Control of electron transfer in nitric-oxide synthases. Swapping of autoinhibitory elements among nitric-oxide synthase isoforms

To clarify the role of the autoinhibitory insert in the endothelial (eNOS) and neuronal (nNOS) nitric-oxide synthases, the insert was excised from nNOS and chimeras with its reductase domain; the eNOS and nNOS inserts were swapped and put into the normally insertless inducible (iNOS) isoform and chimeras with the iNOS reductase domain; and an RRKRK sequence in the insert suggested by earlier peptide studies to be important (Salerno, J. C., Harris, D. E., Irizarry, K., Patel, B., Morales, A. J., Smith, S. M., Martasek, P., Roman, L. J., Masters, B. S., Jones, C. L., Weissman, B. A., Lane, P., Liu, Q., and Gross, S. S. (1997) J. Biol. Chem. 272, 29769-29777) was mutated. Insertless nNOS required calmodulin (CaM) for normal NOS activity, but the Ca(2+) requirement for this activity was relaxed. Furthermore, insert deletion enhanced CaM-free electron transfer within nNOS and chimeras with the nNOS reductase, emphasizing the involvement of the insert in modulating electron transfer. Swapping the nNOS and eNOS inserts gave proteins with normal NOS activities, and the nNOS insert acted normally in raising the Ca(2+) dependence when placed in eNOS. Insertion of the eNOS insert into iNOS and chimeras with the iNOS reductase domain significantly lowered NOS activity, consistent with inhibition of electron transfer by the insert. Mutation of the eNOS RRKRK to an AAAAA sequence did not alter the eNOS Ca(2+) dependence but marginally inhibited electron transfer. The salt dependence suggests that the insert modulates electron transfer within the reductase domain prior to the heme/reductase interface. The results clarify the role of the reductase insert in modulating the Ca(2+) requirement, electron transfer rate, and overall activity of nNOS and eNOS.     

Inhibition of endothelial nitric-oxide synthase by ceruloplasmin.

The plasma copper protein ceruloplasmin (CP) was found to inhibit endothelial nitric-oxide synthase activation in cultured endothelial cells, in line with previous evidence showing that the endothelium-dependent vasorelaxation of the aorta is impaired by physiological concentrations of ceruloplasmin. The data presented here indicate a direct relationship between the extent of inhibition of agonist-triggered endothelial nitric oxide synthase activation and CP-induced enrichment of the copper content of endothelial cells. Copper discharged by CP was mainly localized in the soluble fraction of cells. The subcellular distribution of the metal seems to be of relevance to the inhibitory effect of CP, because it was mimicked by copper chelates, like copper-histidine, able to selectively enrich the cytosolic fraction of cells, but not by copper salts, which preferentially located the metal to the particulate fraction.     

Structural basis for isozyme-specific regulation of electron transfer in nitric-oxide synthase

Three nitric-oxide synthase (NOS) isozymes play crucial, but distinct, roles in neurotransmission, vascular homeostasis, and host defense, by catalyzing Ca(2+)/calmodulin-triggered NO synthesis. Here, we address current questions regarding NOS activity and regulation by combining mutagenesis and biochemistry with crystal structure determination of a fully assembled, electron-supplying, neuronal NOS reductase dimer. By integrating these results, we structurally elucidate the unique mechanisms for isozyme-specific regulation of electron transfer in NOS. Our discovery of the autoinhibitory helix, its placement between domains, and striking similarities with canonical calmodulin-binding motifs, support new mechanisms for NOS inhibition. NADPH, isozyme-specific residue Arg(1400), and the C-terminal tail synergistically repress NOS activity by locking the FMN binding domain in an electron-accepting position. Our analyses suggest that calmodulin binding or C-terminal tail phosphorylation frees a large scale swinging motion of the entire FMN domain to deliver electrons to the catalytic module in the holoenzyme.     

