FMN fluorescence in inducible NOS constructs reveals a series of conformational states involved in the reductase catalytic cycle

Nitric oxide synthases (NOSs) produce NO as a molecular signal in the nervous and cardiovascular systems and as a cytotoxin in the immune response. NO production in the constitutive isoforms is controlled by calmodulin regulation of electron transfer. In the tethered shuttle model for NOS reductase function, the FMN domain moves between NADPH dehydrogenase and oxygenase catalytic centers. Crystal structures of neuronal NOS reductase domain and homologs correspond to an 'input state', with FMN in close contact with FAD. We recently produced two domain 'output state' (oxyFMN) constructs showing calmodulin dependent FMN domain association with the oxygenase domain. FMN fluorescence is sensitive to enzyme conformation and calmodulin binding. The inducible NOS (iNOS) oxyFMN construct is more fluorescent than iNOS holoenzyme. The difference in steady state fluorescence is rationalized by the observation of a series of characteristic states in the two constructs, which we assign to FMN in different environments. OxyFMN and holoenzyme share open conformations with an average lifetime of ~4.3 ns. The majority state in holoenzyme has a short lifetime of ~90 ps, probably because of FAD-FMN interactions. In oxyFMN about 25-30% of the FMN is in a state with a lifetime of 0.9 ns, which we attribute to quenching by heme in the output state. Occupancy of the output state together with our previous kinetic results yields a heme edge to FMN distance estimate of 12-15 ?. These results indicate that FMN fluorescence is a valuable tool to study conformational states involved in the NOS reductase catalytic cycle.     

The function of the small insertion in the hinge subdomain in the control of constitutive mammalian nitric-oxide synthases

Control of nitric oxide (NO) synthesis in the constitutive nitric-oxide synthases (NOS) by calcium/calmodulin is exerted through the regulation of electron transfer from NADPH through the reductase domains. This process has been shown previously to involve the calmodulin binding site, the autoinhibitory insertion in the FMN binding domain, and the C-terminal tail. Smaller sequence elements also appear to correlate with control. Although some of these elements appear well positioned to function in control, they are poorly conserved; their role in control is neither well established nor defined by available information. In this study mutations have been induced in the small insertion of the hinge subdomain, which has been shown recently to form a beta hairpin in structural studies of the neuronal NOS reductase domains adjacent to the calmodulin site and the autoinhibitory element. Modification of the small insertion in neuronal NOS tends to increase cytochrome c reduction but not NO synthetic activity; some modifications or deletions in the corresponding region in endothelial NOS modestly increase activity under some conditions. Unexpectedly, some minor changes in the sequence introduce a loss in the content of heme relative to flavin cofactors. Taken together, these results suggest that the small insertion protects the calmodulin binding site and that it may be a modulator of NOS activity.     

Differential activation of nitric-oxide synthase isozymes by calmodulin-troponin C chimeras

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 (*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 co-expressed with the human iNOS enzyme in Escherichia coli and subsequently purified. Domains 2 and 3 of CaM contain important elements required for the Ca2+/CaM independence of *NO production by the iNOS enzyme. The disparity between cytochrome c reduction and *NO production at low calcium can be attributed to poor association of heme and FMN domains when the bound CaM constructs are depleted of Ca2+. 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.     

Autoinhibition of endothelial nitric-oxide synthase. Identification of an electron transfer control element.

