Mammalian Nitric Oxide Synthase Research Articles

The Role of Heme Peroxo Oxidants in the Rational Mechanistic Modeling of Nitric Oxide Synthase: Characterization of Key Intermediates and Elucidation of the Mechanism.

Mammalian nitric oxide synthase (NOS) mediates the two-step O2 -dependent oxidative degradation of arginine, and has been linked to a medley of disease situations in humans. Nonetheless, its exact mechanism of action still remains unclear. This work presents the first NOS model system where biologically proposed heme superoxo and peroxo intermediates are assessed as active oxidants against oxime substrates. Markedly, heme peroxo intermediates engaged in a bioinspired oxime oxidation reaction pathway, converting oximes to ketones and nitroxyl anions (NO- ). Detailed thermodynamic, kinetic, and mechanistic interrogations all evince a rate-limiting step primarily driven by the nucleophilicity of the heme peroxo moiety. Coherent with other findings, 18 O and 15 N isotope substitution experiments herein suffice compelling evidence toward a detailed mechanism, which draw close parallels to one of the enzymatic proposals. Intriguingly, recent enzymatic studies also lend credence to these findings, and several relevant reaction intermediates have been observed during NOS turnover.

Nitric oxide alleviates salt-induced stress damage by regulating the ascorbate–glutathione cycle and Na+/K+ homeostasis in Nitraria tangutorum Bobr

Nitric oxide (NO) is an important signaling molecule involved in mediation of salt stress induced physiological responses in plants. In this study, we investigated the effect of NO on Nitraria tangutorum seedlings exposed to salt stress. Exogenous application of NO donor, sodium nitroprusside (SNP) increased fresh weight, shoot and root elongation and decreased electrolyte leakage and malondialdehyde (MDA) content in N. tangutorum seedlings under salt stress. Simultaneously, leaf senescence and root damage induced by salt stress were alleviated. SNP effectively increased NO content both in leaves and roots of plants under salt stress. Meanwhile, SNP activated the ascorbate-glutathione (AsA-GSH) cycle by increasing antioxidants contents, antioxidant enzymes activities, and related genes expression, thereby scavenging reactive oxygen species (ROS) and alleviating oxidative damage caused by salt stress. SNP alleviated salt stress induced ion toxicity by promoting Na+ efflux and ion transporter gene expression and reducing Na+ content and the Na+/K+ ratio. In addition, application of NO specific scavenger cPTIO and mammalian NO synthase inhibitor L-NAME sifnificantly aggravated stress damage in plant under salt stress. These results show the beneficial impacts of NO as a stress-signaling molecule that positively regulates defense response in N. tangutorum to salt stress.

Identification of Partner Proteins of the Algae Klebsormidium nitens NO Synthases: Toward a Better Understanding of NO Signaling in Eukaryotic Photosynthetic Organisms.

In animals, NO is synthesized from L-arginine by three isoforms of nitric oxide synthase (NOS) enzyme. NO production and effects have also been reported in plants but the identification of its sources, especially the enzymatic ones, remains one of the critical issues in the field. NOS-like activities have been reported, although there are no homologs of mammalian NOS in the land plant genomes sequenced so far. However, several NOS homologs have been found in algal genomes and transcriptomes. A first study has characterized a functional NOS in the chlorophyte Ostreococcus tauri and the presence of NOS homologs was later confirmed in a dozen algae. These results raise the questions of the significance of the presence of NOS and their molecular diversity in algae. We hypothesize that comparisons among protein structures of the two KnNOS, together with the identification of their interacting partner proteins, might allow a better understanding of the molecular diversification and functioning of NOS in different physiological contexts and, more generally, new insights into NO signaling in photosynthetic organisms. We recently identified two NOS homologs sequences in the genome of the streptophyte Klebsormidium nitens, a model alga in the study of plant adaptation to terrestrial life. The first sequence, named KnNOS1, contains canonical NOS signatures while the second, named KnNOS2, presents a large C-ter extension including a globin domain. In order to identify putative candidates for KnNOSs partner proteins, we draw the protein–protein interaction networks of the three human NOS using the BioGRID database and hypothesized on the biological role of K. nitens orthologs. Some of these conserved partners are known to be involved in mammalian NOSs regulation and functioning. In parallel, our methodological strategy for the identification of partner proteins of KnNOS1 and KnNOS2 by in vitro pull-down assay is presented.