Calmodulin activates intersubunit electron transfer in the neuronal nitric-oxide synthase dimer

Neuronal nitric oxide synthase (nNOS) is composed of an oxygenase domain that binds heme, (6R)-tetrahydrobiopterin, and Arg, coupled to a reductase domain that binds FAD, FMN, and NADPH. Activity requires dimeric interaction between two oxygenase domains and calmodulin binding between the reductase and oxygenase domains, which triggers electron transfer between flavin and heme groups. We constructed four different nNOS heterodimers to determine the path of calmodulin-induced electron transfer in a nNOS dimer. A predominantly monomeric mutant of rat nNOS (G671A) and its Arg binding mutant (G671A/E592A) were used as full-length subunits, along with oxygenase domain partners that either did or did not contain the E592A mutation. The E592A mutation prevented Arg binding to the oxygenase domain in which it was present. It also prevented NO synthesis when it was located in the oxygenase domain adjacent to the full-length subunit. However, it had no effect when present in the full-length subunit (i.e. the subunit containing the reductase domain). The active heterodimer (G671A/E592A full-length subunit plus wild type oxygenase domain subunit) showed remarkable similarity with wild type homodimeric nNOS in its catalytic responses to five different forms and chimeras of calmodulin. This reveals an active involvement of calmodulin in supporting transelectron transfer between flavin and heme groups on adjacent subunits in nNOS. In summary, we propose that calmodulin functions to properly align adjacent reductase and the oxygenase domains in a nNOS dimer for electron transfer between them, leading to NO synthesis by the heme.     

Direct interaction between endothelial nitric-oxide synthase and dynamin-2. Implications for nitric-oxide synthase function

Endothelial nitric-oxide synthase (eNOS) is regulated in part through specific protein interactions. Dynamin-2 is a large GTPase residing within similar membrane compartments as eNOS. Here we show that dynamin-2 binds directly with eNOS thereby augmenting eNOS activity. Double label confocal immunofluorescence demonstrates colocalization of eNOS and dynamin in both Clone 9 cells cotransfected with green fluorescent protein-dynamin and eNOS, as well as in bovine aortic endothelial cells (BAEC) expressing both proteins endogenously, predominantly in a Golgi membrane distribution. Immunoprecipitation of eNOS from BAEC lysate coprecipitates dynamin and, conversely, immunoprecipitation of dynamin coprecipitates eNOS. Additionally, the calcium ionophore, a reagent that promotes nitric oxide release, enhances coprecipitation of dynamin with eNOS in BAEC, suggesting the interaction between the proteins can be regulated by intracellular signals. In vitro studies demonstrate that glutathione S-transferase (GST)-dynamin-2 quantitatively precipitates both purified recombinant eNOS protein as well as in vitro transcribed (35)S-labeled eNOS from solution indicating a direct interaction between the proteins in vitro. Scatchard analysis of binding studies demonstrates an equilibrium dissociation constant (K(d)) of 27.6 nm. Incubation of purified recombinant eNOS protein with GST-dynamin-2 significantly increases eNOS activity as does overexpression of dynamin-2 in ECV 304 cells stably transfected with eNOS-green fluorescent protein. These studies demonstrate a direct protein-protein interaction between eNOS and dynamin-2, thereby identifying a new NOS-associated protein and providing a novel function for dynamin. These events may have relevance for eNOS regulation and trafficking within vascular endothelium.     

Oxygen metabolism by endothelial nitric-oxide synthase

Nitric-oxide synthase (NOS) catalyzes both coupled and uncoupled reactions that generate nitric oxide and reactive oxygen species. Oxygen is often the overlooked substrate, and the oxygen metabolism catalyzed by NOS has been poorly defined. In this paper we focus on the oxygen stoichiometry and effects of substrate/cofactor binding on the endothelial NOS isoform (eNOS). In the presence of both L-arginine and tetrahydrobiopterin, eNOS is highly coupled (>90%), and the measured stoichiometry of O(2)/NADPH is very close to the theoretical value. We report for the first time that the presence of L-arginine stimulates oxygen uptake by eNOS. The fact that nonhydrolyzable L-arginine analogs are not stimulatory indicates that the occurrence of the coupled reaction, rather than the accelerated uncoupled reaction, is responsible for the L-arginine-dependent stimulation. The presence of 5,6,7,8-tetrahydrobiopterin quenched the uncoupled reactions and resulted in much less reactive oxygen species formation, whereas the presence of redox-incompetent 7,8-dihydrobiopterin demonstrates little quenching effect. These results reveal different mechanisms for oxygen metabolism for eNOS as opposed to nNOS and, perhaps, partially explain their functional differences.     