The primary sequences of the three mammalian nitric- oxide synthase (NOS) isoforms differ by the insertion of a 52-55-amino acid loop into the reductase domains of the endothelial (eNOS) and neuronal (nNOS), but not inducible (iNOS). On the basis of studies of peptide derivatives as inhibitors of.NO formation and calmodulin (CaM) binding (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), the insert has been proposed to be an autoinhibitory element. We have examined the role of the insert in its native protein context by deleting the insert from both wild-type eNOS and from chimeras obtained by swapping the reductase domains of the three NOS isoforms. The Ca2+ concentrations required to activate the enzymes decrease significantly when the insert is deleted, consistent with suppression of autoinhibition. Furthermore, removal of the insert greatly enhances the maximal activity of wild-type eNOS, the least active of the three isoforms. Despite the correlation between reductase and overall enzymatic activity for the wild-type and chimeric NOS proteins, the loop-free eNOS still requires CaM to synthesize.NO. However, the reductive activity of the CaM-free, loop-deleted eNOS is enhanced significantly over that of CaM-free wild-type eNOS and approaches the same level as that of CaM-bound wild-type eNOS. Thus, the inhibitory effect of the loop on both the eNOS reductase and. NO-synthesizing activities may have an origin distinct from the loop's inhibitory effects on the binding of CaM and the concomitant activation of the reductase and.NO-synthesizing activities. The eNOS insert not only inhibits activation of the enzyme by CaM but also contributes to the relatively low overall activity of this NOS isoform.     

Chimeric enzymes of cytochrome P450 oxidoreductase and neuronal nitric-oxide synthase reductase domain reveal structural and functional differences

The nitric-oxide synthases (NOSs) are comprised of an oxygenase domain and a reductase domain bisected by a calmodulin (CaM) binding region. The NOS reductase domains share approximately 60% sequence similarity with the cytochrome P450 oxidoreductase (CYPOR), which transfers electrons to microsomal cytochromes P450. The crystal structure of the neuronal NOS (nNOS) connecting/FAD binding subdomains reveals that the structure of the nNOS-connecting subdomain diverges from that of CYPOR, implying different alignments of the flavins in the two enzymes. We created a series of chimeric enzymes between nNOS and CYPOR in which the FMN binding and the connecting/FAD binding subdomains are swapped. A chimera consisting of the nNOS heme domain and FMN binding subdomain and the CYPOR FAD binding subdomain catalyzed significantly increased rates of cytochrome c reduction in the absence of CaM and of NO synthesis in its presence. Cytochrome c reduction by this chimera was inhibited by CaM. Other chimeras consisting of the nNOS heme domain, the CYPOR FMN binding subdomain, and the nNOS FAD binding subdomain with or without the tail region also catalyzed cytochrome c reduction, were not modulated by CaM, and could not transfer electrons into the heme domain. A chimera consisting of the heme domain of nNOS and the reductase domain of CYPOR reduced cytochrome c and ferricyanide at rates 2-fold higher than that of native CYPOR, suggesting that the presence of the heme domain affected electron transfer through the reductase domain. These data demonstrate that the FMN subdomain of CYPOR cannot effectively substitute for that of nNOS, whereas the FAD subdomains are interchangeable. The differences among these chimeras most likely result from alterations in the alignment of the flavins within each enzyme construct.     

Calmodulin-induced Conformational Control and Allostery Underlying Neuronal Nitric Oxide Synthase Activation

Nitric oxide synthase (NOS) is the primary generator of nitric oxide signals controlling diverse physiological processes such as neurotransmission and vasodilation. NOS activation is contingent on Ca^2 +/calmodulin binding at a linker between its oxygenase and reductase domains to induce large conformational changes that orchestrate inter-domain electron transfer. However, the structural dynamics underlying activation of full-length NOS remain ambiguous. Employing hydrogen–deuterium exchange mass spectrometry, we reveal mechanisms underlying neuronal NOS activation by calmodulin and regulation by phosphorylation. We demonstrate that calmodulin binding orders the junction between reductase and oxygenase domains, exposes the FMN subdomain, and elicits a more dynamic oxygenase active site. Furthermore, we demonstrate that phosphorylation partially mimics calmodulin activation to modulate neuronal NOS activity via long-range allostery. Calmodulin binding and phosphorylation ultimately promote a more dynamic holoenzyme while coordinating inter-domain communication and electron transfer.     

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.     

