Regional variations in allergen-induced airway inflammation correspond to changes in soluble guanylyl cyclase heme and expression of heme oxygenase-1.

Asthma is characterized by airway remodeling and hyperreactivity. Our earlier studies determined that the nitric oxide (NO)-soluble guanylyl cyclase (sGC)-cGMP pathway plays a significant role in human lung bronchodilation. However, this bronchodilation is dysfunctional in asthma due to high NO levels, which cause sGC to become heme-free and desensitized to its natural activator, NO. In order to determine how asthma impacts the various lung segments/lobes, we mapped the inflammatory regions of lungs to determine whether such regions coincided with molecular signatures of sGC dysfunction. We demonstrate using murine models of asthma (OVA and CFA/HDM) that the inflamed segments of these murine lungs can be tracked by upregulated expression of HO1 and these regions in turn overlap with regions of heme-free sGC as evidenced by a decreased sGC-α1ß1 heterodimer and an increased response to heme-independent sGC activator, BAY 60-2770, relative to naïve uninflamed regions. We also find that NO generated from iNOS upregulation in the inflamed segments has a higher impact on developing heme-free sGC as increasing iNOS activity correlates linearly with elevated heme-independent sGC activation. This excess NO works by affecting the epithelial lung hemoglobin (Hb) to become heme-free in asthma, thereby causing the Hb to lose its NO scavenging function and exposing the underlying smooth muscle sGC to excess NO, which in turn becomes heme-free. Recognition of these specific lung segments enhances our understanding of the inflamed lungs in asthma with the ultimate aim to evaluate potential therapies and suggest that regional and not global inflammation impacts lung function in asthma.

Visualizing mitochondrial heme flow through GAPDH in living cells and its regulation by NO.

Iron protoporphyrin IX (heme) is a redox-active cofactor that is bound in mammalian cells by GAPDH and allocated by a process influenced by physiologic levels of NO. This impacts the activity of many heme proteins including indoleamine dioxygenase-1 (IDO1), a redox enzyme involved in immune response and tumor growth. To gain further understanding we created a tetra-Cys human GAPDH reporter construct (TC-hGAPDH) which after labeling could indicate its heme binding by fluorescence quenching. When purified or expressed in a human cell line, TC-hGAPDH had properties like native GAPDH and heme binding quenched its fluorescence by 45-65%, allowing it to report on GAPDH binding of mitochondrially-generated heme in live cells in real time. In cells with active mitochondrial heme synthesis, low-level NO exposure increased heme allocation to IDO1 while keeping the TC-hGAPDH heme level constant due to replenishment by mitochondria. When mitochondrial heme synthesis was blocked, low NO caused a near complete transfer of the existing heme in TC-hGAPDH to IDO1 in a process that required IDO1 be able to bind the heme and have an active hsp90 present. Higher NO exposure had the opposite effect and caused IDO1 heme to transfer back to TC-hGAPDH. This demonstrated: (i) flow of mitochondrial heme through GAPDH is tightly coupled to target delivery, (ii) NO up- or down-regulates IDO1 activity by promoting a conserved heme exchange with GAPDH that goes in either direction according to the NO exposure level. The ability to drive a concentration-dependent, reversible protein heme exchange is unprecedented and reveals a new role for NO in biology.

Visualizing Mitochondrial Heme Flow through GAPDH to Targets in Living Cells and its Regulation by NO.

Iron protoporphyrin IX (heme) is an essential cofactor that is chaperoned in mammalian cells by GAPDH in a process regulated by NO. To gain further understanding we generated a tetra-Cys human GAPDH reporter construct (TC-hGAPDH) which after being expressed and labeled with fluorescent FlAsH reagent could indicate heme binding by fluorescence quenching. When purified or expressed in HEK293T mammalian cells, FlAsH-labeled TC-hGAPDH displayed physical, catalytic, and heme binding properties like native GAPDH and its heme binding (2 mol per tetramer) quenched its fluorescence by 45-65%. In live HEK293T cells we could visualize TC-hGAPDH binding mitochondrially-generated heme and releasing it to the hemeprotein target IDO1 by monitoring cell fluorescence in real time. In cells with active mitochondrial heme synthesis, a low-level NO exposure increased heme allocation into IDO1 while keeping steady the level of heme-bound TC-hGAPDH. When mitochondrial heme synthesis was blocked at the time of NO exposure, low NO caused cells to reallocate existing heme from TC-hGAPDH to IDO1 by a mechanism requiring IDO1 be present and able to bind heme. Higher NO exposure had an opposite effect and caused cells to reallocate existing heme from IDO1 to TC-hGAPDH. Thus, with TC-hGAPDH we could follow mitochondrial heme as it travelled onto and through GAPDH to a downstream target (IDO1) in living cells, and to learn that NO acted at or downstream from the GAPDH heme complex to promote a heme reallocation in either direction depending on the level of NO exposure.

