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There is still much that is unknown about how nitric oxide (NO) biosynthesis by NO synthase (NOS) isoform is tightly regulated at the molecular level. This is remarkable because impaired NO production in vivo has been implicated in an increasing number of diseases that currently lack effective treatments, including stroke and cancer. Given the significant public health burden of these diseases, the NOS enzyme family is a key target for development of new pharmaceuticals. Three NOS isoforms, inducible, endothelial and neuronal NOS (iNOS, eNOS and nNOS, respectively), achieve their key biological functions via intriguing regulations of interdomain electron transfer (IET) processes. Unlike iNOS, eNOS and nNOS isoforms are controlled by calmodulin (CaM) through facilitating catalytically significant IET processes. It is proposed that CaM activates NO synthesis in eNOS and nNOS through a conformational change of the flavin mononucleotide (FMN) domain from its shielded electron-accepting (input) state to a new electron-donating (output) state. The FMN–heme IET within the NOS output state is essential for NO synthesis at the catalytic heme. Due to lack of reliable techniques for specifically determining the inter-domain FMN–heme interactions and their direct effects on the catalytic heme center, the molecular mechanism that underlies the output state formation remains elusive. The recent developments in our understanding of mechanisms of the NOS output state formation that are driven by a combination of molecular biology, laser flash photolysis, and spectroscopic techniques are the subject of this perspective.
NOS is the enzyme responsible for the oxidation of L-arginine (Arg) to NO, a signal for vasodilatation and neurotransmission at low concentrations and a defensive cytotoxin at higher concentrations 1, 2. NO’s availability is tightly regulated at the synthesis level by NOS. Aberrant NO synthesis by NOS is associated with an increasing number of human pathologies, including stroke, inflammation, arthritis, and cancer 2, 3. Selective NOS modulators are required for therapeutic intervention because of the ubiquitous nature of NO in mammalian physiology, and the fact that multiple NOS isoforms are each capable of producing NO in vivo. Three NOS isoforms function differently in human health and disease. For example, in acute ischemic stroke, NO produced by the neuronal NOS or inducible NOS (nNOS or iNOS) is detrimental, whereas NO derived from the endothelial NOS (eNOS) and the accompanying dilation are beneficial 4. Previous advances in understanding the structural and functional mechanisms of this enzyme class led to identification of agents that are designed to modulate the various NOS enzymes 5. However, agents that selectively modulate NOS isoform activity remain elusive for clinical usages because of incomplete understanding of NOS regulation at the molecular level. Clearly, molecular mechanisms of NOS regulation, once fully understood, are potentially key targets for development of selective new pharmaceuticals for treating a wide range of diseases that currently lack effective treatments. For example, an exogenous synthetic peptide was designed based upon an internal fragment of eNOS proteins important in controlling electron transfer processes in NOS 6. This peptide enhances pulmonary artery vasorelaxation through directly activating NOS activity, and thus has significant potential therapeutic applications in the regulation of pulmonary vascular function.
There are three mammalian NOS isoforms: eNOS, nNOS, and iNOS. Mammalian NOS is a homodimeric flavo-hemoprotein that catalyzes the oxidation of L-Arg to NO and L-citrulline with NADPH and O2 as co-substrates (Scheme 1) 7.
Each subunit contains a C-terminal electron-supplying reductase domain with binding sites for NADPH (the electron source), flavin adenine dinucleotide (FAD) and FMN, and an N-terminal catalytic heme-containing oxygenase domain. The oxygenase and reductase domains are connected through a CaM-binding region, which is irreversibly bound to CaM in iNOS at all physiological Ca2+ concentrations, whereas reversible CaM binding to the region of nNOS and eNOS requires an increase in intracellular Ca2+ ,8. The major difference between the CaM-regulated isoforms of nNOS/eNOS and the Ca2+-insensitive isoform iNOS is the presence of intrinsic control elements 8, including a unique autoregulatory (AR) insert within the FMN domain of eNOS/nNOS 9 and the C-terminal tail 10.
There are crystal structures available for the oxygenase domains of each of the NOS isoforms 11–14, the reductase constructs 15, 16 and NOS homologues 17, as well as CaM bound to peptides corresponding to the CaM-binding sequence in human eNOS 18 and nNOS (PDB 2O60). The substrate, L-arginine, and a cofactor, (6R)-5,6,7,8 tetrahydrobiopterin, both bind near the heme center in the oxygenase domain 11–13. These crystal structures provide important information on the mechanism for controlling NO synthesis by NOS. Based on the crystal structures, a combination of kinetic methods and redox titrations, along with site-directed mutagenesis, have provided useful information about the roles of specific NOS amino acid residues within the reductase domain 19–24, oxygenase domain 25, 26, and CaM-binding region 27 in modulating NOS function.
