EPR spectroscopy: A powerful technique for the structural and functional investigation of metalloproteins

Moré, C.; Belle, Valérie; Asso, Marcel; Fournel, A.; Roger, G.; Guigliarelli, Bruno; Bertrand, Patrick

doi:10.1002/(sici)1520-6343(1999)5:5+<s3::aid-bspy2>3.0.co;2-p

Cited by 35 publications

(30 citation statements)

References 43 publications

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“…The reduced CYP116B1 spectrum has a rhombic shape and g ‐values of g z = 2.06, g y = 1.97 and g x = 1.88 that fall within the typical ranges reported by More et al. [52], clearly identifying the presence of a reduced 2Fe‐2S cluster. There is no EPR signal that could be clearly assigned to a flavin semiquinone in the reduced CYP116B1.…”

Section: Resultssupporting

confidence: 83%

Characterization of Cupriavidus metallidurans CYP116B1 – A thiocarbamate herbicide oxygenating P450–phthalate dioxygenase reductase fusion protein

et al. 2012

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The novel cytochrome P450 ⁄ redox partner fusion enzyme CYP116B1 from Cupriavidus metallidurans was expressed in and purified from Escherichia coli. Isolated CYP116B1 exhibited a characteristic Fe(II)CO complex with Soret maximum at 449 nm. EPR and resonance Raman analyses indicated lowspin, cysteinate-coordinated ferric haem iron at both 10 K and ambient temperature, respectively, for oxidized CYP116B1. The EPR of reduced CYP116B1 demonstrated stoichiometric binding of a 2Fe-2S cluster in the reductase domain. FMN binding in the reductase domain was confirmed by flavin fluorescence studies. Steady-state reduction of cytochrome c and ferricyanide were supported by both NADPH ⁄ NADH, with NADPH used more efficiently (K m[NADPH] = 0.9 ± 0.5 lM and K m[NADH] = 399.1 ± 52.1 lM). Stopped-flow studies of NAD(P)H-dependent electron transfer to the reductase confirmed the preference for NADPH. The reduction potential of the P450 haem iron was -301 ± 7 mV, with retention of haem thiolate ligation in the ferrous enzyme. Redox potentials for the 2Fe-2S and FMN cofactors were more positive than that of the haem iron. Multiangle laser light scattering demonstrated CYP116B1 to be monomeric. Type I (substrate-like) binding of selected unsaturated fatty acids (myristoleic, palmitoleic and arachidonic acids) was shown, but these substrates were not oxidized by CYP116B1. However, CYP116B1 catalysed hydroxylation (on propyl chains) of the herbicides S-ethyl dipropylthiocarbamate (EPTC) and S-propyl dipropylthiocarbamate (vernolate), and the subsequent N-dealkylation of vernolate. CYP116B1 thus has similar thiocarbamate-oxidizing catalytic properties to Rhodoccocus erythropolis CYP116A1, a P450 involved in the oxidative degradation of EPTC.Abbreviations CO, carbon monoxide; CPR, cytochrome P450 reductase; CYP101A1, Pseudomonas putida camphor hydroxylase P450cam or CYP102A1; CYP116B1, cytochrome P450 (116B1) from Cupriavidus metallidurans CH34; ddH 2 O, distilled, deionized water; EPTC, S-ethyl dipropylthiocarbamate; Fe-S, iron-sulfur; HS, high-spin; ImC10, imidazolyl decanoic acid; ImC11, imidazolyl undecanoic acid; ImC12, imidazolyl dodecanoic acid; IPTG, isopropyl thio-b-D-galactoside; LC, liquid chromatography; MALLS, multi-angle laser light scattering; NHE, normal hydrogen electrode; NO, nitric oxide; P450, cytochrome P450 or CYP; P450 BM3, cytochrome P450 BM3 from Bacillus megaterium or CYP102A1; PDO, phthalate dioxygenase; PDOR, Burkholderia cepacia phthalate dioxygenase reductase; SEC, size-exclusion chromatography; vernolate, S-propyl dipropylthiocarbamate.