Functional maturation of cytochromes P450 3A4 and 2D6 relies on GAPDH- and Hsp90-Dependent heme allocation.

Cytochrome P450 3A4 and 2D6 (EC 1.14.13.97 and 1.14.14.1; CYP3A4 and 2D6) are heme-containing enzymes that catalyze the oxidation of a wide number of xenobiotic and drug substrates and thus broadly impact human biology and pharmacologic therapies. Although their activities are directly proportional to their heme contents, little is known about the cellular heme delivery and insertion processes that enable their maturation to functional form. We investigated the potential involvement of GAPDH and chaperone Hsp90, based on our previous studies linking these proteins to intracellular heme allocation. We studied heme delivery and insertion into CYP3A4 and 2D6 after they were transiently expressed in HEK293T and GlyA CHO cells or when naturally expressed in HEPG2 cells in response to rifampicin, and also investigated their associations with GAPDH and Hsp90 in cells. The results indicate that GAPDH and its heme binding function is involved in delivery of mitochondria-generated heme to apo-CYP3A4 and 2D6, and that cell chaperone Hsp90 is additionally involved in driving their heme insertions. Uncovering how cells allocate heme to CYP3A4 and 2D6 provides new insight on their maturation processes and how this may help to regulate their functions in health and disease.

Regions of Inflammation in mouse asthma correspond to regions of heme-free soluble guanylyl cyclase and can be tracked by marked expression of heme-oxygenase-1.

Asthma is characterized by airway remodeling and hyperreactivity. Our earlier studies determined that the Nitric Oxide (NO)-soluble Guanylyl Cyclase (sGC)-cGMP pathway plays a significant role in human lung bronchodilation. However this bronchodilation is dysfunctional in asthma due to high NO levels which cause sGC to become heme-free and desensitized to its natural activator, NO. In order to determine how asthma impacts the various lung segments/lobes we mapped the inflammatory regions of lungs to determine whether such regions coincided with molecular signatures of sGC dysfunction. We demonstrate using models of mouse asthma (OVA, CFA/HDM) that the inflammed segments of the mouse asthma lungs can be tracked by upregulated expression of HO1 and these regions in-turn overlap with regions of heme-free sGC as evidenced by a decreased sGC-α1ß1 heterodimer and an increased response to heme-independent sGC activator, BAY 60-2770 relative to naïve uninflamed regions. We also find that NO generated from iNOS upregulation in the inflamed segments has a higher impact in developing heme-free sGC as increasing iNOS activity correlates linearly with elevated heme-independent sGC activation. This excess NO works by affecting the epithelial lung hemoglobin (Hb) to become heme-free in asthma thereby causing the Hb to lose its NO scavenging function and exposing the underlying smooth muscle sGC to excess NO, which in-turn becomes heme-free. Recognition of these specific lung segments enhance our understanding of the inflammed lungs in asthma with the ultimate aim to evaluate potential therapies and suggests that regional and not global inflammation impacts lung function in asthma.

A natural heme deficiency exists in biology that allows nitric oxide to control heme protein functions by regulating cellular heme distribution.

A natural heme deficiency that exists in cells outside of the circulation broadly compromises the heme contents and functions of heme proteins in cells and tissues. Recently, we found that the signaling molecule, nitric oxide (NO), can trigger or repress the deployment of intracellular heme in a concentration-dependent hormetic manner. This uncovers a new role for NO and sets the stage for it to shape numerous biological processes by controlling heme deployment and consequent heme protein functions in biology.

Indoleamine dioxygenase and tryptophan dioxygenase activities are regulated through control of cell heme allocation by nitric oxide.