However, no structures of the complete NOS holoenzyme are available; this is presumably due to dynamic structural rearrangements that occur within the reductase domain and CaM-binding domains of the NOS enzymes (see below). As a consequence, the structural basis for the assembly of NOS domains and CaM during catalysis remains unknown. It is thus of current interest to obtain crystal structures of CaM-bound NOS constructs/domains.
Although knowledge of the NOS catalytic mechanism is incomplete 28–32, it is well established that interdomain electron transfer (IET) processes are key steps in NO synthesis by coupling reactions between domains (Fig. 1) 7, 33, 34. Unlike iNOS, eNOS and nNOS synthesize NO in a Ca2+/CaM dependent manner. Here CaM-binding facilitates the IET reaction from the FAD hydroquinone (FADH2) to FMN semiquinone (FMNH•) within subunit A (reaction 1) 35, and enables IET from the FMN hydroquinone (FMNH2) to the catalytic heme iron in the oxygenase domain of subunit B (reaction 2) 36. It is generally accepted that CaM binding has little or no effect on the thermodynamics of redox processes in NOS 37–39, which indicates that the regulation by CaM is accomplished dynamically by controlling redox-linked conformational changes required for effective IET.
Importantly, the CaM-controlled FMN–heme IET (reaction 2, Fig. 1) is essential in coupling electron transfer in the reductase domain with NO synthesis in the oxygenase domain by the delivery of electrons required for O2 activation in the catalytic heme domain, and is thus under extraordinarily tight control.
Control of the IET processes in eNOS/nNOS by CaM has been shown to involve intrinsic control elements in NOS: the CaM binding site, the AR in the FMN binding domain, and the C-terminal tail, which regulate the IET in a concerted manner 9, 10, 40–42. A unique AR (~ 40 amino acids) within the FMN domain of eNOS/nNOS 9, 40 pins the FMN domain to the reductase complex via a network of hydrogen bonds in the CaM-free state 16. Electron transfer modulation appears also to involve a much smaller insertion present in the hinge subdomain 43 as well as C-terminal tail differences 10. Control element deletion studies indicate that the control elements repress electron flow into and out of the NOS reductase domain in the CaM-free state, and CaM-binding relieves the repression 10, 42, 44–47. A recent crystal structure of an intact nNOS reductase domain has revealed key information about how the control elements may repress electron transfer in the CaM-free state (see below) 16.
An “FMN-domain tethered shuttle” model was originally proposed by Salerno and Ghosh 48, 49. This model involves the swinging of the FMN domain from its original electron-accepting (input) state to a new electron-donating (output) state (Fig. 2). This molecular rearrangement facilitates efficient IET between the FMN and the catalytic heme in the oxygenase domain (reaction 2 in Fig. 1). CaM binding unlocks the input state, thereby enabling the FMN domain to shuttle between the two enzyme states. The tethered shuttle model thus involves an input state that corresponds to the structure of a nNOS reductase domain in the CaM-free state (Fig. 3, see below) 16, and an output state in which the FMN binding domain associates with the oxygenase domain.
Crystal structure studies on an intact nNOS reductase domain construct in the CaM-free state 16 reveal a structure in which FMN is in close proximity with FAD, as required for electron transfer in the input state of FMN from NADPH through FAD (Fig. 3). This structure is not as it exists in the output state; FMN cannot be located to within tunneling distance of the heme, and thus electrons cannot be transferred to the heme in this state. This constrained state of FMN is locked through a concerted interplay of control elements such as the C-terminal tail and the AR (Fig. 3)16, 50.
In order for electrons to be transferred from the FMN to heme for activating NO production, the FMN domain must be unlocked via CaM binding, and must move to another position such that it is accessible to the heme. The formation of the output state involves two primary steps: (1) dissociation of the FMN domain from its reductase binding site, triggered by CaM binding, and (2) the subsequent re-association of FMN with the oxygenase domain (Fig. 2). Mechanistic information suggests that the rate-limiting step in NOS catalysis is the formation of the output state, because NAPDH reduction is 3 orders of magnitude faster than NO production, and cytochrome (cyt) c reduction is intermediate between the two 51. The NOS state formed at step (1) is competent to reduce cyt c, which requires accessibility of FMN to cyt c. This is necessary but not sufficient for NO production by eNOS/nNOS, which in addition requires CaM dependent promotion of the oxygenase-FMN domain interactions (step 2).
