Structural basis for endothelial nitric oxide synthase binding to calmodulin

The enzyme nitric oxide synthase (NOS) is exquisitely regulated in vivo by the Ca(2+) sensor protein calmodulin (CaM) to control production of NO, a key signaling molecule and cytotoxin. The differential activation of NOS isozymes by CaM has remained enigmatic, despite extensive research. Here, the crystallographic structure of Ca(2+)-loaded CaM bound to a 20 residue peptide comprising the endothelial NOS (eNOS) CaM-binding region establishes their individual conformations and intermolecular interactions, and suggests the basis for isozyme-specific differences. The alpha-helical eNOS peptide binds in an antiparallel orientation to CaM through extensive hydrophobic interactions. Unique NOS interactions occur with: (i). the CaM flexible central linker, explaining its importance in NOS activation; and (ii). the CaM C-terminus, explaining the NOS-specific requirement for a bulky, hydrophobic residue at position 144. This binding mode expands mechanisms for CaM-mediated activation, explains eNOS deactivation by Thr495 phosphorylation, and implicates specific hydrophobic residues in the Ca(2+) independence of inducible NOS.     

Differential binding of calmodulin domains to constitutive and inducible nitric oxide synthase enzymes

Calmodulin (CaM) is a Ca2+ signal transducing protein that binds and activates many cellular enzymes with physiological relevance, including the mammalian nitric oxide synthase (NOS) isozymes: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). The mechanism of CaM binding and activation to the iNOS enzyme is poorly understood in part due to the strength of the bound complex and the difficulty of assessing the role played by regions outside of the CaM-binding domain. To further elucidate these processes, we have developed the methodology to investigate CaM binding to the iNOS holoenzyme and generate CaM mutant proteins selectively labeled with fluorescent dyes at specific residues in the N-terminal lobe, C-terminal lobe, or linker region of the protein. In the present study, an iNOS CaM coexpression system allowed for the investigation of CaM binding to the holoenzyme; three different mutant CaM proteins with cysteine substitutions at residues T34 (N-domain), K75 (central linker), and T110 (C-domain) were fluorescently labeled with acrylodan or Alexa Fluor 546 C5-maleimide. These proteins were used to investigate the differential association of each region of CaM with the three NOS isoforms. We have also N-terminally labeled an iNOS CaM-binding domain peptide with dabsyl chloride in order to perform FRET studies between Alexa-labeled residues in the N- and C-terminal domains of CaM to determine CaM's orientation when associated to iNOS. Our FRET results show that CaM binds to the iNOS CaM-binding domain in an antiparallel orientation. Our steady-state fluorescence and circular dichroism studies show that both the N- and C-terminal EF hand pairs of CaM bind to the CaM-binding domain peptide of iNOS in a Ca2+-independent manner; however, only the C-terminal domain showed large Ca2+-dependent conformational changes when associated with the target sequence. Steady-state fluorescence showed that Alexa-labeled CaM proteins are capable of binding to holo-iNOS coexpressed with nCaM, but this complex is a transient species and can be displaced with the addition of excess CaM. Our results show that CaM does not bind to iNOS in a sequential manner as previously proposed for the nNOS enzyme. This investigation provides additional insight into why iNOS remains active even under basal levels of Ca2+ in the cell.     

FRET conformational analysis of calmodulin binding to nitric oxide synthase peptides and enzymes

Calmodulin (CaM) is a ubiquitous Ca (2+)-sensor protein that binds and activates the nitric oxide synthase (NOS) enzymes. We have used fluorescence resonance energy transfer (FRET) to examine the conformational transitions of CaM induced by its binding to synthetic nitric oxide synthase (NOS) CaM-binding domain peptides and full length heme-free constitutive NOS (cNOS) enzymes over a range of physiologically relevant free Ca (2+) concentrations. We demonstrate for the first time that the domains of CaM collapse when associated with Ca (2+)-independent inducible NOS CaM-binding domain, similar to the previously solved crystal structures of CaM bound to the Ca (2+)-dependent cNOS peptides. We show that the association of CaM is not detectable with the cNOS peptides at low free Ca (2+) concentrations (<40 nM). In contrast, we demonstrate that CaM associates with the cNOS holo-enzymes in the absence of Ca (2+) and that the Ca (2+)-dependent transition occurs at a lower free Ca (2+) concentration with the cNOS holo-enzymes. Our results suggest that other regions outside of the CaM-binding domain in the cNOS enzymes are involved in the recruitment and binding of CaM. We also demonstrate that CaM binds to the cNOS enzymes in a sequential manner with the Ca (2+)-replete C-lobe binding first followed by the Ca (2+)-replete N-lobe. This novel FRET study helps to clarify some of the observed similarities and differences between the Ca (2+)-dependent/independent interaction between CaM and the NOS isozymes.     