Calmodulin activates electron transfer through neuronal nitric-oxide synthase reductase domain by releasing an NADPH-dependent conformational lock

Neuronal nitric-oxide synthase (nNOS) is activated by the Ca(2+)-dependent binding of calmodulin (CaM) to a characteristic polypeptide linker connecting the oxygenase and reductase domains. Calmodulin binding also activates the reductase domain of the enzyme, increasing the rate of reduction of external electron acceptors such as cytochrome c. Several unusual structural features appear to control this activation mechanism, including an autoinhibitory loop, a C-terminal extension, and kinase-dependent phosphorylation sites. Pre-steady state reduction and oxidation time courses for the nNOS reductase domain indicate that CaM binding triggers NADP(+) release, which may exert control over steady-state turnover. In addition, the second order rate constant for cytochrome c reduction in the absence of CaM was found to be highly dependent on the presence of NADPH. It appears that NADPH induces a conformational change in the nNOS reductase domain, restricting access to the FMN by external electron acceptors. CaM binding reverses this effect, causing a 30-fold increase in the second order rate constant. The results show a startling interplay between the two ligands, which both exert control over the conformation of the domain to influence its electron transfer properties. In the full-length enzyme, NADPH binding will probably close the conformational lock in vivo, preventing electron transfer to the oxygenase domain and the resultant stimulation of nitric oxide synthesis.     

Crucial role of Lys(423) in the electron transfer of neuronal nitric-oxide synthase.

Nitric-oxide synthase (NOS) is composed of an oxygenase domain having cytochrome P450-type heme active site and a reductase domain having FAD- and FMN-binding sites. To investigate the route of electron transfer from the reductase domain to the heme, we generated mutants at Lys(423) in the heme proximal site of neuronal NOS and examined the catalytic activities, electron transfer rates, and NADPH oxidation rates. A K423E mutant showed no NO formation activity (<0.1 nmol/min/nmol heme), in contrast with that (72 nmol/min/nmol heme) of the wild type enzyme. The electron transfer rate (0.01 min(-1)) of the K423E on addition of excess NADPH was much slower than that (>10 min(-1)) of the wild type enzyme. From the crystal structure of the oxygenase domain of endothelial NOS, Lys(423) of neuronal NOS is likely to interact with Trp(409) which lies in contact with the heme plane and with Cys(415), the axial ligand. It is also exposed to solvent and lies in the region where the heme is closest to the protein surface. Thus, it seems likely that ionic interactions between Lys(423) and the reductase domain may help to form a flavin to heme electron transfer pathway.     

Rapid calmodulin-dependent interdomain electron transfer in neuronal nitric-oxide synthase measured by pulse radiolysis

Electron transfer within rat neuronal nitric-oxide synthase (nNOS) was investigated by pulse radiolysis. Radiolytically generated 1-methyl-3-carbamoyl pyridinium (MCP) radical was found to react predominantly with the heme of the enzyme with a second-order rate constant for heme reduction of 3 x 10(8) m(-1) s(-1). In the calmodulin (CaM)-bound enzyme a subsequent first-order phase was observed which had a rate constant of 1.2 x 10(3) s(-1). In the absence of CaM, this phase was absent. Kinetic difference spectra for nNOS reduction indicated that the second phase consisted of heme reoxidation accompanied by formation of a neutral flavin semiquinone, suggesting that it is heme to flavin electron transfer. Experiments with the heme proximal surface mutant, K423E, had no second phase, confirming that the mutation blocks interdomain electron transfer. With the autoinhibitory loop deletion mutant, Delta40, the slow phase was observed even in the absence of CaM consistent with the role of the loop in impeding interdomain electron transfer. The rate of heme to FMN electron transfer observed in the wild-type enzyme is approximately 1000 times faster than the FMN to heme electron transfer rate predicted during catalysis from kinetic modeling, suggesting that the catalytic process is slowed by kinetic gating.     