Direct measurement of nitric oxide (NO) production rates from enzymes using ozone-based gas-phase chemiluminescence (CL)

Nitric oxide (NO) chemiluminescence detectors (CLDs) are specialized and sensitive spectroscopic instruments capable of directly measuring NO flux rates. NO CLDs have been instrumental in the characterization of mammalian nitrite-dependent NO synthases. However, no detailed description of NO flux analysis using NO CLD is available. Herein, a detailed review of the NO CL methodology is provided with guidelines for measuring NO-production rates from aqueous samples, such as isolated enzymes or protein homogenates. Detailed description of the types of signals one can encounter, data processing, and potential pitfalls related to NO flux measurements will also be covered.

Evidence for an arginine-dependent route for the synthesis of NO in the model filamentous fungus Aspergillus nidulans.

Nitric oxide (NO) is a signalling molecule in eukaryotic and prokaryotic organisms. NO levels transiently boost upon induction of conidiation in Aspergillus nidulans. Only one pathway for NO synthesis involving nitrate reductase has been reported in filamentous fungi so far, but this does not satisfy all the NO produced in fungal cells. Here we provide evidence for at least one additional biosynthetic pathway in A. nidulans involving l-arginine or an intermediate metabolite as a substrate. Under certain growth conditions, the addition of l-arginine to liquid media elicited a burst of NO that was not dependent on any of the urea cycle genes. The NO levels were controlled by the metabolically available arginine, which was regulated by mobilization from the vacuoles and during development. In vitro assays with protein extracts and amino acid profiling strongly suggested the existence of an arginine-dependent NO pathway analogous to the mammalian NO synthase. Addition of polyamines induced NO synthesis, and mutations in the polyamine synthesis genes puA and spdA reduced the production of NO. In conclusion, here we report an additional pathway for the synthesis of NO in A. nidulans using urea cycle intermediates.

Nitrogen Depletion Blocks Growth Stimulation Driven by the Expression of Nitric Oxide Synthase in Tobacco.

Nitric oxide (NO) is a messenger molecule widespread studied in plant physiology. Latter evidence supports the lack of a NO-producing system involving a NO synthase (NOS) activity in higher plants. However, a NOS gene from the unicellular marine alga Ostreococcus tauri (OtNOS) was characterized in recent years. OtNOS is a genuine NOS, with similar spectroscopic fingerprints to mammalian NOSs and high NO producing capacity. We are interested in investigating whether OtNOS activity alters nitrogen metabolism and nitrogen availability, thus improving growth promotion conditions in tobacco. Tobacco plants were transformed with OtNOS under the constitutive CaMV 35S promoter. Transgenic tobacco plants expressing OtNOS accumulated higher NO levels compared to siblings transformed with the empty vector, and displayed accelerated growth in different media containing sufficient nitrogen availability. Under conditions of nitrogen scarcity, the growth promoting effect of the OtNOS expression is diluted in terms of total leaf area, protein content and seed production. It is proposed that OtNOS might possess a plant growth promoting effect through facilitating N remobilization and nitrate assimilation with potential to improve crop plants performance.

Importance of Val567 on heme environment and substrate recognition of neuronal nitric oxide synthase.

Nitric oxide (NO) produced by mammalian nitric oxide synthases (mNOSs) is an important mediator in a variety of physiological functions. Crystal structures of mNOSs have shown strong conservation of the active‐site residue Val567 (numbering for rat neuronal NOS, nNOS). NOS‐like proteins have been identified in several bacterial pathogens, and these display striking sequence identity to the oxygenase domain of mNOS (NOSoxy), with the exception of a Val to Ile mutation at the active site. Preliminary studies have highlighted the importance of this Val residue in NO‐binding, substrate recognition, and oxidation in mNOSs. To further elucidate the role of this valine in substrate and substrate analogue recognition, we generated five Val567 mutants of the oxygenase domain of the neuronal NOS (nNOSoxy) and used UV‐visible and EPR spectroscopy to investigate the effects of these mutations on the heme distal environment, the stability of the heme‐FeII‐CO complexes, and the binding of a series of substrate analogues. Our results are consistent with Val567 playing an important role in preserving the integrity of the active site for substrate binding, stability of heme‐bound gaseous ligands, and potential NO production.