Indoleamine-2, 3-dioxygenase (IDO1) and Tryptophan-2, 3-dioxygenase (TDO) catalyze the conversion of L-tryptophan to N-formyl-kynurenine and thus play primary roles in metabolism, inflammation, and tumor immune surveillance. Because their activities depend on their heme contents, which vary in biological settings and go up or down in a dynamic manner, we studied how their heme levels may be impacted by nitric oxide (NO) in mammalian cells. We utilized cells expressing TDO or IDO1 either naturally or via transfection and determined their activities, heme contents, and expression levels as a function of NO exposure. We found NO has a bimodal effect: a narrow range of low NO exposure promoted cells to allocate heme into the heme-free TDO and IDO1 populations and consequently boosted their heme contents and activities 4- to 6-fold, while beyond this range the NO exposure transitioned to have a negative impact on their heme contents and activities. NO did not alter dioxygenase protein expression levels, and its bimodal impact was observed when NO was released by a chemical donor or was generated naturally by immune-stimulated macrophage cells. NO-driven heme allocations to IDO1 and TDO required participation of a GAPDH-heme complex and for IDO1 required chaperone Hsp90 activity. Thus, cells can up- or downregulate their IDO1 and TDO activities through a bimodal control of heme allocation by NO. This mechanism has important biomedical implications and helps explain why the IDO1 and TDO activities in animals go up and down in response to immune stimulation.

Hemoglobin resident in the lung epithelium is protective for smooth muscle soluble guanylate cyclase function.

Hemoglobin (Hb) present in the lung epithelium is of unknown significance. However Hb being an nitric oxide (NO) scavenger can bind to NO and reduce its deleterious effects. Hence we postulated an NO scavenging role for this lung Hb. Doing transwell co-culture with bronchial epithelial cells, A549/16-HBE (apical) and human airway smooth muscle cells (HASMCs as basal), we found that Hb can protect the smooth muscle soluble guanylyl cyclase (sGC) from excess NO. Inducing the apical A549/16-HBE cells with cytokines to trigger iNOS expression and NO generation caused a time dependent increase in SNO-sGC and this was accompanied with a concomitant drop in sGC-α1ß1 heterodimerization. Silencing Hbαß in the apical cells further increased the SNO on sGC with a faster drop in the sGC heterodimer and these effects were additive along with further silencing of thioredoxin 1 (Trx1). Since heme of Hb is critical for NO scavenging we determined the Hb heme in a mouse model of allergic asthma (OVA) and found that Hb in the inflammed OVA lungs was low in heme or heme-free relative to those of naïve lungs. Further we established a direct correlation between the status of the sGC heterodimer and the Hb heme from lung samples of human asthma, iPAH, COPD and cystic fibrosis. These findings present a new mechanism of protection of lung sGC by the epithelial Hb, and suggests that this protection maybe lost in asthma or COPD where lung Hb is unable to scavenge the NO due to it being heme-deprived.

BAY58-2667 Activates Different Soluble Guanylyl Cyclase Species by Distinct Mechanisms that Indicate Its Principal Target in Cells is the Heme-Free Soluble Guanylyl Cyclase-Heat Shock Protein 90 Complex.

Nitric oxide (NO)-unresponsive forms of soluble guanylyl cyclase (sGC) exist naturally and in disease can disable NO-sGC-cGMP signaling. Agonists like BAY58-2667 (BAY58) target these sGC forms, but their mechanisms of action in living cells are unclear. We studied rat lung fibroblast-6 cells and human airway smooth muscle cells that naturally express sGC and HEK293 cells that we transfected to express sGC and variants. Cells were cultured to build up different forms of sGC, and we used fluorescence and FRET-based measures to monitor BAY58-driven cGMP production and any protein partner exchange or heme loss events that may occur for each sGC species. We found that: (i) BAY58 activated cGMP production by the apo-sGCß-Hsp90 species after a 5-8 minute delay that was associated with apo-sGCß exchanging its Hsp90 partner with an sGCα subunit. (ii) In cells containing an artificially constructed heme-free sGC heterodimer, BAY58 initiated an immediate and three times faster cGMP production. However, this behavior was not observed in cells expressing native sGC under any condition. (iii) BAY58 activated cGMP production by ferric heme sGC only after a 30-minute delay, coincident with it initiating a delayed, slow ferric heme loss from sGCß We conclude that the kinetics favor BAY58 activation of the apo-sGCß-Hsp90 species over the ferric heme sGC species in living cells. The protein partner exchange events driven by BAY58 account for the initial delay in cGMP production and also limit the speed of subsequent cGMP production in the cells. Our findings clarify how agonists like BAY58 may activate sGC in health and disease. SIGNIFICANCE STATEMENT: A class of agonists can activate cyclic guanosine monophosphate (cGMP) synthesis by forms of soluble guanylyl cyclase (sGC) that do not respond to NO and accumulate in disease, but the mechanisms of action are unclear. This study clarifies what forms of sGC exist in living cells, which of these can be activated by the agonists, and the mechanisms and kinetics by which each form is activated. This information may help to hasten deployment of these agonists for pharmaceutical intervention and clinical therapy.