The plasmamembrane calmodulin-dependent calcium pump: a major regulator of nitric oxide synthase I

The plasma membrane calcium/calmodulin-dependent calcium ATPase (PMCA) (Shull, G.E., and J. Greeb. 1988. J. Biol. Chem. 263:8646-8657; Verma, A.K., A.G. Filoteo, D.R. Stanford, E.D. Wieben, J.T. Penniston, E.E. Strehler, R. Fischer, R. Heim, G. Vogel, S. Mathews, et al. 1988. J. Biol. Chem. 263:14152-14159; Carafoli, E. 1997. Basic Res. Cardiol. 92:59-61) has been proposed to be a regulator of calcium homeostasis and signal transduction networks of the cell. However, little is known about its precise mechanisms of action. Knock-out of (mainly neuronal) isoform 2 of the enzyme resulted in hearing loss and balance deficits due to severe inner ear defects, affecting formation and maintenance of otoconia (Kozel, P.J., R.A. Friedman, L.C. Erway, E.N. Yamoah, L.H. Liu, T. Riddle, J.J. Duffy, T. Doetschman, M.L. Miller, E.L. Cardell, and G.E. Shull. 1998. J. Biol. Chem. 273:18693-18696). Here we demonstrate that PMCA 4b is a negative regulator of nitric oxide synthase I (NOS-I, nNOS) in HEK293 embryonic kidney and neuro-2a neuroblastoma cell models. Binding of PMCA 4b to NOS-I was mediated by interaction of the COOH-terminal amino acids of PMCA 4b and the PDZ domain of NOS-I (PDZ: PSD 95/Dlg/ZO-1 protein domain). Increasing expression of wild-type PMCA 4b (but not PMCA mutants unable to bind PDZ domains or devoid of Ca2+-transporting activity) dramatically downregulated NO synthesis from wild-type NOS-I. A NOS-I mutant lacking the PDZ domain was not regulated by PMCA, demonstrating the specific nature of the PMCA-NOS-I interaction. Elucidation of PMCA as an interaction partner and major regulator of NOS-I provides evidence for a new dimension of integration between calcium and NO signaling pathways.     

Oxygen dependence of mitochondrial nitric oxide synthase activity

The effect of O(2) concentration on mitochondrial nitric oxide synthase (mtNOS) activity and on O(2)(-) production was determined in rat liver, brain, and kidney submitochondrial membranes. The K(mO(2)) for mtNOS were 40, 73, and 37 microM O(2) and the V(max) were 0.51, 0.49, and 0.42 nmol NO/minmg protein for liver, brain, and kidney mitochondria, respectively. The rates of O(2)(-) production, 0.5-12.8 nmol O(2)(-)/minmg protein, depended on O(2) concentration up to 1.1mM O(2). Intramitochondrial NO, O(2)(-), and ONOO(-) steady-state concentrations were calculated for the physiological level of 20 microM O(2); they were 20-39 nM NO, 0.17-0.33 pM O(2)(-), and 0.6-2.2 nM ONOO(-) for the three organs. These levels establish O(2)/NO ratios of 513-1000 that correspond to physiological inhibitions of cytochrome oxidase by intramitochondrial NO of 16-25%. The production of NO by mtNOS appears as a regulatory process that modulates mitochondrial oxygen uptake and cellular energy production.     

Developmental changes in nitric oxide synthase isoform expression and nitric oxide production in fetal baboon lung

Nitric oxide (NO), produced by NO synthase (NOS), plays a critical role in multiple processes in the lung during the perinatal period. To better understand the regulation of pulmonary NO production in the developing primate, we determined the cell specificity and developmental changes in NOS isoform expression and action in the lungs of third-trimester fetal baboons. Immunohistochemistry in lungs obtained at 175 days (d) of gestation (term = 185 d) revealed that all three NOS isoforms, neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), are primarily expressed in proximal airway epithelium. In proximal lung, there was a marked increase in total NOS enzymatic activity from 125 to 140 d gestation due to elevations in nNOS and eNOS, whereas iNOS expression and activity were minimal. Total NOS activity was constant from 140 to 175 d gestation, and during the latter stage (160-175 d gestation), a dramatic fall in nNOS and eNOS was replaced by a rise in iNOS. Studies done within 1 h of delivery at 125 or 140 d gestation revealed that the principal increase in NOS during the third trimester is associated with an elevation in exhaled NO levels, a decline in expiratory resistance, and greater pulmonary compliance. Thus, there are developmental increases in pulmonary NOS expression and NO production during the early third trimester in the primate that may enhance airway and parenchymal function in the immediate postnatal period.     