Receptor-regulated dynamic S-nitrosylation of endothelial nitric-oxide synthase in vascular endothelial cells

The endothelial isoform of nitric-oxide synthase (eNOS) is regulated by a complex pattern of post-translational modifications. In these studies, we show that eNOS is dynamically regulated by S-nitrosylation, the covalent adduction of nitric oxide (NO)-derived nitrosyl groups to the cysteine thiols of proteins. We report that eNOS is tonically S-nitrosylated in resting bovine aortic endothelial cells and that the enzyme undergoes rapid transient denitrosylation after addition of the eNOS agonist, vascular endothelial growth factor. eNOS is thereafter progressively renitrosylated to basal levels. The receptor-mediated decrease in eNOS S-nitrosylation is inversely related to enzyme phosphorylation at Ser(1179), a site associated with eNOS activation. We also document that targeting of eNOS to the cell membrane is required for eNOS S-nitrosylation. Acylation-deficient mutant eNOS, which is targeted to the cytosol, does not undergo S-nitrosylation. Using purified eNOS, we show that eNOS S-nitrosylation by exogenous NO donors inhibits enzyme activity and that eNOS inhibition is reversed by denitrosylation. We determine that the cysteines of the zinc-tetrathiolate that comprise the eNOS dimer interface are the targets of S-nitrosylation. Mutation of the zinc-tetrathiolate cysteines eliminates eNOS S-nitrosylation but does not eliminate NO synthase activity, arguing strongly that disruption of the zinc-tetrathiolate does not necessarily lead to eNOS monomerization in vivo. Taken together, these studies suggest that eNOS S-nitrosylation may represent an important mechanism for regulation of NO signaling pathways in the vascular wall.     

Characterization of the roles of the 594-645 region in human endothelial nitric-oxide synthase in regulating calmodulin binding and electron transfer

It has been postulated that a segment (residues 594-645) inserted in the FMN subdomain of human endothelial nitric-oxide synthase (eNOS) plays a crucial role in controlling Ca(2+)-dependent CaM binding for eNOS activity. To investigate its functions, we expressed human eNOS in a baculovirus system with deletion of a 45-residue segment from this region (residues 594-606 and 614-645, designated as Delta45eNOS), and characterized the purified mutant enzyme. In contrast with wild-type eNOS, Delta45eNOS exhibited characteristics resembling inducible NOS (iNOS). It contained an endogenously bound CaM, which was essential in folding and stabilizing this mutant enzyme, and retained 60% of L-citrulline formation in 5 mM EGTA. We also produced four N-terminally truncated reductase domains with or without the 45-residue segment, and either including or excluding the CaM-binding sequence. Basal cytochrome c reductase activity of reductase domains without the 45-residue segment was up to 20 fold greater than that of corresponding insert-containing domains, and higher than CaM-stimulated activity of the wild-type enzyme. A series of mutants with smaller fragment deletion in this region such as Delta594-604, Delta605-612, Delta613-625, Delta626-634, Delta632-639, and Delta640-645 mutants were further characterized. The crude lysate of mutants Delta613-625 and Delta632-639 did not show activity in the presence of Ca(2+)/CaM, while other four mutants had activity comparable to that of WTeNOS. The purified Delta594-604 and Delta605-612 proteins had a 3-5-fold higher affinity for Ca(2+)/CaM, but their L-citrulline forming activity was still 80% dependent upon the addition of Ca(2+)/CaM. Both mutants exhibited a low level of the cytochrome c and ferricyanide reductase activities, which either did not respond to (Delta594-604) or slightly enhanced by (Delta605-612) the exogenous CaM. In contrast, activities of Delta626-634 and Delta640-645 like those of WTeNOS were largely Ca(2+)/CaM-dependent. Thus, our findings indicate that the N-terminal half of the 594-645 segment containing residues 594-612 plays a significant role in regulating Ca(2+)/CaM binding.     