Lys842 in Neuronal Nitric-oxide Synthase Enables the Autoinhibitory Insert to Antagonize Calmodulin Binding, Increase FMN Shielding, and Suppress Interflavin Electron Transfer

Neuronal nitric-oxide synthase (nNOS) contains a unique autoinhibitory insert (AI) in its FMN subdomain that represses nNOS reductase activities and controls the calcium sensitivity of calmodulin (CaM) binding to nNOS. How the AI does this is unclear. A conserved charged residue (Lys⁸⁴²) lies within a putative CaM binding helix in the middle of the AI. We investigated its role by substituting residues that neutralize (Ala) or reverse (Glu) the charge at Lys⁸⁴². Compared with wild type nNOS, the mutant enzymes had greater cytochrome c reductase and NADPH oxidase activities in the CaM-free state, were able to bind CaM at lower calcium concentration, and had lower rates of heme reduction and NO synthesis in one case (K842A). Moreover, stopped-flow spectrophotometric experiments with the nNOS reductase domain indicate that the CaM-free mutants had faster flavin reduction kinetics and had less shielding of their FMN subdomains compared with wild type and no longer increased their level of FMN shielding in response to NADPH binding. Thus, Lys⁸⁴² is critical for the known functions of the AI and also enables two additional functions of the AI as newly identified here: suppression of electron transfer to FMN and control of the conformational equilibrium of the nNOS reductase domain. Its effect on the conformational equilibrium probably explains suppression of catalysis by the AI.     

Reductase domain of Drosophila melanogaster nitric-oxide synthase: redox transformations, regulation, and similarity to mammalian homologues

The nitric oxide synthase of Drosophila melanogaster (dNOS) participates in essential developmental and behavioral aspects of the fruit fly, but little is known about dNOS catalysis and regulation. To address this, we expressed a construct comprising the dNOS reductase domain and its adjacent calmodulin (CaM) binding site (dNOSr) and characterized the protein regarding its catalytic, kinetic, and regulatory properties. The Ca2+ concentration required for CaM binding to dNOSr was between that of the mammalian endothelial and neuronal NOS enzymes. CaM binding caused the cytochrome c reductase activity of dNOSr to increase 4 times and achieve an activity comparable to that of mammalian neuronal NOS. This change was associated with decreased shielding of the FMN cofactor from solvent and an increase in the rate of NADPH-dependent flavin reduction. Flavin reduction in dNOSr was relatively slow following the initial 2-electron reduction, suggesting a slow inter-flavin electron transfer, and no charge-transfer complex was observed between bound NADP+ and reduced FAD during the process. We conclude that dNOSr catalysis and regulation is most similar to the mammalian neuronal NOS reductase domain, although differences exist in their flavin reduction behaviors. The apparent conservation between the fruit fly and mammalian enzymes is consistent with dNOS operating in various signal cascades that involve NO.     

Crystal structure of the FAD/NADPH-binding domain of rat neuronal nitric-oxide synthase. Comparisons with NADPH-cytochrome P450 oxidoreductase

Nitric-oxide synthase (NOS) is composed of a C-terminal, flavin-containing reductase domain and an N-terminal, heme-containing oxidase domain. The reductase domain, similar to NADPH-cytochrome P450 reductase, can be further divided into two different flavin-containing domains: (a) the N terminus, FMN-containing portion, and (b) the C terminus FAD- and NADPH-binding portion. The crystal structure of the FAD/NADPH-containing domain of rat neuronal nitric-oxide synthase, complexed with NADP(+), has been determined at 1.9 A resolution. The protein is fully capable of reducing ferricyanide, using NADPH as the electron donor. The overall polypeptide fold of the domain is very similar to that of the corresponding module of NADPH-cytochrome P450 oxidoreductase (CYPOR) and consists of three structural subdomains (from N to C termini): (a) the connecting domain, (b) the FAD-binding domain, and (c) the NADPH-binding domain. A comparison of the structure of the neuronal NOS FAD/NADPH domain and CYPOR reveals the strict conservation of the flavin-binding site, including the tightly bound water molecules, the mode of NADP(+) binding, and the aromatic residue that lies at the re-face of the flavin ring, strongly suggesting that the hydride transfer mechanisms in the two enzymes are very similar. In contrast, the putative FMN domain-binding surface of the NOS protein is less positively charged than that of its CYPOR counterpart, indicating a different nature of interactions between the two flavin domains and a different mode of regulation in electron transfer between the two flavins involving the autoinhibitory element and the C-terminal 33 residues, both of which are absent in CYPOR.     

