Role of Acidic Residues in the Interaction of NADPH-Cytochrome P450 Oxidoreductase with Cytochrome P450 and Cytochrome c

Shen, Anna L.; Kasper, Charles B.

doi:10.1074/jbc.270.46.27475

Cited by 133 publications

(121 citation statements)

References 47 publications

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“…4). Previous results have provided evidence that the binding of P450 reductase to CYP2B1 (26), CYP2B4 (29,30), and CYP1A2 (28) is facilitated by interactions of positively charged residues on the hemoproteins with negatively charged carboxylic acid residues on P450 reductase (27). The present results with hHO-1 265 are consistent with the findings for cytochrome P450 enzymes.…”

Section: Discussionsupporting

confidence: 82%

The Binding Sites on Human Heme Oxygenase-1 for Cytochrome P450 Reductase and Biliverdin Reductase

Wang

Montellano

2003

Journal of Biological Chemistry

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Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. (2000 Heme oxygenase oxidizes heme, 1 a pro-oxidant and toxic species, to biliverdin, CO, and free iron (1). Each of the three metabolites formed in the reaction is thought to be physiologically important. Biliverdin is converted by biliverdin reductase to bilirubin, which must then be conjugated with glucuronic acid to be excreted (2). However, bilirubin, albeit toxic at high concentrations, has potent antioxidant properties that may contribute to the anti-inflammatory activity of the heme oxygenases (3, 4). Although still controversial, CO appears to behave as a gaseous signaling molecule akin to nitric oxide (5-7). Finally, the iron released from heme is largely recycled and is critical for iron homeostasis, since less than 3% of the body's daily iron needs are met by absorption from the diet (8). As a result of these activities, heme oxygenase has been shown to have, inter alia, important anti-inflammatory (9) and antiatherosclerotic (10) functions.Human heme oxygenase-1 (hHO-1) is a 33-kDa membranebound protein of 288 amino acid residues that binds heme and uses the bound entity as both the prosthetic group and substrate. A soluble form of hHO-1 (hHO-1 265 ) has been obtained by heterologous expression in E. coli of a truncated version of 265 amino acids that lacks the membrane binding domain (11). The crystal structures of hHO-1 truncated to a length of 233 amino acids (12), a rat homologue truncated to a length of 267 residues (13), and an inherently soluble full-length microbial heme oxygenase (14) have been determined. As recently reviewed, mechanistic studies have established that the transformation of heme to biliverdin is a three-step process that consumes three molecules of oxygen and seven electrons (15, 16). In the first step, ferric heme is oxidized to ␣-meso-hydroxyheme in a reaction that consumes one molecule of oxygen and two electrons. The ferric ␣-meso-hydroxyheme is then converted to ferrous verdoheme in a reaction that consumes a further molecule of oxygen and two electrons and releases CO. In the final stage of the reaction sequence, the verdoheme is converted to ferrous biliverdin with the consumption of a further molecule of oxygen and three electrons. The ferrous iron is then released, followed by dissociation of the biliverdin. Single turnover studies have shown that the rate-limiting step is the release of biliverdin, but in the presence of biliverdin reductase this step is accelerated, and one of the electron transfer steps becomes rate-limiting (17).The electrons required for the catalytic turnover of heme oxygenase are provided by NADPH-cytochrome P450 reductase (1,19,20), a 78-kDa membrane-bound flavoprotein that incorpo...

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Section: Discussionsupporting

confidence: 82%

The Binding Sites on Human Heme Oxygenase-1 for Cytochrome P450 Reductase and Biliverdin Reductase

Wang

Montellano

2003

Journal of Biological Chemistry

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“…5C and Table S1). The E660R mutation may also disrupt the interaction of cytochrome c with the FMN subdomain (28). In addition, a decreased efficiency of electron transfer from FAD to FMN was predicted to uncouple NADPH oxidation from NO formation.…”

Section: Resultsmentioning

confidence: 99%

Molecular architecture of mammalian nitric oxide synthases

Campbell

Smith

Potter

et al. 2014

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

122

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NOSs are homodimeric multidomain enzymes responsible for producing NO. In mammals, NO acts as an intercellular messenger in a variety of signaling reactions, as well as a cytotoxin in the innate immune response. Mammals possess three NOS isoformsinducible, endothelial, and neuronal NOS-that are composed of an N-terminal oxidase domain and a C-terminal reductase domain. Calmodulin (CaM) activates NO synthesis by binding to the helical region connecting these two domains. Although crystal structures of isolated domains have been reported, no structure is available for full-length NOS. We used high-throughput single-particle EM to obtain the structures and higher-order domain organization of all three NOS holoenzymes. The structures of inducible, endothelial, and neuronal NOS with and without CaM bound are similar, consisting of a dimerized oxidase domain flanked by two separated reductase domains. NOS isoforms adopt many conformations enabled by three flexible linkers. These conformations represent snapshots of the continuous electron transfer pathway from the reductase domain to the oxidase domain, which reveal that only a single reductase domain participates in electron transfer at a time, and that CaM activates NOS by constraining rotational motions and by directly binding to the oxidase domain. Direct visualization of these large conformational changes induced during electron transfer provides significant insight into the molecular underpinnings governing NO formation.heme | flavin | electron microscopy | conformational heterogeneity N O has emerged as an integral signaling molecule in biology.NOSs are the only enzymes responsible for NO production in mammals. Disruptions in NO signaling are linked to hypertension, erectile dysfunction, neurodegeneration, stroke, and heart disease (1-3). Three NOS isoforms, which vary slightly in size and composition from 260 to 321 kDa for the NOS homodimer, are present in mammals; each is a distinct gene product differing in subcellular localization, tissue distribution, and mode of regulation. The constitutively expressed NOS isoforms are found primarily in endothelial cells [endothelial NOS (eNOS)] and neuronal cells [neuronal NOS (nNOS)], and NO produced by these isoforms initiates diverse signaling processes, including vasodilation, platelet aggregation, myocardial functions, and neurotransmission (4). The activity of the constitutive NOS isoforms is dependent on intracellular Ca 2+ concentrations. The third isoform, inducible NOS (iNOS), is transcriptionally controlled and produces cytotoxic concentrations of NO at sites of infection or inflammation. Unlike eNOS and nNOS, the activity of iNOS is independent of the intracellular Ca 2+ concentration.Mammalian NOS isoforms use a complex assembly of domains and cofactors to convert L-arginine to L-citrulline and NO, via the intermediate N-hydroxy-L-arginine. Mammalian NOS isoforms are active as homodimers and composed of two primary domains: the N-terminal oxidase domain and the C-terminal reductase domain (Fig. 1A). The re...

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“…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.…”

Section: Introductionmentioning

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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Role of Acidic Residues in the Interaction of NADPH-Cytochrome P450 Oxidoreductase with Cytochrome P450 and Cytochrome c

Cited by 133 publications

References 47 publications

The Binding Sites on Human Heme Oxygenase-1 for Cytochrome P450 Reductase and Biliverdin Reductase

The Binding Sites on Human Heme Oxygenase-1 for Cytochrome P450 Reductase and Biliverdin Reductase

Molecular architecture of mammalian nitric oxide synthases

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

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