Functional interactions between NADPH oxidase 5 and actin.

NADPH oxidase 5 (NOX5) is a transmembrane oxidative signaling enzyme which produces superoxide in response to intracellular calcium flux. Increasing evidence indicates that NOX5 is involved in a variety of physiological processes as well as human disease, however, details of NOX5 signaling pathways and targets of NOX5 mediated oxidative modifications remain poorly resolved. Actin dynamics have previously been shown to be modulated by oxidative modification, however, a direct connection to NOX5 expression and activity has not been fully explored. Here we show that NOX5 and actin interact in the cell, and each modulate the activity of the other. Using actin effector molecules jasplakinolide, cytochalasin D and latrunculin A, we show that changes in actin dynamics affect NOX5 superoxide production. In tandem, NOX5 oxidatively modifies actin, and shifts the ratio of filamentous to monomeric actin. Finally, we show that knockdown of NOX5 in the pancreatic cancer cell line PSN-1 impairs cell migration. Together our findings indicate an important link between actin dynamics and oxidative signaling through NOX5.

Heme delivery to heme oxygenase-2 involves glyceraldehyde-3-phosphate dehydrogenase.

Heme regulatory motifs (HRMs) are found in a variety of proteins with diverse biological functions. In heme oxygenase-2 (HO2), heme binds to the HRMs and is readily transferred to the catalytic site in the core of the protein. To further define this heme transfer mechanism, we evaluated the ability of GAPDH, a known heme chaperone, to transfer heme to the HRMs and/or the catalytic core of HO2. Our results indicate GAPDH and HO2 form a complex in vitro. We have followed heme insertion at both sites by fluorescence quenching in HEK293 cells with HO2 reporter constructs. Upon mutation of residues essential for heme binding at each site in our reporter construct, we found that HO2 binds heme at the core and the HRMs in live cells and that heme delivery to HO2 is dependent on the presence of GAPDH that is competent for heme binding. In sum, GAPDH is involved in heme delivery to HO2 but, surprisingly, not to a specific site on HO2. Our results thus emphasize the importance of heme binding to both the core and the HRMs and the interplay of HO2 with the heme pool via GAPDH to maintain cellular heme homeostasis.

New roles for GAPDH, Hsp90, and NO in regulating heme allocation and hemeprotein function in mammals.

The intracellular trafficking of mitochondrial heme presents a fundamental challenge to animal cells. This article provides some background on heme allocation, discusses some of the concepts, and then reviews research done over the last decade, much in the author's laboratory, that is uncovering unexpected and important roles for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), heat shock protein 90 (hsp90), and nitric oxide (NO) in enabling and regulating the allocation of mitochondrial heme to hemeproteins that mature and function outside of the mitochondria. A model for how hemeprotein functions can be regulated in cells through the coordinate participation of GAPDH, hsp90, and NO in allocating cellular heme is presented.

Low levels of nitric oxide promotes heme maturation into several hemeproteins and is also therapeutic.