Calcium-binding sites of calmodulin and electron transfer by inducible nitric oxide synthase

Like that of the neuronal nitric oxide synthase (nNOS), the binding of Ca(2+)-bound calmodulin (CaM) also regulates the activity of the inducible isoform (iNOS). However, the role of each of the four Ca(2+)-binding sites of CaM in the activity of iNOS is unclear. Using a series of single-point mutants of Drosophila melanogaster CaM, the effect that mutating each of the Ca(2+)-binding sites plays in the transfer of electrons within iNOS has been examined. The same Glu (E) to Gln (Q) mutant series of CaM used previously [Stevens-Truss, R., Beckingham, K., and Marletta, M. A. (1997) Biochemistry 36, 12337-12345] to study the role of the Ca(2+)-binding sites in the activity of nNOS was used for these studies. We demonstrate here that activity of iNOS is dependent on Ca(2+) being bound to sites II (B2Q) and III (B3Q) of CaM. Nitric oxide ((*)NO) producing activity (as measured using the hemoglobin assay) of iNOS bound to the B2Q and B3Q CaMs was found to be 41 and 43% of the wild-type activity, respectively. The site I (B1Q) and site IV (B4Q) CaM mutants only minimally affected (*)NO production (95 and 90% of wild-type activity, respectively). These results suggest that NOS isoforms, although all possessing a prototypical CaM binding sequence and requiring CaM for activity, interact with CaM differently. Moreover, iNOS activation by CaM, like nNOS, is not dependent on Ca(2+) being bound to all four Ca(2+)-binding sites, but has specific and distinct requirements. This novel information, in addition to helping us understand NOS, should aid in our understanding of CaM target activation.     

一氧化氮合酶的遗传、生理研究进展

一氧化氮具有广泛的生理功能,哺乳动物体内的NO是由NO合酶(NOS)氧化L-精氨酸而合成的,合成后的NO迅速跨膜扩散释放,NO合成失调能介导多种疾病。催化NO生物合成的NOS有三种亚型:神经元型NOS(nNOS)、内皮型NOS(eNOS)和诱导型NOS(iNOS),目前,人的三型NOS已纯化并且已分子克隆成功,对一氧化氮合酶的遗传研究确认了NOS家族的基因结构和染色体定位。     

Physiologic pH changes modulate calcium ion dependence of brain nitric oxide synthase in Carassius auratus

Species of the fish genus Carassius survive prolonged anoxia. Nitric oxide (NO) regulates cerebral blood flow in these fish during normoxic conditions whereas adenosine is the main vasoregulating molecule during anoxia. We investigated the calcium ion dependence of Carassius auratus brain NO synthase (NOS) as a function of pH. The physiological pH decrease from 7.2 to 6.8, which takes place during anoxia, greatly decreases NOS activity. This strong pH dependence is mainly due to variation of the calcium sensitivity of the enzyme. The EC(50) is 0.15 microM at pH 7.2 and 2.1 microM at pH 6.8 for the soluble enzyme. The particulate enzyme is also dependent on pH variations. The reduced sensitivity to calcium ions at acidic pH decreases both NO and H(2)O(2) production, saving the cells by suppression of the formation of potentially toxic nitrogen and oxygen species. Modulation of NOS activity by variation of its calcium affinity within the range of physiological pH constitutes an important and rapid mechanism to control the formation of NO and H(2)O(2) during normoxia-anoxia and anoxia-normoxia transitions.     