Deciphering mechanism of conformationally controlled electron transfer in nitric oxide synthases.

Electron transfer is a fundamental process in life that is very often coupled to catalysis within redox enzymes through a stringent control of protein conformational movements. Mammalian nitric oxide synthase (NOS) proteins are redox flavo-hemoproteins consisting of multiple modular domains. The NOS enzyme is exquisitely regulated in vivo by its partner, the Ca2+ sensing protein calmodulin (CaM), to control production of nitric oxide (NO). The importance of functional domain motion in NOS regulation has been increasingly recognized. The significant size and flexibility of NOS is a tremendous challenge to the mechanistic studies. Herein recent applications of modern biophysical techniques to NOS problems have been critically analyzed. It is important to note that any current biophysical technique alone can only probe partial aspects of the conformational dynamics due to limitations in the technique itself and/or the sample preparations. It is necessary to combine the latest methods to comprehensively quantitate the key conformational aspects (conformational states and distribution, conformational change rates, and domain interacting interfaces) governing the electron transfer. This is to answer long-standing central questions about the NOS isoforms by defining how specific CaM-NOS interactions and regulatory elements underpin the distinct conformational behavior of the NOS isoform, which in turn determine unique electron transfer and NO synthesis properties. This review is not intended as comprehensive, but as a discussion of prospects that promise impact on important questions in the NOS enzymology field.

Exploring the conformations of nitric oxide synthase with fluorescence.

Multi-domain oxidoreductases are a family of enzymes that catalyze oxidation-reduction reactions through a series of electron transfers. Efficient electron transfer requires a sequence of protein conformations that position electron donor and acceptor domains in close proximity to each other so that electron transfer can occur efficiently. An example is mammalian nitric oxide synthase (NOS), which consists of an N-terminal oxygenase domain containing heme and a C-terminal reductase domain containing NADPH/FAD and FMN subdomains. We describe the use of time-resolved and single-molecule fluorescence to detect and characterize the conformations and conformational dynamics of the neuronal and endothelial isoforms of NOS. Fluorescence signals are provided by a fluorescent dye attached to the Ca2+-signaling protein calmodulin (CaM), which regulates NOS activity. Time-resolved fluorescence decays reveal the presence of at least four underlying conformational states that are differentiated by the extent of fluorescence quenching. Single-molecule fluorescence displays transitions between conformational states on the time scales of milliseconds to seconds. This review describes the type of information available by analysis of time-resolved and single-molecule fluorescence experiments.

Emerging Roles of Nitric Oxide Synthase in Bacterial Physiology.

Nitric oxide (NO) is a potent inhibitor of diverse cellular processes in bacteria. Therefore, it was surprising to discover that several bacterial species, primarily Gram-positive organisms, harboured a gene encoding nitric oxide synthase (NOS). Recent attempts to characterize bacterial NOS (bNOS) have resulted in the discovery of structural features that may allow it to function as a NO dioxygenase and produce nitrate in addition to NO. Consistent with this characterization, investigations into the biological function of bNOS have also emphasized a role for NOS-dependent nitrate and nitrite production in aerobic and microaerobic respiration. In this review, we aim to compare, contrast, and summarize the structure, biochemistry, and biological role of bNOS with mammalian NOS and discuss how recent advances in our understanding of bNOS have enabled efforts at designing inhibitors against it.

Subcellular Targeting of Nitric Oxide Synthases Mediated by Their N-Terminal Motifs.

From a catalytic point of view, the three mammalian nitric oxide synthases (NOSs) function in an almost identical way. The N-terminal oxygenase domain catalyzes the conversion of l-arginine to l-citrulline plus ·NO in two sequential oxidation steps. Once l-arginine binds to the active site positioned above the heme moiety, two consecutive monooxygenation reactions take place. In the first step, l-arginine is hydroxylated to make Nω-hydroxy-l-arginine in a process that requires 1 molecule of NADPH and 1 molecule of O2 per mol of l-arginine reacted. In the second step, Nω-hydroxy-l-arginine, never leaving the active site, is oxidized to ·NO plus l-citrulline and 1 molecule of O2 and 0.5 molecules of NADPH are consumed. Since nitric oxide is an important signaling molecule that participates in a number of biological processes, including neurotransmission, vasodilation, and immune response, synthesis and release of ·NO in vivo must be exquisitely regulated both in time and in space. Hence, NOSs have evolved introducing in their amino acid sequences subcellular targeting motifs, most of them located at their N-termini. Deletion studies performed on recombinant, purified NOSs have revealed that part of the N-terminus of all three NOS can be eliminated with the resulting mutant enzymes still being catalytically active. Likewise, NOS isoforms lacking part of their N-terminus when transfected in cells render mislocalized, active proteins. In this review we will comment on the current knowledge of these subcellular targeting signals present in nNOS, iNOS, and eNOS.

