Three-dimensional structure of NADPH–cytochrome P450 reductase: Prototype for FMN- and FAD-containing enzymes

Wang, Ming; Roberts, David L.; Paschke, Rosemary; Shea, Thomas M.; Masters, Bettie Sue Siler; Kim, Jung Ja P.

doi:10.1073/pnas.94.16.8411

Cited by 731 publications

(973 citation statements)

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“…Since further reduction of the flavin is rate-limited by NADP + release (B C), this would account for the slower rates of flavin reduction (and NADPH oxidation) at relatively high coenzyme concentrations. Nicotinamide coenzymes bind to CPR in a bipartite fashion ; electron density for the ribose-nicotinamide portion is poorly defined in the structure of rat CPR, whereas the 2hP-AMP portion is clear [9]. This bipartite recognition in CPR might account for the attenuation of flavin reduction rate at high nicotinamide coenzyme concentrations.…”

Section: Concentration-dependence Of Flavin Reduction and Charge-tranmentioning

confidence: 99%

“…They are dimeric flavohaem enzymes : each monomer comprises a C-terminal diflavin reductase domain and an N-terminal oxygenase domain [4][5][6][7]. The reductase domain contains one mole equivalent of FAD and FMN, binds NADPH and is related structurally and functionally to cytochrome P450 reductase (CPR) [8,9], methionine synthase reductase (' MSR ' [10]) and protein NR1 [11]. The N-terminal oxygenase domain of NOS contains one mole equivalent of haem and possesses binding sites for L-arginine and (6R)-5,6,7,8-tetrahydrobiopterin (BH % [12]).…”

Section: Introductionmentioning

confidence: 99%

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Stopped-flow kinetic studies of electron transfer in the reductase domain of neuronal nitric oxide synthase: re-evaluation of the kinetic mechanism reveals new enzyme intermediates and variation with cytochrome P450 reductase

Knight

Scrutton

2002

Biochemical Journal

138

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The reduction by NADPH of the FAD and FMN redox centres in the isolated flavin reductase domain of calmodulin-bound rat neuronal nitric oxide synthase (nNOS) has been studied by anaerobic stopped-flow spectroscopy using absorption and fluorescence detection. We show by global analysis of time-dependent photodiode array spectra, single wavelength absorption and NADPH fluorescence studies, that at least four resolvable steps are observed in stopped-flow studies with NADPH and that flavin reduction is reversible. The first reductive step represents the rapid formation of an equilibrium between an NADPH-enzyme charge-transfer species and two-electron-reduced enzyme bound to NADP + . The second and third steps represent further reduction of the enzyme flavins and NADP + release. The fourth step is attributed to the slow accumulation of an enzyme species that is inferred not to be relevant catalytically in steady-state reactions. Stopped-flow flavin fluorescence studies indicate the presence of slow kinetic phases, the timescales of which correspond to the slow phase observed in absorption and NADPH fluorescence transients. By analogy with stopped-flow studies of

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Section: Concentration-dependence Of Flavin Reduction and Charge-tranmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Stopped-flow kinetic studies of electron transfer in the reductase domain of neuronal nitric oxide synthase: re-evaluation of the kinetic mechanism reveals new enzyme intermediates and variation with cytochrome P450 reductase

Knight

Scrutton

2002

Biochemical Journal

138

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“…For electron transfer, CPR and its redox partners need to associate with each other. Indeed, the molecular surface of HO-1 surrounding the exposed δ-meso edge of heme is positively charged, and can associate, through electrostatic interaction, with the negatively charged surface of CPR [5]. In contrast to cytochrome P450, HO activity can be reconstructed with the soluble forms of HO-1 and CPR, both of which are lacking their membrane binding regions, C-terminal 22 amino acids and Nterminal 57 amino acids, respectively [6].…”

mentioning

confidence: 99%

“…The crystal structures of rat CPR [5] and rat BVR [14,15] have been also reported. However, less is known about how the HO protein interacts with these two reductases.…”

mentioning

confidence: 99%

Mass spectrometric identification of lysine residues of heme oxygenase-1 that are involved in its interaction with NADPH-cytochrome P450 reductase