Drug-protein interactions: binding of chlorpromazine to calmodulin, calmodulin fragments, and related calcium binding proteins

The quantitative binding of a phenothiazine drug to calmodulin, calmodulin fragments, and structurally related calcium binding proteins was measured under conditions of thermodynamic equilibrium by using a gel filtration method. Plant and animal calmodulins, troponin C, S100 alpha, and S100 beta bind chlorpromazine in a calcium-dependent manner with different stoichiometries and affinities for the drug. The interaction between calmodulin and chlorpromazine appears to be a complex, calcium-dependent phenomenon. Bovine brain calmodulin bound approximately 5 mol of drug per mol of protein with apparent half-maximal binding at 17 microM drug. Large fragments of calmodulin had limited ability to bind chlorpromazine. The largest fragment, containing residues 1-90, retained only 5% of the drug binding activity of the intact protein. A reinvestigation of the chlorpromazine inhibition of calmodulin stimulation of cyclic nucleotide phosphodiesterase further indicated a complex, multiple equilibrium among the reaction components and demonstrated that the order of addition of components to the reaction altered the drug concentration required for half-maximal inhibition of the activity over a 10-fold range. These results confirm previous observations using immobilized phenothiazines [Marshak, D.R., Watterson, D.M., & Van Eldik, L.J. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6793-6797] that indicated a subclass of calcium-modulated proteins bound phenothiazines in a calcium-dependent manner, demonstrate that the interaction between phenothiazines and calmodulin is more complex than previously assumed, and suggest that extended regions of the calmodulin molecule capable of forming the appropriate conformation are required for specific, high-affinity, calcium-dependent drug binding activity.     

Role of substrate functional groups in binding to nitric oxide synthase

The interactions between the heme CO ligand in the oxygenase domain of nitric oxide synthase and a set of substrate analogues were determined by measuring the resonance Raman spectra of the Fe-C-O vibrational modes. Substrates were selected that have variations in all the functional units: the guanidino group, the amino acid site and the number of methylene units connecting the two ends. In comparison to the substrate free form of the enzyme, Interactions of the analogues with the CO moiety caused the Fe-CO stretching and the Fe-C-O bending modes to shift in frequency due to the electrostatic environment. An unmodified guanidino group interacted with the CO in a similar fashion despite changes in the amino acid end. However, an unmodified amino acid end is required for catalysis owing to the H-bonding network involving the substrate, the heme and the pterin cofactor.     

Thermodynamic analysis of L-arginine and N omega-hydroxy-L-arginine binding to nitric oxide synthase

Isothermal titration calorimetry has been used to determine thermodynamic parameters of substrate binding to the oxygenase domain of neuronal nitric oxide synthase (nNOS(oxy)) in the presence of the cofactor tetrahydrobiopterin. The intermediate N(omega)-hydroxy-L-arginine (NHA) has a larger affinity than L-Arginine (L-Arg) for nNOS(oxy), with K(d)=0.4+/-0.1 microM and 1.7+/-0.3 microM at 25 degrees C, respectively. nNOS(oxy) binds NHA and L-Arg with DeltaH -4.1+/-0.2 and -1.0+/-0.1 kcal/mol and DeltaS=15 and 23 cal/Kmol respectively. NHA binding is more exothermic probably due to formation of an extra hydrogen bond in the active site compared to L-Arg. The changes in heat capacity (DeltaC(p)) are relatively small for binding of both NHA and L-Arg (-53+/-18 and -95+/-23 cal/L mol, respectively), which indicates that hydrophobic interactions contribute little to binding.     

Conformational states and fluctuations in endothelial nitric oxide synthase under calmodulin regulation

Mechanisms that regulate nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions and is activated by calmodulin (CaM) binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two NOS electron transfer domains in an FRET dye-labeled endothelial NOS reductase domain (eNOSr) and to understand how CaM affects the dynamics to regulate catalysis by shaping the spatial and temporal conformational behaviors of eNOSr. In addition, we developed and applied a new imaging approach capable of recording three-dimensional FRET efficiency versus time images to characterize the impact on dynamic conformal states of the eNOSr enzyme by the binding of CaM, which identifies clearly that CaM binding generates an extra new open state of eNOSr, resolving more detailed NOS conformational states and their fluctuation dynamics. We identified a new output state that has an extra open conformation that is only populated in the CaM-bound eNOSr. This may reveal the critical role of CaM in triggering NOS activity as it gives conformational flexibility for eNOSr to assume the electron transfer output FMN-heme state. Our results provide a dynamic link to recently reported EM static structure analyses and demonstrate a capable approach in probing and simultaneously analyzing all of the conformational states, their fluctuations, and the fluctuation dynamics for understanding the mechanism of NOS electron transfer, involving electron transfer among FAD, FMN, and heme domains, during nitric oxide synthesis.     