Intraprotein electron transfer in inducible nitric oxide synthase holoenzyme

Intraprotein electron transfer (IET) from flavin mononucleotide (FMN) to heme is essential in NO synthesis by NO synthase (NOS). Our previous laser flash photolysis studies provided a direct determination of the kinetics of the FMN–heme IET in a truncated two-domain construct (oxyFMN) of murine inducible NOS (iNOS), in which only the oxygenase and FMN domains along with the calmodulin (CaM) binding site are present (Feng et al. J. Am. Chem. Soc. 128, 3808–3811, 2006). Here we report the kinetics of the IET in a human iNOS oxyFMN construct, a human iNOS holoenzyme, and a murine iNOS holoenzyme, using CO photolysis in comparative studies on partially reduced NOS and a NOS oxygenase construct that lacks the FMN domain. The IET rate constants for the human and murine iNOS holoenzymes are 34 ± 5 and 35 ± 3 s⁻¹, respectively, thereby providing a direct measurement of this IET between the catalytically significant redox couples of FMN and heme in the iNOS holoenzyme. These values are approximately an order of magnitude smaller than that in the corresponding iNOS oxyFMN construct, suggesting that in the holoenzyme the rate-limiting step in the IET is the conversion of the shielded electron-accepting (input) state to a new electron-donating (output) state. The fact that there is no rapid IET component in the kinetic traces obtained with the iNOS holoenzyme implies that the enzyme remains mainly in the input state. The IET rate constant value for the iNOS holoenzyme is similar to that obtained for a CaM-bound neuronal NOS holoenzyme, suggesting that CaM activation effectively removes the inhibitory effect of the unique autoregulatory insert in neuronal NOS. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

NADPH analog binding to constitutive nitric oxide activates electron transfer and NO synthesis.

We report here that NADPH analogs such as 2'5'ADP, ATP, and 2'AMP paradoxically activate constitutive calcium/calmodulin regulated nitric oxide synthases (cNOS), including the endothelial isoform (eNOS) and the neuronal isoform (nNOS). These activators compete with NADPH by filling the binding site of the adenine moiety of NADPH, but do not occupy the entire NADPH binding domain. Effects of these analogs on cNOS's include increasing the electron transfer rate to external acceptors, as assessed by cytochrome c reductase activity in the absence of calmodulin. In addition, NO synthase activity in the presence of calmodulin (with or without added calcium) was increased by the addition of NADPH analogs. In contrast, the same NADPH analogs inhibit iNOS, the calcium insensitive inducible isoform, which lacks control elements found in constitutive isoforms. Because ATP and ADP are among the effective activators of cNOS isoforms, these effects may be physiologically relevant.     

Structure and function of NADPH-cytochrome P450 reductase and nitric oxide synthase reductase domain

NADPH-cytochrome P450 reductase (CPR) and the nitric oxide synthase (NOS) reductase domains are members of the FAD-FMN family of proteins. The FAD accepts two reducing equivalents from NADPH (dehydrogenase flavin) and FMN acts as a one-electron carrier (flavodoxin-type flavin) for the transfer from NADPH to the heme protein, in which the FMNH*/FMNH2 couple donates electrons to cytochrome P450 at constant oxidation-reduction potential. Although the interflavin electron transfer between FAD and FMN is not strictly regulated in CPR, electron transfer is activated in neuronal NOS reductase domain upon binding calmodulin (CaM), in which the CaM-bound activated form can function by a similar mechanism to that of CPR. The oxygenated form and spin state of substrate-bound cytochrome P450 in perfused rat liver are also discussed in terms of stepwise one-electron transfer from CPR. This review provides a historical perspective of the microsomal mixed-function oxidases including CPR and P450. In addition, a new model for the redox-linked conformational changes during the catalytic cycle for both CPR and NOS reductase domain is also discussed.     