Nitric oxide (NO) is a signal molecule and plays a critical role in the regulation of vascular tone, displays anti-platelet and anti-inflammatory properties. While our earlier and current studies found that low NO doses trigger a rapid heme insertion into immature heme-free soluble guanylyl cyclase ß subunit (apo-sGCß), resulting in a mature sGC-αß heterodimer, more recent evidence suggests that low NO doses can also trigger heme-maturation of hemoglobin and myoglobin. This low NO phenomena was not only limited to sGC and the globins, but was also found to occur in all three nitric oxide synthases (iNOS, nNOS and eNOS) and Myeloperoxidase (MPO). Interestingly high NO doses were inhibitory to heme-insertion for these hemeproteins, suggesting that NO has a dose-dependent dual effect as it can act both ways to induce or inhibit heme-maturation of key hemeproteins. While low NO stimulated heme-insertion of globins required the presence of the NO-sGC-cGMP signal pathway, iNOS heme-maturation also required the presence of an active sGC. These effects of low NO were significantly diminished in the tissues of double (n/eNOS-/-) and triple (n/i/eNOS-/-) NOS knock out mice where lung sGC was found be heme-free and the myoglobin or hemoglobin from the heart/lungs were found be low in heme, suggesting that loss of endogenous NO globally impacts the whole animal and that this impact of low NO is both essential and physiologically relevant for hemeprotein maturation. Effects of low NO were also found to be protective against ischemia reperfusion injury on an ex vivo lung perfusion (EVLP) system prior to lung transplant, which further suggests that low NO levels are also therapeutic.

Indoleamine dioxygenase and tryptophan dioxygenase activities are regulated through GAPDH- and Hsp90-dependent control of their heme levels.

Indoleamine-2, 3-dioxygenase (IDO1) and Tryptophan-2, 3-dioxygense (TDO) are heme-containing dioxygenases that catalyze the conversion of tryptophan to N-formyl-kynurenine and thus enable generation of l-kynurenine and related metabolites that govern the immune response and broadly impact human biology. Given that TDO and IDO1 activities are directly proportional to their heme contents, it is important to understand their heme delivery and insertion processes. Early studies established that TDO and IDO1 heme levels are sub-saturating in vivo and subject to change but did not identify the cellular mechanisms that provide their heme or enable dynamic changes in their heme contents. We investigated the potential involvement of GAPDH and chaperone Hsp90, based on our previous studies linking these proteins to intracellular heme allocation. We studied heme delivery and insertion into IDO1 and TDO expressed in both normal and heme-deficient HEK293T cells and into IDO1 naturally expressed in HeLa cells in response to IFN-γ, and also investigated the interactions of TDO and IDO1 with GAPDH and Hsp90 in cells and among their purified forms. We found that GAPDH delivered both mitochondrially-generated and exogenous heme to apo-IDO1 and apo-TDO in cells, potentially through a direct interaction with either enzyme. In contrast, we found Hsp90 interacted with apo-IDO1 but not with apo-TDO, and was only needed to drive heme insertion into apo-IDO1. By uncovering the cellular processes that allocate heme to IDO1 and TDO, our study provides new insight on how their activities and l-kynurenine production may be controlled in health and disease.

NO rapidly mobilizes cellular heme to trigger assembly of its own receptor.

Nitric oxide (NO) signaling in biology relies on its activating cyclic guanosine monophosphate (cGMP) production by the NO receptor soluble guanylyl cyclase (sGC). sGC must obtain heme and form a heterodimer to become functional, but paradoxically often exists as an immature heme-free form in cells and tissues. Based on our previous finding that NO can drive sGC maturation, we investigated its basis by utilizing a fluorescent sGC construct whose heme level can be monitored in living cells. We found that NO generated at physiologic levels quickly triggered cells to mobilize heme to immature sGC. This occurred when NO was generated within cells or by neighboring cells, began within seconds of NO exposure, and led cells to construct sGC heterodimers and thus increase their active sGC level by several-fold. The NO-triggered heme deployment involved cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-heme complexes and required the chaperone hsp90, and the newly formed sGC heterodimers remained functional long after NO generation had ceased. We conclude that NO at physiologic levels triggers assembly of its own receptor by causing a rapid deployment of cellular heme. Redirecting cellular heme in response to NO is a way for cells and tissues to modulate their cGMP signaling and to more generally tune their hemeprotein activities wherever NO biosynthesis takes place.

GAPDH is involved in the heme-maturation of myoglobin and hemoglobin.