Revisiting the Val/Ile Mutation in Mammalian and Bacterial Nitric Oxide Synthases: A Spectroscopic and Kinetic Study.

Nitric oxide is produced in mammals by the nitric oxide synthase (NOS) isoforms at a catalytic site comprising a heme associated with a biopterin cofactor. Through genome sequencing, proteins that are highly homologous to the oxygenase domain of NOSs have been identified, in particular in bacteria. The active site is highly conserved except for a valine residue in the distal pocket that is replaced with an isoleucine in bacteria. This switch was previously reported to influence the kinetics of the reaction. We have used the V346I mutant of the mouse inducible NOS (iNOS) as well as the I224V mutant of the NOS from Bacillus subtilis (bsNOS) to study their spectroscopic signatures in solution and look for potential structural differences compared to their respective wild types. Both mutants seem destabilized in the absence of substrate and cofactor. When both substrate and cofactor are present, small differences can be detected with Nω-hydroxy-l-arginine compared to arginine, which is likely due to the differences in the hydrogen bonding network of the distal pocket. Stopped-flow experiments evidence significant changes in the kinetics of the reaction due to the mutation as was already known. We found these effects particularly marked for iNOS. On the basis of these results, we performed rapid freeze-quench experiments to trap the biopterin radical and found the same results that we had obtained for the wild types. Despite differences in kinetics, a radical could be trapped in both steps for the iNOS mutant but only for the first step in the mutant of bsNOS. This strengthens the hypothesis that mammalian and bacterial NOSs may have a different mechanism during the second catalytic step.

Regulatory mechanism of the flavoprotein Tah18-dependent nitric oxide synthesis and cell death in yeast

Nitric oxide (NO) is a ubiquitous signaling molecule involved in the regulation of a large number of cellular functions. The regulatory mechanism of NO generation in unicellular eukaryotic yeast cells is poorly understood due to the lack of mammalian and bacterial NO synthase (NOS) orthologues, even though yeast produces NO under oxidative stress conditions. Recently, we reported that the flavoprotein Tah18, which was previously shown to transfer electrons to the iron-sulfur cluster protein Dre2, is involved in NOS-like activity in the yeast Saccharomyces cerevisiae. On the other hand, Tah18 was reported to promote apoptotic cell death after exposure to hydrogen peroxide (H2O2). Here, we showed that NOS-like activity requiring Tah18 induced cell death upon treatment with H2O2. Our experimental results also indicate that Tah18-dependent NO production and cell death are suppressed by enhancement of the interaction between Tah18 and its molecular partner Dre2. Our findings indicate that the Tah18-Dre2 complex regulates cell death as a molecular switch via Tah18-dependent NOS-like activity in response to environmental changes.

Towards the free energy landscape for catalysis in mammalian nitric oxide synthases.

The general requirement for conformational sampling in biological electron transfer reactions catalysed by multi-domain redox systems has been emphasized in recent years. Crucially, we lack insight into the extent of the conformational space explored and the nature of the energy landscapes associated with these reactions. The nitric oxide synthases (NOS) produce the signalling molecule NO through a series of complex electron transfer reactions. There is accumulating evidence that protein domain dynamics and calmodulin binding are implicated in regulating electron flow from NADPH, through the FAD and FMN cofactors, to the haem oxygenase domain, where NO is generated. Simple models based on static crystal structures of the isolated reductase domain have suggested a role for large-scale motions of the FMN-binding domain in shuttling electrons from the reductase domain to the oxygenase domain. However, detailed insight into the higher-order domain architecture and dynamic structural transitions in NOS enzymes during enzyme turnover is lacking. In this review, we discuss the recent advances made towards mapping the catalytic free energy landscapes of NOS enzymes through integration of both structural techniques (e.g. cryo-electron microscopy) and biophysical techniques (e.g. pulsed-electron paramagnetic resonance). The general picture that emerges from these experiments is that NOS enzymes exist in an equilibrium of conformations, comprising a 'rugged' or 'frustrated' energy landscape, with a key regulatory role for calmodulin in driving vectorial electron transfer by altering the conformational equilibrium. A detailed understanding of these landscapes may provide new opportunities for discovery of isoform-specific inhibitors that bind at the dynamic interfaces of these multi-dimensional energy landscapes.