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.     

Azo reduction of methyl red by neuronal nitric oxide synthase: the important role of FMN in catalysis

Nitric oxide synthase (NOS) is composed of an oxygenase domain and a reductase domain. The reductase domain has NADPH, FAD, and FMN binding sites. Wild-type nNOS reduced the azo bond of methyl red with a turnover number of approximately 130 min(-1) in the presence of Ca(2+)/calmodulin (CaM) and NADPH under anaerobic conditions. Diphenyleneiodonium chloride (DPI), a flavin/NADPH binding inhibitor, completely inhibited azo reduction. The omission of Ca(2+)/CaM from the reaction system decreased the activity to 5%. The rate of the azo reduction with an FMN-deficient mutant was also 5% that of the wild type. NADPH oxidation rates for the wild-type and mutant enzymes were well coupled with azo reduction. Thus, we suggest that electrons delivered from the FMN of the nNOS enzyme reduce the azo bond of methyl red and that this reductase activity is controlled by Ca(2+)/CaM.     

Nitric-oxide synthase output state. Design and properties of nitric-oxide synthase oxygenase/FMN domain constructs

Mammalian nitric-oxide synthases are large modular enzymes that evolved from independently expressed ancestors. Calmodulin-controlled isoforms are signal generators; calmodulin activates electron transfer from NADPH through three reductase domains to an oxygenase domain. Structures of the reductase unit and its homologs show FMN and FAD in contact but too isolated from the protein surface to permit exit of reducing equivalents. To study states in which FMN/heme electron transfer is feasible, we designed and produced constructs including only oxygenase and FMN binding domains, eliminating strong internal reductase complex interactions. Constructs for all mammalian isoforms were expressed and purified as dimers. All synthesize NO with peroxide as the electron donor at rates comparable with corresponding oxygenase constructs. All bind cofactors nearly stoichiometrically and have native catalytic sites by spectroscopic criteria. Modest differences in electrochemistry versus independently expressed heme and FMN binding domains suggest interdomain interactions. These interactions can be convincingly demonstrated via calmodulin-induced shifts in high spin ferriheme EPR spectra and through mutual broadening of heme and FMNH. radical signals in inducible nitric-oxide synthase constructs. Blue neutral FMN semiquinone can be readily observed; potentials of one electron couple (in inducible nitric-oxide synthase oxygenase FMN, FMN oxidized/semiquinone couple = +70 mV, FMN semiquinone/hydroquinone couple = -180 mV, and heme = -180 mV) indicate that FMN is capable of serving as a one electron heme reductant. The construct will serve as the basis for future studies of the output state for NADPH derived reducing equivalents.     

Differences in a conformational equilibrium distinguish catalysis by the endothelial and neuronal nitric-oxide synthase flavoproteins