GAPDH, a heme chaperone, has been previously implicated in the incorporation of heme into iNOS and soluble guanylyl cyclase (sGC). Since sGC is critical for myoglobin (Mb) heme-maturation, we investigated the role of GAPDH in the maturation of this globin, as well as hemoglobins α, ß, and γ. Utilizing cell culture systems, we found that overexpression of wild-type GAPDH increased, whereas GAPDH mutants H53A and K227A decreased, the heme content of Mb and Hbα and Hbß. Overexpression of wild-type GAPDH fully recovered the heme-maturation inhibition observed with the GAPDH mutants. Partial rescue was observed by overexpression of sGCß1 but not by overexpression of a sGCΔß1 deletion mutant, which is unable to bind the sGCα1 subunit required to form the active sGCα1ß1 complex. Wild type and mutant GAPDH was found to be associated in a complex with each of the globins and Hsp90. GAPDH at endogenous levels was found to be associated with Mb in differentiating C2C12 myoblasts, and with Hbγ or Hbα in differentiating HiDEP-1 erythroid progenitor cells. Knockdown of GAPDH in C2C12 cells suppressed Mb heme-maturation. GAPDH knockdown in K562 erythroleukemia cells suppressed Hbα and Hbγ heme-maturation as well as Hb dimerization. Globin heme incorporation was not only dependent on elevated sGCα1ß1 heterodimer formation, but also influenced by iron provision and magnitude of expression of GAPDH, d-aminolevulinic acid, and FLVCR1b. Together, our data support an important role for GAPDH in the maturation of myoglobin and γ, ß, and α hemoglobins.

Inactivation of soluble guanylyl cyclase in living cells proceeds without loss of haem and involves heterodimer dissociation as a common step.

BACKGROUND AND PURPOSE: Nitric oxide (NO) activates soluble guanylyl cyclase (sGC) for cGMP production, but in disease, sGC becomes insensitive towards NO activation. What changes occur to sGC during its inactivation in cells is not clear. EXPERIMENTAL APPROACH: We utilized HEK293 cells expressing sGC proteins to study the changes that occur regarding its haem content, heterodimer status and sGCß protein partners when the cells were given the oxidant ODQ or the NO donor NOC12 to inactivate sGC. Haem content of sGCß was monitored in live cells through use of a fluorescent-labelled sGCß construct, whereas sGC heterodimer status and protein interactions were studied by Western blot analysis. Experiments with purified proteins were also performed. KEY RESULTS: ODQ- or NOC12-driven inactivation of sGC in HEK293 cells was associated with haem oxidation (by ODQ), S-nitrosation of the sGCß subunit (by NOC12), sGC heterodimer breakup and association of the freed sGCß subunit with cell chaperone Hsp90. These changes occurred without detectable loss of haem from the sGCß reporter construct. Treating a purified ferrous haem-containing sGCß with ODQ or NOC12 caused it to bind with Hsp90 without showing any haem loss. CONCLUSION AND IMPLICATIONS: Oxidative (ODQ) or nitrosative (NOC12) inactivation of cell sGC involves sGC heterodimer dissociation and rearrangement of the sGCß protein partners without any haem loss from sGCß. Clarifying what changes do and do not occur to sGC during its inactivation in cells may direct strategies to preserve or recover NO-dependent cGMP signalling in health and disease. LINKED ARTICLES: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

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

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

Nitric oxide and heme-NO stimulate superoxide production by NADPH oxidase 5.

Nitric oxide (NO) is a ubiquitous cell signaling molecule which mediates widespread and diverse processes in the cell. These NO dependent effects often involve activation (e.g. NO binding to the heme group of soluble guanylyl cyclase for cGMP production) or inactivation (e.g. S-nitrosation) of protein targets. We studied the effect of NO and heme-NO on the transmembrane signaling enzyme NADPH oxidase 5 (NOX5), a heme protein which produces superoxide in response to increases in intracellular calcium. We found that treatment with NO donors increases NOX5 activity through heme-dependent effects, and that this effect could be recapitulated by the addition of heme-NO. This work adds to our understanding of NOX5 regulation in the cell but also provides a framework for understanding how NO could cause widespread changes in hemeprotein activity based on different affinities for heme v. heme-NO, and helps explain the opposing roles NO plays in activation and inactivation of hemeprotein targets.