28 and 0.20 ppm, respectively, implying that d-camphor moves to the heme-iron by 0.15-0.7 Å. Based on the radical rebound mechanism, the approach of d-camphor to the heme-iron could promote the oxygen transfer reaction. On the other hand, the downfield shift of the resonance from the ␥-methyl group of Thr-252 reflects the movement of the side chain away from the heme-iron by ϳ0.25 Å. Because Thr-252 regulates the heterolysis of the dioxygen bond, the positional rearrangement of Thr-252 might assist the scission of the dioxygen bond. We, therefore, conclude that putidaredoxin induces the specific heme environmental changes of P450cam, which would facilitate the oxygen activation and the oxygen transfer reaction.Cytochrome P450 (P450) 1 is a heme-containing monooxygenase, which catalyzes the hydroxylation reactions of a wide variety of natural and unnatural substrates such as steroids, fatty acids, hydrocarbons, and xenobiotics (1). The P450 catalysis reaction requires two electrons from NADH or NADPH through the redox-linked proteins. In microsomal P450s, the electron transfer (ET) from NADPH to P450s is mediated by NADPH-P450 reductase containing FMN and FAD as the redox centers. In contrast, the electron from NADH or NADPH is sequentially transferred to ferredoxin reductase, ferredoxin, and finally P450 in mitochondrial and bacterial systems. The ET reaction between P450 and its redox partner is essential for the subsequent activation of molecular oxygen and the monooxygenation reaction.To understand the mechanism of the ET reaction in P450 and its redox partner system, the ET reaction between cytochrome P450cam (P450cam) from Pseudomonas putida, one of the bacterial P450s, and its redox partner, putidaredoxin (Pdx), has intensively been investigated. P450cam catalyzes the regio-and stereo-specific hydroxylation of its substrate, d-camphor (1, 2) by accepting two electrons from NADH. The ET from NADH to P450cam is sequentially mediated by a flavin group of putidaredoxin reductase and a [2Fe-2S] center of putidaredoxin (Pdx). With the availability of the P450cam (3) and Pdx (4, 5) structures, many investigators have performed the kinetic (6 -9), mutational (10 -14), and theoretical (15, 16) studies to clarify the molecular mechanism for the ET reaction ...

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A structure-based model for cytochrome P450cam-putidaredoxin interactions

Cited by 103 publications

References 36 publications

Comparison of the Complexes Formed by Cytochrome P450_c_am with Cytochrome b₅ and Putidaredoxin, Two Effectors of Camphor Hydroxylase Activity

Comparison of the Complexes Formed by Cytochrome P450_c_am with Cytochrome b₅ and Putidaredoxin, Two Effectors of Camphor Hydroxylase Activity

Adrenodoxin Reductase-Adrenodoxin Complex Structure Suggests Electron Transfer Path in Steroid Biosynthesis

NMR Study on the Structural Changes of Cytochrome P450cam upon the Complex Formation with Putidaredoxin

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A structure-based model for cytochrome P450cam-putidaredoxin interactions

Cited by 103 publications

References 36 publications

Comparison of the Complexes Formed by Cytochrome P450cam with Cytochrome b5 and Putidaredoxin, Two Effectors of Camphor Hydroxylase Activity

Comparison of the Complexes Formed by Cytochrome P450cam with Cytochrome b5 and Putidaredoxin, Two Effectors of Camphor Hydroxylase Activity

Adrenodoxin Reductase-Adrenodoxin Complex Structure Suggests Electron Transfer Path in Steroid Biosynthesis

NMR Study on the Structural Changes of Cytochrome P450cam upon the Complex Formation with Putidaredoxin

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Comparison of the Complexes Formed by Cytochrome P450_c_am with Cytochrome b₅ and Putidaredoxin, Two Effectors of Camphor Hydroxylase Activity

Comparison of the Complexes Formed by Cytochrome P450_c_am with Cytochrome b₅ and Putidaredoxin, Two Effectors of Camphor Hydroxylase Activity