Higashimoto

Sugishima

Satō

et al. 2008

Biochemical and Biophysical Research Communications

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The lysine residues of rat heme oxygenase-1 (HO-1) were acetylated by acetic anhydride in the absence and presence of NADPH-cytochrome P450 reductase (CPR) or biliverdin reductase (BVR). Nine acetylated peptides were identified by MALDI-TOF mass spectrometry in the tryptic fragments obtained from HO-1 acetylated without the reductases (referred to as the fully acetylated HO-1). The presence of CPR prevented HO-1 from acetylation of lysine residues, Lys-149 and Lys-153, located in the F-helix. The heme degradation activity of the fully acetylated HO-1 in the NADPH/CPRsupported system was significantly reduced, whereas almost no inactivation was detected in HO-1 in the presence of CPR, which prevented acetylation of Lys-149 and Lys-153. On the other hand, the presence of BVR showed no protective effect on the acetylation of HO-1. The interaction of HO-1 with CPR or BVR is discussed based on the acetylation pattern and on molecular modeling. KeywordsHeme oxygenase; NADPH-cytochrome P450 reductase; biliverdin reductase; MALDI-TOF mass spectrometry; chemical modification; protein-protein interaction Heme oxygenase (HO, EC 1.14.99.3) is a membrane-bound microsomal enzyme that catalyzes the degradation of heme to biliverdin, carbon monoxide (CO), and free iron [1,2]. Biliverdin is subsequently converted to bilirubin by a soluble cytosolic enzyme, biliverdin IXα reductase (BVR, EC 1.2.1.24) [3]. The electrons required for HO catalysis in mammals are provided by NADPH-cytochrome P450 reductase (CPR, EC 1.6.2.4), a 78-kDa membrane-bound * Correspondence should be addressed to: Prof. Masato Noguchi, Department of Medical Biochemistry, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan, Tel.: +81-942-31-7544; fax: +81-942-31-4377. E-mail address: mnoguchi@med.kurume-u.ac.jp (M. Noguchi).Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Recently, using site-specific mutation and docking modeling, we found that Arg-185 and Lys-149 of rat HO-1 are important in both the HO activity and its association with CPR [16]. In our model, the guanidino group of Arg-185 interacts electrostatically with 2′-phosphate of NADPH bound to CPR. On the other hand, Lys-149 is close to the acidic amino acid clusters near the FMN binding site of CPR; mutation of the acidic clusters causes reduction of the electron transfer rates from CPR to cytochrome P450s [17]. Thus, Arg-185 and Lys-149 appear to interact with CPR in such a way as to orient the redox partners for optimal electron transfer from CPR to the heme of HO-1. The model also suggested that the electrons from CPR are ...

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“…NOSred belongs to a large protein family, including NADPHdependent cytochrome P450 reductase and sulfite reductase flavoprotein. An α-helical connecting domain (CD) orients flanking FMN-and FAD-binding domains, to align the two flavins (Wang et al, 1997). Electron transfer proceeds from NADPH, to the FAD, to FMN and then to the oxygenase heme (Adak et al, 1999).…”

Section: Ros and Nitric Oxide Synthasementioning

confidence: 99%

Developing master keys to brain pathology, cancer and aging from the structural biology of proteins controlling reactive oxygen species and DNA repair

2007

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This review is focused on proteins with key roles in pathways controlling either reactive oxygen species or DNA damage responses, both of which are essential for preserving the nervous system. An imbalance of reactive oxygen species or inappropriate DNA damage response likely causes mutational or cytotoxic outcomes, which may lead to cancer and/or aging phenotypes. Moreover, individuals with hereditary disorders in proteins of these cellular pathways have significant neurological abnormalities. Mutations in a superoxide dismutase, which removes oxygen free radicals, may cause the neurodegenerative disease amyotrophic lateral sclerosis. Additionally, DNA repair disorders that affect the brain to varying extents include ataxia-telangiectasia-like disorder, Cockayne syndrome or Werner syndrome. Here, we highlight recent advances gained through structural biochemistry studies on enzymes linked to these disorders and other related enzymes acting within the same cellular pathways. We describe the current understanding of how these vital proteins coordinate chemical steps and integrate cellular signaling and response events. Significantly, these structural studies may provide a set of master keys to developing a unified understanding of the survival mechanisms utilized after insults by reactive oxygen species and genotoxic agents, and also provide a basis for developing an informed intervention in brain tumor and neurodegenerative disease progression.

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Three-dimensional structure of NADPH–cytochrome P450 reductase: Prototype for FMN- and FAD-containing enzymes

Cited by 731 publications

References 46 publications

Stopped-flow kinetic studies of electron transfer in the reductase domain of neuronal nitric oxide synthase: re-evaluation of the kinetic mechanism reveals new enzyme intermediates and variation with cytochrome P450 reductase

Stopped-flow kinetic studies of electron transfer in the reductase domain of neuronal nitric oxide synthase: re-evaluation of the kinetic mechanism reveals new enzyme intermediates and variation with cytochrome P450 reductase

Mass spectrometric identification of lysine residues of heme oxygenase-1 that are involved in its interaction with NADPH-cytochrome P450 reductase

Developing master keys to brain pathology, cancer and aging from the structural biology of proteins controlling reactive oxygen species and DNA repair

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