Nitric oxide (NO) is a physiological mediator synthesized by NO synthases (NOS). Despite their structural similarity, endothelial NOS (eNOS) has a 6-fold lower NO synthesis activity and 6-16-fold lower cytochrome c reductase activity than neuronal NOS (nNOS), implying significantly different electron transfer capacities. We utilized purified reductase domain constructs of either enzyme (bovine eNOSr and rat nNOSr) to investigate the following three mechanisms that may control their electron transfer: (i) the set point and control of a two-state conformational equilibrium of their FMN subdomains; (ii) the flavin midpoint reduction potentials; and (iii) the kinetics of NOSr-NADP+ interactions. Although eNOSr and nNOSr differed in their NADP(H) interaction and flavin thermodynamics, the differences were minor and unlikely to explain their distinct electron transfer activities. In contrast, calmodulin (CaM)-free eNOSr favored the FMN-shielded (electron-accepting) conformation over the FMN-deshielded (electron-donating) conformation to a much greater extent than did CaM-free nNOSr when the bound FMN cofactor was poised in each of its three possible oxidation states. NADPH binding only stabilized the FMN-shielded conformation of nNOSr, whereas CaM shifted both enzymes toward the FMN-deshielded conformation. Analysis of cytochrome c reduction rates measured within the first catalytic turnover revealed that the rate of conformational change to the FMN-deshielded state differed between eNOSr and nNOSr and was rate-limiting for either CaM-free enzyme. We conclude that the set point and regulation of the FMN conformational equilibrium differ markedly in eNOSr and nNOSr and can explain the lower electron transfer activity of eNOSr.     

Energy Landscapes and Catalysis in Nitric-oxide Synthase

Nitric oxide (NO) plays diverse roles in mammalian physiology. It is involved in blood pressure regulation, neurotransmission, and immune response, and is generated through complex electron transfer reactions catalyzed by NO synthases (NOS). In neuronal NOS (nNOS), protein domain dynamics and calmodulin binding are implicated in regulating electron flow from NADPH, through the FAD and FMN cofactors, to the heme oxygenase domain, the site of NO generation. Simple models based on crystal structures of nNOS reductase have invoked a role for large scale motions of the FMN-binding domain in shuttling electrons from the FAD-binding domain to the heme oxygenase domain. However, molecular level insight of the dynamic structural transitions in NOS enzymes during enzyme catalysis is lacking. We use pulsed electron-electron double resonance spectroscopy to derive inter-domain distance relationships in multiple conformational states of nNOS. These distance relationships are correlated with enzymatic activity through variable pressure kinetic studies of electron transfer and turnover. The binding of NADPH and calmodulin are shown to influence interdomain distance relationships as well as reaction chemistry. An important effect of calmodulin binding is to suppress adventitious electron transfer from nNOS to molecular oxygen and thereby preventing accumulation of reactive oxygen species. A complex landscape of conformations is required for nNOS catalysis beyond the simple models derived from static crystal structures of nNOS reductase. Detailed understanding of this landscape advances our understanding of nNOS catalysis/electron transfer, and could provide new opportunities for the discovery of small molecule inhibitors that bind at dynamic protein interfaces of this multidimensional energy landscape.     

Nitric oxide synthase is a cytochrome P-450 type hemoprotein.

Nitric oxide has emerged as an important mammalian metabolic intermediate involved in critical physiological functions such as vasodilation, neuronal transmission, and cytostasis. Nitric oxide synthase (NOS) catalyzes the five-electron oxidation of L-arginine to citrulline and nitric oxide. Cosubstrates for the reaction include molecular oxygen and NADPH. In addition, there is a requirement for tetrahydrobiopterin. NOS also contains the coenzymes FAD and FMN and demonstrates significant amino acid sequence homology to NADPH-cytochrome P-450 reductase. Herein we report the identification of the inducible macrophage NOS as a cytochrome P-450 type hemoprotein. The pyridine hemochrome assay showed that the NOS contained a bound protoporphyrin IX heme. The reduced carbon monoxide binding spectrum shows an absorption maximum at 447 nm indicative of a cytochrome P-450 hemoprotein. A mixture of carbon monoxide and oxygen (80%/20%) potently inhibited the reaction (73-79%), showing that the heme functions directly in the oxidative conversion of L-arginine to nitric oxide and citrulline. Additionally, partially purified NOS from rat cerebellum was inhibited by CO, suggesting that this isoform may also contain a P-450-type heme. NOS is the first example of a soluble cytochrome P-450 in eukaryotes. In addition, the presence of FAD and FMN indicates that this is the first catalytically self-sufficient mammalian P-450 enzyme, containing both a reductase and a heme domain on the same polypeptide.