Nitric oxide as an antimicrobial molecule against Vibrio harveyi infection in the hepatopancreas of Pacific white shrimp, Litopenaeus vannamei

Nitric oxide (NO) is a key effector molecule produced in the innate immune systems of many species for antimicrobial defense. However, how NO production is regulated during bacterial infection in invertebrates, especially crustaceans, remains poorly understood. Vibrio harveyi, a Gram-negative marine pathogen, is among the most prevalent and serious threats to the world's shrimp culture industry. Its virulence typically manifests itself through shrimp hepatopancreas destruction. In the current study, we found that NO generated by an in vitro donor system (NOC-18) could rapidly and effectively kill V. harveyi. In addition, injection of heat-killed V. harveyi increased the concentration of NO/nitrite and the mRNA expression of nitric oxide synthase (NOS) in the hepatopancreas of Pacific white shrimp (Litopenaeus vannamei), the commercially most significant shrimp species. Live V. harveyi challenge also induced NO/nitrite production and NOS gene expression in primary L. vannamei hepatopancreatic cells in a time- and dose-dependent manner. Co-incubation of l-NAME, an inhibitor selective for mammalian constitutive NOSs, dose-dependently blocked V. harveyi-induced NO/nitrite production, without affecting V. harveyi-induced NOS mRNA expression. Furthermore, l-NAME treatment significantly increased the survival rate of infecting V. harveyi in cultured primary hepatopancreatic cells of L. vannamei. As a whole, we have demonstrated that endogenous NO produced by L. vannamei hepatopancreatic cells occurs in enzymatically regulated manners and is sufficient to act as a bactericidal molecule for V. harveyi clearance.

Nitric oxide production by necrotrophic pathogen Macrophomina phaseolina and the host plant in charcoal rot disease of jute: complexity of the interplay between necrotroph-host plant interactions.

M. phaseolina, a global devastating necrotrophic fungal pathogen causes charcoal rot disease in more than 500 host plants. With the aim of understanding the plant-necrotrophic pathogen interaction associated with charcoal rot disease of jute, biochemical approach was attempted to study cellular nitric oxide production under diseased condition. This is the first report on M. phaseolina infection in Corchorus capsularis (jute) plants which resulted in elevated nitric oxide, reactive nitrogen species and S nitrosothiols production in infected tissues. Time dependent nitric oxide production was also assessed with 4-Amino-5-Methylamino-2′,7′-Difluorofluorescein Diacetate using single leaf experiment both in presence of M. phaseolina and xylanases obtained from fungal secretome. Cellular redox status and redox active enzymes were also assessed during plant fungal interaction. Interestingly, M. phaseolina was found to produce nitric oxide which was detected in vitro inside the mycelium and in the surrounding medium. Addition of mammalian nitric oxide synthase inhibitor could block the nitric oxide production in M. phaseolina. Bioinformatics analysis revealed nitric oxide synthase like sequence with conserved amino acid sequences in M. phaseolina genome sequence. In conclusion, the production of nitric oxide and reactive nitrogen species may have important physiological significance in necrotrophic host pathogen interaction.

Molecular architecture of mammalian nitric oxide synthases.

NOSs are homodimeric multidomain enzymes responsible for producing NO. In mammals, NO acts as an intercellular messenger in a variety of signaling reactions, as well as a cytotoxin in the innate immune response. Mammals possess three NOS isoforms--inducible, endothelial, and neuronal NOS--that are composed of an N-terminal oxidase domain and a C-terminal reductase domain. Calmodulin (CaM) activates NO synthesis by binding to the helical region connecting these two domains. Although crystal structures of isolated domains have been reported, no structure is available for full-length NOS. We used high-throughput single-particle EM to obtain the structures and higher-order domain organization of all three NOS holoenzymes. The structures of inducible, endothelial, and neuronal NOS with and without CaM bound are similar, consisting of a dimerized oxidase domain flanked by two separated reductase domains. NOS isoforms adopt many conformations enabled by three flexible linkers. These conformations represent snapshots of the continuous electron transfer pathway from the reductase domain to the oxidase domain, which reveal that only a single reductase domain participates in electron transfer at a time, and that CaM activates NOS by constraining rotational motions and by directly binding to the oxidase domain. Direct visualization of these large conformational changes induced during electron transfer provides significant insight into the molecular underpinnings governing NO formation.