The structure of the functional output state has not yet been determined. This makes studies that focus on crucial interaction sites in regulating the formation of the output state very important. A promising strategy is to use a combined kinetics (in particular kinetics of discrete IET steps), spectroscopic and site-directed mutagenesis approach to probe the interdomain FMN-heme interactions within the NOS output state.
Our laboratories have been using laser flash photolysis to determine the kinetics of the FMN–heme IET in the output state for NO production. In this Perspective, we will first briefly review the laser flash photolysis approach that has allowed the attainment of new levels of insight into the IET between the FMN and Fe centers in NOS enzymes. We will review IET studies on NOS bi-domain oxygenase/FMN constructs that are validated models of the NOS output state. The IET kinetic studies on full-length NOS enzymes and the roles of control elements on the FMN–heme IET in the nNOS holoenzyme will also be discussed. Finally, we will discuss recent kinetics and thermodynamics studies by other researchers that are closely relevant to the NOS output state of the tethered shuttle mechanism.
Laser flash photolysis is a powerful technique to study rapid IET kinetics in proteins 52, 53. We have successfully developed a new CO photolysis approach (Scheme 2) to directly measure the FMN–heme IET in the NOS enzymes 49, 54–57.
Scheme 2 summarizes the processes that take place after photolysis of the Fe(II)–CO complex in a partially reduced form (i.e. [Fe(II)–CO][FMNH•]) of the nNOS isoform. In the Scheme, species in dashed boxes are CO-bound forms, whereas those in solid boxes are CO-free and participate in the FMN–heme IET. The laser-induced CO dissociation results in a drop of the midpoint potential of the heme and rapidly converts a good electron acceptor (the Fe(II)–CO complex) into an electron donor (the free Fe(II) species), favoring the IET from Fe(II) to FMNH•. In the absence of added CaM, CO rebinding to Fe(II) (reaction 2) competes well with the slow IET between the heme and FMN domains (reaction 3), whereas in the presence of CaM, CO rebinding is a poor competitor for efficient IET (reaction 4). A CO/Ar mixture is used to retard the CO rebinding rate (reaction 2), and thus favor the IET from Fe(II) to FMNH• in the CaM-bound nNOS (reaction 4). This makes the loss of FMNH• observable as a bleaching at 580–600 nm (Fig. 4). Briefly, the NOS solution in the presence of deazariboflavin is illuminated for an appropriate period of time (~90 seconds) to obtain [Fe(II)–CO][FMNH•], and the partially reduced protein is subsequently flashed with 450 nm laser excitation to dissociate CO from Fe(II)–CO, and generate a transient Fe(II) species that is able to transfer one electron to the FMNH• intramolecularly to produce FMNH2 and Fe(III).
It is important to note that we can individually time-resolve the consumption of FMNH• (due to the IET to Fe(II)) and CO rebinding, and thereby reliably measure the rapid FMN–heme IET (Fig. 4) followed by a much slower CO rebinding process (Fig. 5). Heme reduction in the NOS isoforms was indirectly probed by formation of the Fe(II)–CO complex using a stopped-flow approach, which is unable to distinguish these two reactions as only formation of the Fe(II)–CO complex was observed 36, 58. Thus our CO photolysis approach offers clear advantages since it allows us to observe both processes directly. Most importantly, our recent results 55 demonstrate that the FMN–heme IET kinetics can be used as a direct measure for formation of the NOS output state, and the CO photolysis method is thus a powerful approach in studying the roles of specific regions in formation of the output state (see below).
Our experiments do not involve any step other than the IET between the FMN and heme domains, and therefore provide the first direct determination of the kinetics of the following IET process between catalytically significant redox couples of the FMN and heme centers (eq 1):
Note that the FMN–heme IET is reversible because the redox couples of Fe(III)/Fe(II) and FMNH2/FMNH• are nearly iso-potential 59, and therefore the IET rate constant of the backward reaction (i.e. heme reduction) is about one-half of the observed rate constant, which is the sum of the forward and backward rate constants.
