Chimeric Enzymes of Cytochrome P450 Oxidoreductase and Neuronal Nitric-oxide Synthase Reductase Domain Reveal Structural and Functional Differences

Roman, Linda J.; McLain, Jennifer; Masters, Bettie Sue Siler

doi:10.1074/jbc.m212309200

Cited by 31 publications

(34 citation statements)

References 35 publications

(39 reference statements)

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“…Separate roles for FMN and FAD in catalysis by liver microsomal NADPH-cytochrome P450 reductase and NOS (32,51,52) have already been proposed and demonstrated, so the addition of the C termini had the predicted effect. The reduction of potassium ferricyanide, however, has been shown to be mediated through the FAD domain of these enzymes (15). As a result, the addition of the shortest (iNOS) C terminus had no effect on ferricyanide reduction, whereas the addition of either the nNOS or eNOS C terminus produced minimal effects.…”

Section: Resultsmentioning

confidence: 84%

“…The search for differences in the oxygenase domains among the different NOS isoforms led to the elucidation of the heme domain structures of all three (18,(20)(21)(22). The remarkable similarity of these structures has redirected the interest of our laboratory (14,15,(23)(24)(25) and the laboratories of others (26)(27)(28)(29) to the role of the flavoprotein domains of NOS isoforms in their intrinsic and extrinsic regulation. Because the proximal electron donor and acceptor are on the same polypeptide chain in the NOSs, and thus each potential acceptor is linked to its donor, unlike CYPOR and the cytochromes P450, regulation of electron transfer by the reductase domain is reasonable.…”

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confidence: 99%

“…The proposal that this diflavin enzyme, CYPOR, is in turn the precursor of further fusion products that gain new functions is a subject of intensive research (13)(14)(15). The family of nitric oxide synthases (NOS) combines the unique properties of heme-and flavin-containing proteins into one polypeptide chain to achieve production, by very intricately controlled processes, of the gaseous messenger, NO.…”

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confidence: 99%

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Recruitment of governing elements for electron transfer in the nitric oxide synthase family

Jáchymová

Martásek

Panda

et al. 2005

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

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At least three building blocks are responsible for the molecular basis of the modulation of electron transfer in nitric oxide synthase (NOS) isoforms: the calmodulin-binding sequence, the C-terminal extension, and the autoregulatory loop in the reductase domain. We have attempted to impart the control conferred by the C termini of NOS to cytochrome P450 oxidoreductase (CYPOR), which contains none of these regulatory elements. The effect of these C termini on the properties of CYPOR sheds light on the possible evolutionary origin of NOS and addresses the recruitment of new peptides on the development of new functions for CYPOR. The C termini of NOSs modulate flavoprotein-mediated electron transfer to various electron acceptors. The reduction of the artificial electron acceptors cytochrome c, 2,6-dichlorophenolindophenol, and ferricyanide was inhibited by the addition of any of these C termini to CYPOR, whereas the reduction of molecular O 2 was increased. This suggests a shift in the rate-limiting step, indicating that the NOS C termini interrupt electron flux between flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) and͞or the electron acceptors. The modulation of CYPOR by the addition of the NOS C termini is also supported by flavin reoxidation and fluorescence-quenching studies and antibody recognition of the C-terminal extension. These experiments support the origin of the NOS enzymes from modules consisting of a heme domain and CYPOR or ferredoxin-NADP ؉ reductase-and flavodoxin-like subdomains that constitute CYPOR, followed by further recruitment of smaller modulating elements into the flavin-binding domains.flavoprotein ͉ NADPH-cytochrome P450 reductase ͉ protein evolution

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Section: Resultsmentioning

confidence: 84%

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confidence: 99%

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confidence: 99%

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Recruitment of governing elements for electron transfer in the nitric oxide synthase family