Effects of Ca(2+)-activated calmodulin on neuronal nitric oxide synthase reductase activity and binding of substrates: pH dependence of kinetic parameters.

The pH dependence of basal and calmodulin- (CaM-) stimulated neuronal nitric oxide synthase (nNOS) reduction of 2,6-dichloroindophenol (DCIP) and cytochrome c(3+) was investigated. The wave-shaped log V versus pH profile revealed that optimal DCIP reduction occurred when a group, pK(a) of 7.6-7.8, was ionized. The (V/K)(NADPH) and (V/K)(DCIP) versus pH profiles increased with the protonation of a group with a pK(a) of 6.5 or 5.9 and the ionization of two groups with the same pK(a) of 7.5 or 7.0, respectively. (V/K)(DCIP) decreased with the ionization of a group, pK(a) of 9.0. Similar V, (V/K)(NADPH), and (V/K)(DCIP) versus pH profiles for DCIP reduction were obtained with and without CaM, indicating that CaM does not influence ionizable groups involved in catalysis or substrate binding. In contrast, CaM affected the pH dependence of cytochrome c(3+) reduction. The wave-shaped log V versus pH profile for basal cytochrome c(3+) reduction revealed that ionization of a group, pK(a) of 8.6, increased catalysis. Log V for CaM-stimulated cytochrome c(3+) reduction displayed a bell-shaped pH dependence with the protonation of a group with a pK(a) of 6.4 and the ionization of a group with a pK(a) of 9.3, resulting in a loss of activity. The log(V/K)(cytc) versus pH profiles with and without CaM were bell-shaped with the ionization of a group at pK(a) of 7.1 or 7.6 (CaM) or pK(a) of 9.4 or 9.6 (CaM), increasing and decreasing (V/K)(cytc). These results suggest that CaM may change the nature of the rate-limiting catalytic steps or ionizable groups involved in cytochrome c(3+) reduction.     

Binding and activation of nitric oxide synthase isozymes by calmodulin EF hand pairs

Calmodulin (CaM) is a cytosolic Ca(2+) signal-transducing protein that binds and activates many different cellular enzymes with physiological relevance, including the nitric oxide synthase (NOS) isozymes. CaM consists of two globular domains joined by a central linker; each domain contains an EF hand pair. Four different mutant CaM proteins were used to investigate the role of the two CaM EF hand pairs in the binding and activation of the mammalian inducible NOS (iNOS) and the constitutive NOS (cNOS) enzymes, endothelial NOS (eNOS) and neuronal NOS (nNOS). The role of the CaM EF hand pairs in different aspects of NOS enzymatic function was monitored using three assays that monitor electron transfer within a NOS homodimer. Gel filtration studies were used to determine the effect of Ca(2+) on the dimerization of iNOS when coexpressed with CaM and the mutant CaM proteins. Gel mobility shift assays were performed to determine binding stoichiometries of CaM proteins to synthetic NOS CaM-binding domain peptides. Our results show that the N-terminal EF hand pair of CaM contains important binding and activating elements for iNOS, whereas the N-terminal EF hand pair in conjunction with the central linker region is required for cNOS enzyme binding and activation. The iNOS enzyme must be coexpressed with wild-type CaM in vitro because of its propensity to aggregate when residues of the highly hydrophobic CaM-binding domain are exposed to an aqueous environment. A possible role for iNOS aggregation in vivo is also discussed.     

Stopped-flow analysis of substrate binding to neuronal nitric oxide synthase.

The kinetics of binding L-arginine and three alternative substrates (homoarginine, N-methylarginine, and N-hydroxyarginine) to neuronal nitric oxide synthase (nNOS) were characterized by conventional and stopped-flow spectroscopy. Because binding these substrates has only a small effect on the light absorbance spectrum of tetrahydrobiopterin-saturated nNOS, their binding was monitored by following displacement of imidazole, which displays a significant change in Soret absorbance from 427 to 398 nm. Rates of spectral change upon mixing Im-nNOS with increasing amounts of substrates were obtained and found to be monophasic in all cases. For each substrate, a plot of the apparent rate versus substrate concentration showed saturation at the higher concentrations. K(-)(1), k(2), k(-)(2), and the apparent dissociation constant were derived for each substrate from the kinetic data. The dissociation constants mostly agreed with those calculated from equilibrium spectral data obtained by titrating Im-nNOS with each substrate. We conclude that nNOS follows a two-step, reversible mechanism of substrate binding in which there is a rapid equilibrium between Im-nNOS and the substrate S followed by a slower isomerization process to generate nNOS'-S: Im-nNOS + S if Im-nNOS-S if nNOS'-S + Im. All four substrates followed this general mechanism, but differences in their kinetic values were significant and may contribute to their varying capacities to support NO synthesis.     