We have also conducted similar CO photolysis experiments on the NOS oxygenase construct, which does not contain FMN. Therefore by carefully comparing the absorption changes of NOS oxyFMN and oxygenase constructs occurring upon a 450 nm laser pulse, we can distinguish the process of FMN–heme IET from rebinding of CO to Fe(II). For example, in Fig. 4, the 600 nm trace of nNOS oxyFMN is a bleaching (i.e. loss of absorbance), whereas the trace of nNOS oxygenase stays above the baseline. This comparison gives us definite evidence that the bleaching is due to consumption of FMNH• triggered by formation of free Fe(II), i.e. the FMN–heme IET (eq 1).
Another important control experiment is that we always collect traces with different amplitude of signals, i.e. at different intermediate protein concentrations generated by the flash. We thus confirm that this process is protein concentration independent, which indicates an intra-protein process. This is important because other inter-protein processes may confound the absorbance changes, and if so, the observed rate would depend upon protein concentration. Therefore, the kinetics must be determined at various protein concentrations to be sure that we are studying the intra-protein IET.
To favor observation of the output state of the shuttle mechanism, Ghosh and Salerno designed two-domain NOS oxyFMN constructs in which only the oxygenase and FMN domains along with the CaM-binding region are expressed (Fig. 6) to favor the interaction of the FMN binding domain with the oxygenase domain over interactions within the reductase unit 59. The absence of the NADPH-FAD binding domain removes the dominant input state complex from the conformational repertoire of the construct. This provides us with a greatly enhanced opportunity to observe the putative output state, in which the FMN binding site is closely associated with the oxygenase active site rather than with the NADPH-FAD binding domain. The homologous dimeric oxyFMN construct is active and stable, binds cofactors nearly stoichiometrically, and has characteristic absorption and EPR spectra similar to the holoenzyme 59. Potentiometric titration studies have shown that the redox potentials of the oxyFMN construct are comparable to the holoenzyme 59. Therefore the truncated construct appears to be a reasonable approximation of the holoenzyme. Our IET kinetics data have further demonstrated that these oxyFMN constructs are well-validated models of the NOS output state (see below).
The kinetics of FMN–heme IET in a rat nNOS oxyFMN construct in the presence and absence of added CaM 49, in a murine iNOS oxyFMN 57 and in a human iNOS oxyFMN 54 were directly determined using laser flash photolysis of CO dissociation in comparative studies on partially reduced oxyFMN and single domain heme-containing oxygenase constructs. For nNOS oxyFMN, the IET rate constant in the presence of CaM (262 s−1) was increased approximately ten-fold compared to that in the absence of CaM (22 s−1) 49. We believe that the slow but still substantial rate of IET in the absence of CaM is real and may reflect the different conformations of the CaM-bound and CaM-free proteins.
The effect of CaM on inter-domain interactions was further evidenced by EPR spectra; upon adding CaM, changes in the line shape and g values of the high spin heme EPR signal have been observed 49. This distortion is probably due to the CaM-driven association of the FMN and heme domains in the nNOS oxyFMN construct. Such distortion effects have not yet been reported in the holoenzyme, probably because most of the holoenzyme is in the input state. Collectively, these kinetic and EPR results provide the first direct evidence of CaM control of electron transfer between FMN and heme domains through facilitation of the interdomain FMN–heme interactions in the output state. Therefore, CaM controls IET between heme and FMN domains by a conformational gated mechanism. This is essential in coupling electron transfer in the reductase domain in NOS with NO synthesis in the oxygenase domain.
Previously, CaM control of NOS electron transfer was believed to be primarily localized within the reductase portion of the enzyme 60. Evidence for multiple steps of CaM control was attributed to separate CaM modulation of FMN reduction and heme reduction (32, 33). Our recent experiments with the oxyFMN constructs have clearly demonstrated CaM dependence outside the reductase complex, i.e. CaM-dependent formation of the output state, through the FMN domain interacting with the heme in the oxygenase domain 49. This may be the original role of CaM in the evolution of NOS proteins, and could predate the evolution of the modern control elements.
We have further extended our CO photolysis approach to measure the FMN–heme IET kinetics in a rat nNOS holoenzyme56, and in murine and human iNOS holoenzymes 54. The IET kinetics for the NOS holoenzymes are approximately an order of magnitude slower than the corresponding NOS oxyFMN constructs. This fact suggests that in the holoenzyme the rate-limiting step in the IET reaction is the conversion of the input state to the output state (i.e., the first step in the following scheme, in which the FMN becomes accessible to the heme), and that the role of CaM is to allow this conversion to occur. The IET reaction scheme in the holoenzyme can thus be represented as follows:
where sq represents “semiquinone”, hq represents “hydroquinone”, red represents “reduced”, and ox represents “oxidized”.