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Section: Resultssupporting

confidence: 83%

Characterization of Cupriavidus metallidurans CYP116B1 – A thiocarbamate herbicide oxygenating P450–phthalate dioxygenase reductase fusion protein

et al. 2012

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“…Importantly, in both cases, the semiquinone species interact at the Q D site via a H-bond to the His66 residue. The g z parameter of b-type cytochromes with histidine axial ligands is known to be highly sensitive to their environment and in particular to the distortion at the heme center controlled by the orientation of the imidazole rings (37,38). In support of this, we observed a decrease of heterogeneity of the heme b D EPR signature upon delipidation of the enzyme complex.…”

Section: Discussionsupporting

confidence: 75%

Cardiolipin-based respiratory complex activation in bacteria

Arias-Cartin

Grimaldi

Pommier

et al. 2011

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

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Anionic lipids play a variety of key roles in membrane function, including functional and structural effects on respiratory complexes. However, little is known about the molecular basis of these lipid-protein interactions. In this study, NarGHI, an anaerobic respiratory complex of Escherichia coli, has been used to investigate the relations in between membrane-bound proteins with phospholipids. Activity of the NarGHI complex is enhanced by anionic phospholipids both in vivo and in vitro. The anionic cardiolipin tightly associates with the NarGHI complex and is the most effective phospholipid to restore functionality of a nearly inactive detergent-solubilized enzyme complex. A specific cardiolipin-binding site is identified on the basis of the available X-ray diffraction data and of site-directed mutagenesis experiment. One acyl chain of cardiolipin is in close proximity to the heme b D center and is responsible for structural adjustments of b D and of the adjacent quinol substrate binding site. Finally, cardiolipin binding tunes the interaction with the quinol substrate. Together, our results provide a molecular basis for the activation of a bacterial respiratory complex by cardiolipin.bioenergetics | EPR spectroscopy | metalloprotein | molybdenum

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“…The spectroscopic features of the low-spin species are consistent with those expected for a bis-histidine species [21,22], but this assignment is problematic for hIDO in which there is no active-site histidine residue (see above). Upon addition of L-tryptophan to ferric hTDO, the formation of a new lowspin species (g values of 2.63, 2.20, 1.84 [12]) was observed that was also observed for ferric hIDO in the presence of the same substrate (g values of 2.53, 2.19, 1.86 [14]).…”

Section: Binding Of L-and D-tryptophanmentioning

confidence: 60%

Oxidation of L-tryptophan in biology: a comparison between tryptophan 2,3-dioxygenase and indoleamine 2,3-dioxygenase

Rafice

Chauhan

Efimov

et al. 2009

Biochemical Society Transactions

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The family of haem dioxygenases catalyse the initial oxidative cleavage of L-tryptophan to N-formylkynurenine, which is the first, rate-limiting, step in the L-kynurenine pathway. In the present paper, we discuss and compare structure and function across the family of haem dioxygenases by focusing on TDO (tryptophan 2,3-dioxygenase) and IDO (indoleamine 2,3-dioxygenase), including a review of recent structural information for both enzymes. The present paper describes how the recent development of recombinant expression systems has informed our more detailed understanding of the substrate binding, catalytic activity and mechanistic properties of these haem dioxygenases.

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EPR spectroscopy: A powerful technique for the structural and functional investigation of metalloproteins

Cited by 35 publications

References 43 publications

Characterization of Cupriavidus metallidurans CYP116B1 – A thiocarbamate herbicide oxygenating P450–phthalate dioxygenase reductase fusion protein

Characterization of Cupriavidus metallidurans CYP116B1 – A thiocarbamate herbicide oxygenating P450–phthalate dioxygenase reductase fusion protein

Cardiolipin-based respiratory complex activation in bacteria

Oxidation of L-tryptophan in biology: a comparison between tryptophan 2,3-dioxygenase and indoleamine 2,3-dioxygenase

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