The interactions of neuronal nitric-oxide synthase (nNOS) with calmodulin (CaM) and mutant forms of CaM, including CaM-troponin C chimeras, have been previously reported, but there has been no comparable investigation of CaM interactions with the other constitutively expressed NOS (cNOS), endothelial NOS (eNOS), or the inducible isoform (iNOS). The present study was designed to evaluate the role of the four CaM EF hands in the activation of eNOS and iNOS. To assess the role of CaM regions on aspects of enzymatic function, three distinct activities associated with NOS were measured: NADPH oxidation, cytochrome c reduction, and nitric oxide ( ⅐ NO) generation as assessed by the oxyhemoglobin capture assay. CaM activates the cNOS enzymes by a mechanism other than stimulating electron transfer into the oxygenase domain. Interactions with the reductase moiety are dominant in cNOS activation, and EF hand 1 is critical for activation of both nNOS and eNOS. Although the activation patterns for nNOS and eNOS are clearly related, effects of the chimeras on all the reactions are not equivalent. We propose that cytochrome c reduction is a measure of the release of the FMN domain from the reductase complex. In contrast, cytochrome c reduction by iNOS is readily activated by each of the chimeras examined here and may be constitutive. Each of the chimeras were coexpressed with the human iNOS enzyme in Escherichia coli and subsequently purified. Domains 2 and 3 of CaM contain important elements required for the Ca 2؉ /CaM independence of ⅐ NO production by the iNOS enzyme. The disparity between cytochrome c reduction and ⅐ NO production at low calcium can be attributed to poor association of heme and FMN domains when the bound CaM constructs are depleted of Ca 2؉ . In general cNOSs are much more difficult to activate than iNOS, which can be attributed to their extra sequence elements, which are adjacent to the CaM-binding site and associated with CaM control.
The three mammalian nitric-oxide synthases produce NO from arginine in a reaction requiring 3 electrons per NO, which are supplied to the catalytic center from NADPH through reductase domains incorporating FAD and FMN cofactors. The isoforms share a common reaction mechanism and requirements for reducing equivalents but differ in regulation; the endothelial and neuronal isoforms are controlled by calcium/calmodulin modulation of the electron transfer system, while the inducible isoform binds calmodulin at all physiological Ca 2؉ concentrations and is always on. The thermodynamics of electron transfer through the flavin domains in all three isoforms are basically similar. The major flavin states are FMN, FMNH ⅐ , FMNH 2 , FAD, FADH ⅐ , and FADH 2 . The FMN/FMNH ⅐ couple is high potential (ϳ100 mV) in all three isoforms and is unlikely to be catalytically competent; the other three flavin couples form a nearly isopotential group clustered around ؊250 mV. Reduction of the flavins by the pyridine nucleotide couple at ؊325 mV is thus moderately thermodynamically favorable. The ferri/ferroheme couple in all three isoforms is ϳ؊270 mV in the presence of saturating arginine. Ca 2؉ /calmodulin has no effect on the potentials of any of the couples in endothelial nitric-oxide synthase (eNOS) or neuronal nitric-oxide synthase (nNOS). The pH dependence of the flavin couples suggests the presence of ionizable groups coupled to the flavin redox/ protonation states. Nitric oxide (NO)1 is an important molecular messenger in a variety of signal transduction pathways (1-4). Nitric-oxide synthases (NOS) comprise a family of complex, modular enzymes, including three isoforms expressed in mammals. The endothelial and neuronal isoforms (eNOS and nNOS) are signal generators under the control of calcium/calmodulin (5); a third isoform, iNOS, is induced during immune response (6 -9) and produces much higher levels of NO even at basal levels of calcium.The generation of NO from arginine and oxygen by nitricoxide synthases requires the delivery of three pyridine nucleotide derived electrons to the catalytic site per mol of NO formed. The electron transfer system in endothelial nitric-oxide synthase (eNOS, NOSIII) utilizes one flavin mononucleotide (FMN) and one flavin adenine dinucleotide (FAD) cofactor (10 -15) to deliver electrons derived from reduced nicotinamide dinucleotide phosphate (NADPH) to the site of oxygen chemistry, a protoporphyrin IX-derived heme with cysteinyl thiolate axial ligation (11,12,16,17). The electron transfer pathway is closely related to the NADPH P450 reductase/P450 system, and significant sequence homology exists between the flavoprotein binding domains in the two systems (10).The details of electron transfer differ significantly in FAD/ FMN flavoprotein reductases related by homology and function. The two flavin cofactors have the capacity to accept four electrons. In mammalian P450 reductase, extensive studies have revealed that the system operates between the one and three electron reduced states (18,19). T...