Jáchymová

Martásek

Panda

et al. 2005

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

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“…for ferricyanide of 32 M, the K m of nNOS for ferricyanide is in the 1-2 mM range in the absence of CaM but is decreased by about 50% (ϳ750 M) in its presence (38). Concentrations of ferricyanide over 2 mM have very high absorbance, making it difficult to assay reduction at large enough concentrations of ferricyanide, so turnover numbers must be extrapolated from plots of initial rate versus substrate concentration, which also will yield apparent K m values.…”

Section: Resultsmentioning

confidence: 99%

Electron Transfer by Neuronal Nitric-oxide Synthase Is Regulated by Concerted Interaction of Calmodulin and Two Intrinsic Regulatory Elements

Roman

Masters

2006

Journal of Biological Chemistry

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110

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The nitric-oxide synthases (NOSs) are modular, cofactorcontaining enzymes, divided into a heme-containing oxygenase domain and an FMN-and FAD-containing reductase domain. The domains are connected by a calmodulin (CaM)-binding sequence, occupancy of which is required for nitric oxide (NO) production. Two additional CaM-modulated regulatory elements are present in the reductase domains of the constitutive isoforms, the autoregulatory region (AR) and the C-terminal tail region. Deletion of the AR reduces CaM stimulation of electron flow through the reductase domain from 10-fold in wild-type nNOS to 2-fold in the mutant. Deletion of the C terminus yields an enzyme with greatly enhanced reductase activity in the absence of CaM but with activity equivalent to that of wild-type enzyme in its presence. A mutant in which both the AR and C terminus were deleted completely loses CaM modulation through the reductase domain. Thus, transduction of the CaM effect through the reductase domain of nNOS is dependent on these elements. Formation of nitric oxide is, however, still stimulated by CaM in all three mutants. A CaM molecule in which the N-terminal lobe was replaced by the C-terminal lobe (CaM-CC) supported NO synthesis by the deletion mutants but not by wild-type nNOS. We propose a model in which the AR, the C-terminal tail, and CaM interact directly to regulate the conformational state of the reductase domain of nNOS.The nitric-oxide synthases (NOSs) 2 comprise a family of enzymes that catalyze the formation of nitric oxide and L-citrulline from L-arginine through a series of monooxygenation steps using NADPH as the electron donor. There are three different isoforms encoded by different genes, neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). Nitric oxide has been implicated in hemodynamic control, neurotransmission, and the immune response, depending on the cell type in which it is being expressed (for review see Refs. 1-8).All three NOS isoforms are modular, cofactor-containing enzymes. They can be roughly divided into an oxygenase domain, containing heme, tetrahydrobiopterin, and the arginine-binding site, and a reductase domain, containing the flavins FMN and FAD, as well as the NADPH-binding site. These two domains are connected by a calmodulin (CaM)-binding site, which is always occupied under physiological conditions by CaM in iNOS, whereas CaM binding to nNOS and eNOS requires an increase in intracellular calcium.CaM controls NOS activity by regulating the rate of electron flux, by enhancing electron flow through the flavin domain as well as switching on transfer of electrons from the flavin to the heme domain. The flow of electrons is in the form of a hydride ion from NADPH to FAD, from FAD to FMN, and as an electron from FMN to the final acceptor, i.e. the heme domain of the adjacent monomer in the NOS dimer (9 -11) or exogenously added cytochrome c. Recent models (12, 13) propose that bound NADPH locks the enzyme into a state in which the FMN domain is closely associated with the FAD and NADPH bindin...

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“…CaM-stimulated NO synthesis and NADPH oxidation by ⌬14eNOS approach those of CaM-bound nNOS (21); thus the ⌬14-segment can be inferred to negatively control electron transfer through enzymes (36) that contributes to the relatively low catalytic activities of eNOS. Analysis of an x-ray crystal structure of the nNOS FAD domain lacking the Cterminal 22 residues (48) has revealed that the adjacent Cterminal 10 residues are disordered in the crystal structure, suggesting that the C-terminal tail probably lies between the flavins and/or between the FAD and NADPH, modulating the interflavin distance (49). As both orientation and distance between each redox partner influence the electron transfer rate, modification at the C-tail may either change the relative orientation or the distance between the two redox partners, resulting in a greater increase in electron flux.…”