Subcellular targeting and differential S-nitrosylation of endothelial nitric-oxide synthase

Endothelial nitric-oxide synthase (eNOS) undergoes a complex pattern of post-translational modifications that regulate its activity. We have recently reported that eNOS is constitutively S-nitrosylated in endothelial cells and that agonists promote eNOS denitrosylation concomitant with enzyme activation (Erwin, P. A., Lin, A. J., Golan, D. E., and Michel, T. (2005), J. Biol. Chem. 280, 19888-19894). In the present studies, we use mass spectrometry to confirm that the zinc-tetrathiolate cysteines of eNOS are S-nitrosylated. eNOS targeting to the plasma membrane is necessary for enzyme S-nitrosylation, and we report that translocation between cellular compartments is necessary for dynamic eNOS S-nitrosylation. We transfected cells with cDNA encoding wild-type eNOS, which is membrane-targeted, or with acylation-deficient mutant eNOS (Myr-), which is expressed solely in the cytosol. While wild-type eNOS is robustly S-nitrosylated, we found that S-nitrosylation of the Myr- eNOS mutant is nearly abolished. When we transfected cells with a fusion protein in which Myr- eNOS is ligated to the CD8-transmembrane domain (CD8-Myr-), we found that CD8-Myr- eNOS, which does not undergo dynamic subcellular translocation, is hypernitrosylated relative to wild-type eNOS. Furthermore, we found that when endothelial cells transfected with wild-type or CD8-Myr- eNOS are stimulated with eNOS agonist, only wild-type eNOS is denitrosylated; CD8-Myr- eNOS S-nitrosylation is unchanged. These findings indicate that subcellular targeting is a critical determinant of eNOS S-nitrosylation. Finally, we show that eNOS S-nitrosylation can be detected in intact arterial preparations from mouse and that eNOS S-nitrosylation is a dynamic agonist-modulated process in intact blood vessels. These studies suggest that receptor-regulated eNOS S-nitrosylation may represent an important determinant of NO-dependent signaling in the vascular wall.     

Lysophosphatidic acid and receptor-mediated activation of endothelial nitric-oxide synthase

Both lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are platelet-derived phospholipids that elicit diverse biological responses. In endothelial cells, S1P stimulates the EDG-1 receptor-mediated activation of the endothelial isoform of nitric oxide synthase (eNOS), but the role of LPA in eNOS regulation is less well understood. We now report that LPA treatment of bovine aortic endothelial cells (BAEC) activates eNOS enzyme activity in a pathway that involves phosphorylation of eNOS on serine 1179 by protein kinase Akt. In contrast to the cellular responses elicited by S1P in COS-7 cells, LPA can stimulate the activation of eNOS and Akt independently of EDG-1 receptor transfection. LPA-stimulated enzyme activation was significantly attenuated in an eNOS mutant lacking the site that is phosphorylated by kinase Akt (eNOS S1179A). In BAEC, activation of eNOS by LPA is completely blocked by pertussis toxin, by the intracellular calcium chelator BAPTA (1,2-bis(aminophenoxy) ethane-N,N,N',N'-tetraacetic acid), and by the phosphoinositide 3-kinase (PI3-K) inhibitor wortmannin, but is unaffected by U0126, an inhibitor of mitogen-activated protein (MAP) kinase pathways. Analysis of the LPA dose response for eNOS activation reveals an EC(50) of approximately 40 nM, a concentration well below the potency of LPA at the EDG-1 receptor. Taken together, these results indicate that LPA potently activates eNOS in BAEC in a pathway distinct from the EDG-1 receptor, but mediated by a similar receptor-mediated pathway dependent on pertussis toxin-sensitive G proteins and involving activation of the PI3-K/Akt pathway. These studies have identified a role for the phospholipid LPA in eNOS activation, and point out the complementary role of distinct platelet-derived lipids in endothelial signaling pathways.     