Photo-control of nitric oxide synthase activity using a caged isoform specific inhibitor

Nitric oxide (NO) plays a critical role in a number of physiological processes and is produced in mammalian cells by nitric oxide synthase (NOS) isozymes. Because of the diverse functions of NO, pharmaceutical interventions which seek to abrogate adverse effects of excess NOS activity must not interfere with the normal regulation of NO levels in the body. A method has been developed for the control of NOS enzyme activity using the localized photochemical release of a caged isoform-specific NOS inhibitor. The caged form of an iNOS inhibitor has been synthesized and tested for photosensitivity and potency. UV and multiphoton uncaging were verified using a hemoglobin-based assay. IC(50) values were determined for the inhibitor (70+/-11 nM), the caged inhibitor (1098+/-172 nM), the UV uncaged inhibitor (67+/-26 nM) and the multiphoton uncaged inhibitor (73+/-11 nM). UV irradiation of the caged inhibitor resulted in a 86% reduction in iNOS activity after 5 min. Multiphoton uncaging had an apparent first order time constant of 0.007+/-0.001 min(-1). A therapeutic range exists, with molar excess of inhibitor to enzyme from 3- to 7-fold, over which the full dynamic range of the inhibition can be exploited.     

Calcium-deficient calmodulin binding and activation of neuronal and inducible nitric oxide synthases

The nitric oxide synthase (NOS) enzymes are bound and activated by the Ca(2+)-binding protein, calmodulin (CaM). We have utilized CaM mutants deficient in binding Ca(2+) with mutations in the N-lobe (CaM(12)), the C-lobe (CaM(34)), or both lobes of CaM (CaM(1234)) to determine their effect on the binding and activation of the Ca(2+)-dependent neuronal (nNOS) and Ca(2+)-independent inducible NOS (iNOS) isoforms. Four different kinetic assays were employed to monitor the effect of these CaM mutants on electron transfer rates in NOS. Protein-protein interactions between CaM and NOS were studied using steady-state fluorescence and spectropolarimetry to monitor the binding of these CaM mutants to nNOS and iNOS CaM-binding domain peptides. The CaM mutants were unable to activate nNOS, however, our CD results show that the C-terminal lobe of CaM is capable of binding to nNOS peptide in the presence of Ca(2+). Our results prove for the first time without the use of chelators that apo-CaM is capable of binding to iNOS peptides and holoenzymes.     

Binding kinetics of calmodulin with target peptides of three nitric oxide synthase isozymes

Efficient electron transfer from reductase domain to oxygenase domain in nitric oxide synthase (NOS) is dependent on the binding of calmodulin (CaM). Rate constants for the binding of CaM to NOS target peptides was only determined previously by surface plasmon resonance (SPR) (Biochemistry 35, 8742-8747, 1996) suggesting that the binding of CaM to NOSs is slow and does not support the fast electron transfer in NOSs measured in previous and this studies. To resolve this contradiction, the binding rates of holo Alexa 350 labeled T34C/T110W CaM (Alexa-CaM) to target peptides from three NOS isozymes were determined using fluorescence stopped-flow. All three target peptides exhibited fast k_on constants at 4.5 °C: 6.6 × 10⁸ M^− 1 s^− 1 for nNOS_726-749, 2.9 × 10⁸ M^− 1 s^− 1 for eNOS_492-511 and 6.1 × 10⁸ M^− 1 s^− 1 for iNOS_507-531, 3-4 orders of magnitude faster than those determined previously by SPR. Dissociation rates of NOS target peptides from Alexa-CaM/peptide complexes were measured by Ca²⁺ chelation with ETDA: 3.7 s^− 1 for nNOS_726-749, 4.5 s^− 1 for eNOS_492-511, and 0.063 s^− 1 for iNOS_507-531. Our data suggest that the binding of CaM to NOS is fast and kinetically competent for efficient electron transfer and is unlikely rate-limiting in NOS catalysis. Only iNOS_507-531 was able to bind apo Alexa-CaM, but in a very different conformation from its binding to holo Alexa-CaM.