Note that in the iNOS oxyFMN and nNOS oxyFMN (with added CaM) constructs, the IET rate constants are much larger and not equal in the oxyFMN constructs, although they are similar in order of magnitude (850±50, 320±45 and 262±40 s−1, for oxyFMN constructs of murine iNOS 57, human iNOS, and rat nNOS 56, respectively). Therefore the rate-limiting factor in the nNOS and iNOS holoenzyme is not the FMN–heme IET per se. As a preliminary interpretation, we propose that at least some of the interactions that constrain the holoenzyme to be in the input state are lost in these oxyFMN constructs, making it easier for the truncated protein to achieve the output state, thereby increasing the IET rate constant. Structural studies will be required in order to better understand this difference.
The fact that there is no rapid IET component in the traces obtained with the iNOS holoenzyme implies that the holoenzyme is mainly in the input state, despite having CaM bound to it; the iNOS holoenzyme is still constrained by the contacts that exist which control the rate constant for the IET 54.
CaM unlocks the input state, thereby enabling the FMN domain to shuttle between the two enzyme states, and thus make contacts with the heme domain (Fig. 2); the CaM effect is primarily kinetic, although there may be a thermodynamic component as well 61. CaM does not affect the kinetics of the rate-limiting step (i.e. conversion of the input state to output state, see above); the evidence for this is that the IET rate constant for the holoenzyme is smaller than that for the oxyFMN construct. Thus in the holoenzyme, the IET rate constant is still controlled by the conversion step, even when CaM is present. The IET in the oxyFMN constructs are faster because it has lost contacts with the FAD domain that constrain this motion.
It is interesting that the reduction of oxygenase heme and of cyt c both require release of the FMN domain from the input state, but that Vmax for cyt c reduction 10 is an order of magnitude faster than the reduction of heme measured by laser flash photolysis. For cyt c reduction one can postulate a reaction first order in ferric cyt c and in accessible FMN domains; the rate of formation of the complex with cyt c is then the second order rate constant kcf (cyt c complex formation) times the concentration of enzyme molecules with accessible FMN binding domains. The rapid rate of cyt c reduction could be accounted for by a large value of kcf, but it is more informative to consider the ‘free’ FMN binding domain as partitioning into a range of conformational states. If a much large number of these states are accessible to cyt c than to the oxygenase domain, one would expect much more rapid reduction of cyt c under saturated cyt c concentration. This is a reasonable expectation given the small size of cyt c and the conformational constraints placed on the oxygenase/FMN complex by the dimeric structure of NOS and the connecting polypeptides linking the domains.
Increasing evidence shows that the AR exerts its regulatory function by stabilizing certain NOS states via interdomain interactions. Identified originally from sequence alignments and modeling and postulated to restrict alignment of the FMN binding domain with the oxygenase and/or FAD binding domains 9, this AR insert within the FMN domain, in the absence of CaM, locks the FMN binding domain to the reductase complex via a network of hydrogen bonds so as to obstruct CaM binding and enzyme activation (Fig. 3) 16. When CaM binds to the linker between the FMN and oxygenase domains at high [Ca2+], the AR insert is proposed to be displaced so that the enzyme can be activated. Evidence for a conformational change in this AR region upon CaM binding came from both proteolysis and fluorescence experiments 9. Importantly, recent studies by Roman and Masters suggest that the AR insert may also be involved in stabilizing the output state of the FMN domain for NO synthesis in the presence of CaM 23. Indeed, in the crystal structure of the nNOS reductase 16, the AR insertion is well positioned to interact with the oxygenase or FMN domain; 28 of the 42 residues in the AR insert are not observable, suggesting flexibility of the AR insert.
We have conducted comparative CO photolysis kinetic studies on wild type and the AR insert-deletion mutant of full-length rat nNOS proteins, to directly investigate the role of the unique AR insert in the FMN–heme IET 55. Although the amplitude of the IET kinetic traces was decreased two- to three-fold, the AR deletion did not change the rate constant for the calmodulin-controlled IET. This suggests that the rate-limiting conversion of the electron-accepting state to a new electron-donating (output) state does not involve interactions with the AR insert, but that the AR may stabilize the output state once it is formed. Our results indicate an important role of the AR insert in stabilizing interdomain FMN–heme interactions in the output state, rather than simply playing the role of an inhibitory element 9. This provides new information on the role of the AR insert in regulating electron transfer in the nNOS isoform, and further demonstrates that the IET kinetics measured by our CO photolysis approach can be used as a direct measure for the output state formation in full-length NOS proteins, i.e. the efficiency of the FMN domain docking onto the heme domain for effective IET.