Electron transfer, oxygen binding, and nitric oxide feedback inhibition in endothelial nitric-oxide synthase

We studied steps that make up the initial and steady-state phases of nitric oxide (NO) synthesis to understand how activity of bovine endothelial NO synthase (eNOS) is regulated. Stopped-flow analysis of NADPH-dependent flavin reduction showed the rate increased from 0. 13 to 86 s(-1) upon calmodulin binding, but this supported slow heme reduction in the presence of either Arg or N(omega)-hydroxy-l-arginine (0.005 and 0.014 s(-1), respectively, at 10 degrees C). O(2) binding to ferrous eNOS generated a transient ferrous dioxy species (Soret peak at 427 nm) whose formation and decay kinetics indicate it can participate in NO synthesis. The kinetics of heme-NO complex formation were characterized under anaerobic conditions and during the initial phase of NO synthesis. During catalysis heme-NO complex formation required buildup of relatively high solution NO concentrations (>50 nm), which were easily achieved with N(omega)-hydroxy-l-arginine but not with Arg as substrate. Heme-NO complex formation caused eNOS NADPH oxidation and citrulline synthesis to decrease 3-fold and the apparent K(m) for O(2) to increase 6-fold. Our main conclusions are: 1) The slow steady-state rate of NO synthesis by eNOS is primarily because of slow electron transfer from its reductase domain to the heme, rather than heme-NO complex formation or other aspects of catalysis. 2) eNOS forms relatively little heme-NO complex during NO synthesis from Arg, implying NO feedback inhibition has a minimal role. These properties distinguish eNOS from the other NOS isoforms and provide a foundation to better understand its role in physiology and pathology.     

The 42-amino acid insert in the FMN domain of neuronal nitric-oxide synthase exerts control over Ca(2+)/calmodulin-dependent electron transfer.

The neuronal and endothelial nitric-oxide synthases (nNOS and eNOS) differ from inducible NOS in their dependence on the intracellular Ca(2+) concentration. Both nNOS and eNOS are activated by the reversible binding of calmodulin (CaM) in the presence of Ca(2+), whereas inducible NOS binds CaM irreversibly. One major divergence in the close sequence similarity between the NOS isoforms is a 40-50-amino acid insert in the middle of the FMN-binding domains of nNOS and eNOS. It has previously been proposed that this insert forms an autoinhibitory domain designed to destabilize CaM binding and increase its Ca(2+) dependence. To examine the importance of the insert we constructed two deletion mutants designed to remove the bulk of it from nNOS. Both mutants (Delta40 and Delta42) retained maximal NO synthesis activity at lower concentrations of free Ca(2+) than the wild type enzyme. They were also found to retain 30% of their activity in the absence of Ca(2+)/CaM, indicating that the insert plays an important role in disabling the enzyme when the physiological Ca(2+) concentration is low. Reduction of nNOS heme by NADPH under rigorous anaerobic conditions was found to occur in the wild type enzyme only in the presence of Ca(2+)/CaM. However, reduction of heme in the Delta40 mutant occurred spontaneously on addition of NADPH in the absence of Ca(2+)/CaM. This suggests that the insert regulates activity by inhibiting electron transfer from FMN to heme in the absence of Ca(2+)/CaM and by destabilizing CaM binding at low Ca(2+) concentrations, consistent with its role as an autoinhibitory domain.     

C-terminal tail residue Arg1400 enables NADPH to regulate electron transfer in neuronal nitric-oxide synthase