Section: Discussionmentioning

confidence: 99%

Structural Elements Contribute to the Calcium/Calmodulin Dependence on Enzyme Activation in Human Endothelial Nitric-oxide Synthase

Chen

2003

Journal of Biological Chemistry

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Two regions, located at residues 594 -606/614 -645 and residues 1165-1178, are present in the reductase domain of human endothelial nitric-oxide synthase (eNOS) but absent in its counterpart, inducible nitric-oxide synthase (iNOS). We previously demonstrated that removing residues 594 -606/614 -645 resulted in an enzyme (⌬45) containing an intrinsic calmodulin (CaM) purified from an Sf9/baculovirus expression system (Chen, P.-F., and Wu, K.K. (2000) J. Biol. Chem. 275, 13155-13163). Here we have further elucidated the differential requirement of Ca 2؉ / CaM for enzyme activation between eNOS and iNOS by either deletion of residues 1165-1178 (⌬14) or combined deletions of residues 594 -606/614 -645 and 1165-1178 (⌬45/ ⌬14) from eNOS to mimic iNOS. We measured the catalytic rates using purified proteins completely free of CaM. Steady-state analysis indicated that the ⌬45 supported NO synthesis in the absence of CaM at 60% of the rate in its presence, consistent with our prior result that CaMbound ⌬45 retained 60% of its activity in the presence of 10 mM EGTA. Mutant ⌬14 displayed a 1.5-fold reduction of EC 50 for Ca 2؉ /CaM-dependence in L-citrulline formation, and a 2-4-fold increase in the rates of NO synthesis, NADPH oxidation, and cytochrome c reduction relative to the wild type. The basal rates of double mutant ⌬45/⌬14 in NO production, NADPH oxidation, and cytochrome c reduction were 3-fold greater than those of CaM-stimulated wild-type eNOS. Interestingly, all three activities of ⌬45/ ⌬14 were suppressed rather than enhanced by Ca /CaM-dependent catalytic activity of eNOS appears to be conferred mainly by these two structural elements, and the interdomain electron transfer from reductase to oxygenase domain does not require Ca 2؉ /CaM when eNOS lacks these two segments. Nitric-oxide synthase (NOS)1 catalyzes the synthesis of NO through a series of electron transfers from the C-terminal reductase domain, which harbors the FAD and FMN cofactors and the NADPH binding site, to the N-terminal oxygenase domain, which contains the heme catalytic center, the H 4 B cofactor, and the arginine binding sites (1-8). An electron generated by NADPH is transferred in tandem to FAD and FMN and then to the heme, which has been proposed to be facilitated by CaM bound to a site situated between these two domains (9). The NOS family comprises three isoforms that share domain structures, sequence homology, and catalytic properties (10 -12). Despite these similarities, there are considerable differences among the NOS isoforms with respect to their cellular expressions, Ca 2ϩ -dependent CaM activation, and rate of electron transfer. Two isoforms, i.e. nNOS and eNOS, are constitutively expressed NOSs (cNOSs), but their expressed enzymes are latent until CaM binding is elicited by an elevated intracellular calcium level (13). In contrast, iNOS is absent or expressed in low abundance at the resting state, and its expression is induced by cytokines and endotoxins. The expressed iNOS is catalytically active, thought to be due to its hi...

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Chimeric Enzymes of Cytochrome P450 Oxidoreductase and Neuronal Nitric-oxide Synthase Reductase Domain Reveal Structural and Functional Differences

Cited by 31 publications

References 35 publications

Recruitment of governing elements for electron transfer in the nitric oxide synthase family

Recruitment of governing elements for electron transfer in the nitric oxide synthase family

Electron Transfer by Neuronal Nitric-oxide Synthase Is Regulated by Concerted Interaction of Calmodulin and Two Intrinsic Regulatory Elements

Structural Elements Contribute to the Calcium/Calmodulin Dependence on Enzyme Activation in Human Endothelial Nitric-oxide Synthase

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