De-Etiolation of Wheat Seedling Leaves: Cross Talk between Heme Oxygenase/Carbon Monoxide and Nitric Oxide

Greening of etiolated plants is predominantly stimulated by light but the complete molecular mechanism is still unknown. Multiple studies currently focus on the important physiological effects of heme oxygenase (HO)/carbon monoxide (CO) in plants. In this report, firstly, the role of HO/CO in light-induced de-etiolation process was investigated. We discovered that light could significantly increase HO activities, HO-1 gene expression, CO release, and chlorophyll accumulation, all of which were sensitive to zinc protoporphyrin (ZnPPIX), the potent inhibitor of HO-1, respectively. Both HO-1 inducer hematin (H) and CO aqueous solution were able to relieve etiolation in wheat seedling leaves under completely darkness by up-regulating endogenous HO/CO system, so as nitric oxide (NO) donor sodium nitroprusside (SNP) did. Similarly, endogenous NO production was also boost in response to light, SNP, hematin and CO aqueous solution in wheat seedling leaves. Additionally, the restoration of chlorophyll contents was blocked, when the inhibitor of mammalian nitric oxide synthase NG-nitro-L-arginine methylester hydrochloride (L-NAME) or the specific scavenger of NO 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO) was added, respectively. Furthermore, the inducible effects of light were different from those of SNP, hematin, and CO on Pfr accumulation and PHYA transcripts. However, all of sodium nitroprusside (SNP), hematin, and CO could accelerate NO emission, which suggested that HO/CO in wheat seedlings de-etiolation under dark-light transition may have a cross talk with NO.

R Is for Arginine

and provided its distinction as 1 of the 3 basic amino acids. Abundant in many types of food, synthesized from simple precursors, or recycled in different tissues in the body, arginine is classified as a nonessential amino acid; except under extreme conditions, nutritional states of arginine deficiency are rare. Yet how much more complicated and interesting has arginine become in the past 3 decades! The role of arginine as a humdrum amino acid was forever transformed by the discovery that this amino acid is a substrate for the formation of nitric oxide by all mamma-lian nitric oxide synthases.

Regulatory Role of Glu546 in Flavin Mononucleotide — Heme Electron Transfer in Human Inducible Nitric Oxide Synthase

Nitric oxide (NO) production by mammalian NO synthase (NOS) is believed to be regulated by the docking of the flavin mononucleotide (FMN) domain in one subunit of the dimer onto the heme domain of the adjacent subunit. Glu546, a conserved charged surface residue of the FMN domain in human inducible NOS (iNOS), is proposed to participate in the interdomain FMN/heme interactions [Sempombe et al. Inorg. Chem.2011, 50, 6869-6861]. In the present work, we further investigated the role of the E546 residue in the FMN-heme interdomain electron transfer (IET), a catalytically essential step in the NOS enzymes. Laser flash photolysis was employed to directly measure the FMN-heme IET kinetics for the E546N mutant of human iNOS oxygenase/FMN (oxyFMN) construct. The temperature dependence of the IET kinetics was also measured over the temperature range of 283-304 K to determine changes in the IET activation parameters. The E546N mutation was found to retard the IET by significantly raising the activation entropic barrier. Moreover, pulsed electron paramagnetic resonance data showed that the geometry of the docked FMN/heme complex in the mutant is basically the same as in the wild type construct, whereas the probability of formation of such a complex is about twice lower. These results indicate that the retarded IET in the E546N mutant is not caused by an altered conformation of the docked FMN/heme complex, but by a lower population of the IET-active conformation. In addition, the negative activation entropy of the mutant is still substantially lower than that of the holoenzyme. This supports a mechanism by which the FMN domain can modify the IET through altering probability of the docked state formation.