Neuronal nitric-oxide synthase (NOS) and endothelial NOS are constitutive NOS isoforms that are activated by binding calmodulin in response to elevated intracellular calcium. In contrast, the inducible NOS isoform binds calmodulin at low basal levels of calcium in resting cells. Primary sequence comparisons show that each constitutive NOS isozyme contains a polypeptide segment within its reductase domain, which is absent in the inducible NOS enzyme. To study a possible link between the presence of these additional polypeptide segments in constitutive NOS enzymes and their calcium-dependent calmodulin activation, three deletion mutants were created. The putative inhibitory insert was removed from the FMN binding regions of the neuronal NOS holoenzyme and from two truncated neuronal NOS reductase enzymes in which the calmodulin binding region was either included or deleted. All three mutant enzymes showed reduced incorporation of FMN and required reconstitution with exogenous FMN for activity. The combined removal of both the calmodulin binding domain and the putative inhibitory insert did not result in a calmodulin-independent neuronal NOS reductase. Thus, although the putative inhibitory element has an effect on the calcium-dependent calmodulin activation of neuronal NOS, it does not have the properties of the typical autoinhibitory domain found in calmodulin-activated enzymes.The mammalian nitric-oxide synthases (NOSs, 1 EC 1.14.13.39) all produce nitric oxide (NO) and the by-product L-citrulline from the substrates L-arginine, NADPH, and molecular oxygen (1, 2). The constitutively expressed neuronal NOS (nNOS, NOS-1) and endothelial NOS (eNOS, NOS-3) produce NO as an intercellular signal within the nervous and endothelial systems, respectively. These two constitutive NOS (cNOS) isoforms are controlled by the calcium-dependent binding of the regulatory protein calmodulin (CaM). The inducible NOS isoform (iNOS, NOS-2) binds CaM at basal levels of calcium (Ca 2ϩ
Several calmodulin (CaM) mutants were engineered in an effort to identify the functional implications of the oxidation of individual methionines in CaM on the activity of the constitutive isoforms of nitric oxide synthase (NOS). Site-directed mutagenesis was used to substitute the majority of methionines with leucines. Substitution of all nine methionine residues in CaM with leucines had minimal effects on the binding affinity or maximal enzyme activation for either the neuronal (nNOS) or endothelial (eNOS) isoform. Selective substitution permitted determination of the functional consequences of the site-specific oxidation of Met(144) and Met(145) on the regulation of electron transfer within nNOS and eNOS. Site-specific oxidation of Met(144) and Met(145) resulted in changes in the CaM concentration necessary for half-maximal activation of nNOS and eNOS, suggesting that these side chains are involved in stabilizing the productive association between CaM and NOS. However, the site-specific oxidation of Met(144) and Met(145) had essentially no effect on the maximal extent of eNOS activation in the presence of saturating concentrations of CaM. In contrast, the site-specific oxidation of Met(144) (but not Met(145)) resulted in a reduction in the level of nNOS activation that was associated with decreased rates of electron transfer within the reductase domain. Thus, nNOS and eNOS exhibit different functional sensitivities to conditions of oxidative stress that are expected to oxidize CaM. This may underlie some aspects of the observed differences in the sensitivities of proteins in vasculature and neuronal tissues to nitration that are linked to NOS activation and the associated generation of peroxynitrite.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.