The neuronal nitric-oxide synthase (nNOS) flavoprotein domain (nNOSr) contains regulatory elements that repress its electron flux in the absence of bound calmodulin (CaM). The repression also requires bound NADP(H), but the mechanism is unclear. The crystal structure of a CaM-free nNOSr revealed an ionic interaction between Arg(1400) in the C-terminal tail regulatory element and the 2'-phosphate group of bound NADP(H). We tested the role of this interaction by substituting Ser and Glu for Arg(1400) in nNOSr and in the full-length nNOS enzyme. The CaM-free nNOSr mutants had cytochrome c reductase activities that were less repressed than in wild-type, and this effect could be mimicked in wild-type by using NADH instead of NADPH. The nNOSr mutants also had faster flavin reduction rates, greater apparent K(m) for NADPH, and greater rates of flavin auto-oxidation. Single-turnover cytochrome c reduction data linked these properties to an inability of NADP(H) to cause shielding of the FMN module in the CaM-free nNOSr mutants. The full-length nNOS mutants had no NO synthesis in the CaM-free state and had lower steady-state NO synthesis activities in the CaM-bound state compared with wild-type. However, the mutants had faster rates of ferric heme reduction and ferrous heme-NO complex formation. Slowing down heme reduction in R1400E nNOS with CaM analogues brought its NO synthesis activity back up to normal level. Our studies indicate that the Arg(1400)-2'-phosphate interaction is a means by which bound NADP(H) represses electron transfer into and out of CaM-free nNOSr. This interaction enables the C-terminal tail to regulate a conformational equilibrium of the FMN module that controls its electron transfer reactions in both the CaM-free and CaM-bound forms of nNOS.     

Exploring the electron transfer properties of neuronal nitric-oxide synthase by reversal of the FMN redox potential

In nitric-oxide synthase (NOS) the FMN can exist as the fully oxidized (ox), the one-electron reduced semiquinone (sq), or the two-electron fully reduced hydroquinone (hq). In NOS and microsomal cytochrome P450 reductase the sq/hq redox potential is lower than that of the ox/sq couple, and hence it is the hq form of FMN that delivers electrons to the heme. Like NOS, cytochrome P450BM3 has the FAD/FMN reductase fused to the C-terminal end of the heme domain, but in P450BM3 the ox/sq and sq/hq redox couples are reversed, so it is the sq that transfers electrons to the heme. This difference is due to an extra Gly residue found in the FMN binding loop in NOS compared with P450BM3. We have deleted residue Gly-810 from the FMN binding loop in neuronal NOS (nNOS) to give Delta G810 so that the shorter binding loop mimics that in cytochrome P450BM3. As expected, the ox/sq redox potential now is lower than the sq/hq couple. Delta G810 exhibits lower NO synthase activity but normal levels of cytochrome c reductase activity. However, unlike the wild-type enzyme, the cytochrome c reductase activity of Delta G810 is insensitive to calmodulin binding. In addition, calmodulin binding to Delta G810 does not result in a large increase in FMN fluorescence as in wild-type nNOS. These results indicate that the FMN domain in Delta G810 is locked in a unique conformation that is no longer sensitive to calmodulin binding and resembles the "on" output state of the calmodulin-bound wild-type nNOS with respect to the cytochrome c reduction activity.     

Intra-subunit and inter-subunit electron transfer in neuronal nitric-oxide synthase: effect of calmodulin on heterodimer catalysis

In neuronal nitric-oxide synthase (nNOS), calmodulin (CaM) binding is thought to trigger electron transfer from the reductase domain to the heme domain, which is essential for O(2) activation and NO formation. To elucidate the electron-transfer mechanism, we characterized a series of heterodimers consisting of one full-length nNOS subunit and one oxygenase-domain subunit. The results support an inter-subunit electron-transfer mechanism for the wild type nNOS, in that electrons for catalysis transfer in a Ca(2+)/CaM-dependent way from the reductase domain of one subunit to the heme of the other subunit, as proposed for inducible NOS. This suggests that the two different isoforms form similar dimeric complexes. In a series of heterodimers containing a Ca(2+)/CaM-insensitive mutant (delta40), electrons transferred from the reductase domain to both hemes in a Ca(2+)/CaM-independent way. Thus, in the delta40 mutant electron transfer from the reductase domains to the heme domains can occur via both inter-subunit and intra-subunit mechanisms. However, NO formation activity was exclusively linked to inter-subunit electron transfer and was observed only in the presence of Ca(2+)/CaM. This suggests that the mechanism of activation of nNOS by CaM is not solely dependent on the activation of electron transfer to the nNOS hemes but may involve additional structural factors linked to the catalytic action of the heme domain.