In the iNOS holoenzyme the rate constant for the IET between heme and FMN is indistinguishable from our previously reported rate for CaM activated nNOS holoenzyme 56. This is a remarkable observation considering that in the nNOS holoenzyme the IET in the absence of CaM is too slow to measure 56. Control of the IET processes in eNOS/nNOS by CaM has been shown to mainly involve the CaM binding site 62, and the unique AR insert within the FMN binding domain 9, 40. The AR insert within the FMN domain, in the absence of CaM, locks the FMN binding domain to the reductase complex via a network of hydrogen bonds so as to obstruct CaM binding and enzyme activation 16. When CaM binds to the linker between the FMN and oxygenase domains at high [Ca2+], the AR insert is proposed to be displaced so that the enzyme can be activated. Indeed, our results suggest that in the nNOS holoenzyme CaM activation effectively removes the restraints imposed by the nNOS unique AR insert on the release of the FMN binding domain, at least in single turnover.
The tethered shuttle model is also strongly supported by recent kinetic studies by other researchers 19, 63–66. Interdomain FAD/FMN interactions have been shown to be also important in modulating electron transfer from FAD to the heme via FMN motions 19, 66. Moreover, it has been suggested that regulation of the FMN conformational equilibrium differs markedly in reductase domain constructs of eNOS and nNOS, and this difference can explain the lower electron transfer activity of the eNOS reductase domain construct 64. Interestingly, a single mutation in the FMN-binding loop of nNOS leads to a FMN conformation that resembles the output state of the CaM-bound nNOS with respect to the cyt c reduction activity 21.
Another recent interesting study showed that the hinge region, which tethers the FMN domain to the connecting domain between the FMN and FAD domains, is important in restricting the activity of eNOS relative to other NOS enzymes.65 This information strongly supports the tethered shuttle model in which FMN motion controlled by CaM binding is critical in regulating NO production in NOS isoforms. More importantly, the fact that the hinge substitution did not completely convert eNOS to nNOS (regarding the NO synthesis activity) implies that other structural elements of the eNOS reductase domain must also help restrict electron flux to the heme.65 These results support a synergistic control mechanism of formation of the NOS output state through CaM binding, and intrinsic control elements such as the AR insert, and C-terminal tail.
The aim of the present review is to highlight current mechanistic information about the NOS output state. It has become clear from these studies that our understanding of NOS enzymes will be moved forward by increasing applications of a combined approach of molecular biology, rapid kinetics (laser flash photolysis and stopped flow), redox titrations, advanced spectroscopy, protein crystallography, and computational modeling. This is clearly a fertile area for future study. Although considerable progress has been obtained, some outstanding questions remain unanswered, and certainly deserve further investigation at the molecular level. These can be summarized as follows.
The NOS output state is a complex between the FMN domain and the catalytic heme domain, and it thus facilitates the catalytically significant IET between the FMN and heme centers. Recent kinetic studies have strongly supported the important role of FMN motion in interdomain electron transfer processes during NOS catalysis. Evidence is rapidly accumulating to show that CaM controls formation of the output state through facilitation of the interdomain FMN–heme interactions. Nonetheless, the molecular mechanism that underlies the CaM-modulated output state formation remains elusive. There is a clear need to understand how the FMN–heme IET is modulated at the molecular level, and how this IET step specifically regulates catalytic activity of the various NOS isoforms. A combined kinetic, spectroscopic and site-directed mutagenesis approach will permit detailed investigation of molecular mechanisms that underly formation of the NOS output state for NO production.
We thank many colleagues who have worked with us on NOS enzymes. In particular we are grateful to Dr. Dipak Ghosh at Duke University, Prof. John Salerno at Kennesaw State University and Prof. Guy Guillemette at University of Waterloo for providing oxyFMN constructs, and Prof. Bettie Sue Masters and Dr. Linda Roman at University of Texas Health Science Center at San Antonio for providing full-length NOS proteins/mutants, as well as for critical discussions during our collaborations. The research was supported by grant GM081811 and HL091280 to CF and NM-INBRE P20RR016480.