Dennis J Stuehr

Cleveland Clinic · Department of Pathobiology

Heme is an iron-containing cofactor essential for life. In eukaryotes heme is generated in the mitochondria and must leave this organelle to reach protein targets in other cell compartments. Mitochondrial heme binding by cytosolic GAPDH was recently found essential for heme distribution in eukaryotic cells. Here, we sought to uncover how mitochondr...

Heme is an iron-containing cofactor essential for life. In eukaryotes heme is generated in the mitochondria and must leave this organelle to reach protein targets in other cell compartments. Mitochondrial heme binding by cytosolic GAPDH was recently found essential for heme distribution in eukaryotic cells. Here, we sought to uncover how mitochondr...

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 desens...

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 w...

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 quenc...

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...

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 desensit...

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 uncover...

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 manne...

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 RFL-6 cells and human airway smooth muscle cells that naturally express sGC and HEK293 cells that we...

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 modificatio...

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 range from 30- 60% heme-saturated in biological settings and go...

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...

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 β subunit (apo-sGCβ), resulting in a mature sGC-αβ...

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-p...

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, β, and γ. Utilizing cell culture systems, we found that overexpress...

Significance Nitric oxide (NO) performs many biological functions, but how it operates at the molecular and cellular levels is not fully understood. We discovered that cell NO generation at physiologic levels triggers a rapid redeployment of intracellular heme, an iron-containing cofactor, and we show that this drives the assembly of the natural NO...

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 direct...

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 d...

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-N...

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 regardi...

Background Idiopathic pulmonary arterial hypertension (IPAH) is a rapidly progressive disease with several treatment options. Long-term mortality remains high with great heterogeneity in treatment response. Even though most of the pathology of IPAH is observed in the lung, there is systemic involvement. Platelets from IPAH patients have characteris...

Soluble guanylate cyclase (sGC) is a heme-containing heterodimeric enzyme that generates many molecules of cGMP in response to its ligand NO; sGC thereby acts as an amplifier in NO-driven biological signaling cascades. Because sGC helps regulate the cardiovascular, neuronal, and gastrointestinal systems through its cGMP production, boosting sGC act...

Inflammation Cell Signaling Keywords Bronchodilation Smooth muscle Cytochrome b5 reductase Catalase Thioredoxin Reactive oxygen Nitric oxide ABSTRACT A subset of asthmatics develop a severe form of the disease whose etiology involves airway inflammation along with inherent drivers that remain ill-defined. To address this, we studied human airway sm...

NADPH oxidase 5 (NOX5) is a transmembrane signaling enzyme that produces superoxide in response to elevated cytosolic calcium. In addition to its association with numerous human diseases, NOX5 has recently been discovered to play crucial roles in the immune response and cardiovascular system. Details of NOX5 maturation, and specifically its respons...

Idiopathic pulmonary arterial hypertension (IPAH) is a progressive and devastating disease characterized by vascular smooth muscle and endothelial cell proliferation leading to a narrowing of the vessels in the lung. The increased resistance in the lung and the higher pressures generated result in right heart failure. Nitric Oxide (NO) deficiency i...

Soluble guanylyl cyclase (sGC) is a key component of nitric oxide (NO)-cGMP signaling in mammals. Although heme must bind in the sGC β1 subunit (sGCβ) for sGC to function, how heme is delivered to sGCβ remains unknown. Given that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) displays properties of a heme chaperone for inducible NO synthase, we i...

Significance Endothelial nitric oxide synthase (eNOS) is a fundamental mediator of vascular function influencing cardiovascular homeostasis through the generation of nitric oxide (NO). eNOS activity can be regulated by protein–protein interactions and here using unbiased proteomics, we uncover plasminogen activator inhibitor-1 (PAI-1) as a potent n...

Nitric oxide (NO) is an important signal and effector molecule in animal physiology. NO is generated from l-arginine by the NO synthases (NOSs). Three NOSs have been characterized in animals: neuronal NOS (nNOS, type I), cytokine-inducible NOS (iNOS, type II), and endothelial NOS (eNOS, type III). Importantly, the activities of all three isoforms d...

Production of reactive oxygen species due to dysregulated endothelial nitric oxide synthase (eNOS) activity is linked to vascular dysfunction. eNOS is a major target protein of the primary calcium-sensing protein calmodulin. Calmodulin is often modified by the main biomarker of nitroxidative stress, 3-nitrotyrosine (nitroTyr). Despite nitroTyr bein...

Hemeproteins are essential for life and heme insertion is an essential step in their maturation. Maturation of hemeprotein requires that they incorporate heme and become active, but knowledge of this essential cellular process remains incomplete. However recent studies on chaperon Hsp90 has revealed that it drives functional heme insertion in vital...

The soluble guanylyl cyclase (sGC)-cyclic guanosine monophosphate signaling pathway evokes vascular smooth muscle relaxation; whether this pathway mediates airway smooth muscle relaxation remains controversial. We posit that sGC activators are equi-effective as β-agonists in reversing contractile agonist-induced airway smooth muscle shortening. To...

The enzyme soluble guanylyl cyclase (sGC) is a heterodimer comprised of an α subunit and a heme-containing β subunit. It participates in signaling by generating cGMP in response to nitric oxide (NO). Heme insertion into the β1 subunit of sGC (sGCβ) is critical for function, and heat shock protein 90 (HSP90) associates with heme-free sGCβ (apo-sGCβ)...

The superoxide-generating activity of Nox5 is regulated by Ca²⁺ flux, primarily through its self-contained calcium binding domain (EFD). Upon Ca²⁺ binding, Nox5’s EFD undergoes a conformational change that exposes its buried hydrophobic residues. Previously, we determined the Ca²⁺ binding constants of the N-terminal half domain (N-EFD). Here we per...

Myoglobin (Mb) maturation involves heme incorporation as a final step. We investigated a role for heat shock protein (hsp) 90 in Mb maturation in C2C12 skeletal muscle myoblasts and cell lines. We found the following: 1) Hsp90 directly interacts preferentially with heme‐free Mb both in purified form and in cells. 2) Hsp90 drives heme insertion into...

Nitric oxide (NO) synthases (NOSs) catalyze the formation of NO from L-arginine. We have shown previously that the NOS enzyme catalytic cycle involves a large number of reactions but can be characterized by a global model with three main ratelimiting steps. These are the rate of heme reduction by the flavin domain (kr), of dissociation of NO from t...

This review briefly summarizes what was known about NOS enzymology at the time of the Nobel Prize award in 1998 and then discusses from the author's perspective some of the advances in NOS enzymology over the subsequent 20 years, focused on five aspects: the maturation process of NOS enzymes and its regulation; the mechanism of NO synthesis; the re...

Cellular heme is thought to be distributed between a pool of sequestered heme that is tightly bound within heme proteins and a labile heme pool required for signaling and transfer into proteins. A heme chaperone that can hold and allocate labile heme within cells has long been proposed but never been identified. Here, we show that the glycolytic pr...

Asthma is a chronic inflammatory disease that is known to cause changes in the extracellular matrix, including changes in hyaluronan (HA) deposition. However, little is known about the factors that modulate its deposition or the potential consequences. Asthmatics with high levels of exhaled nitric oxide (NO) are characterized by greater airway reac...

NO synthase (NOS) enzymes perform inter-domain electron transfer reactions during catalysis that may rely on complementary charge interactions at domain-domain interfaces. Guided by our previous results and a computer-generated domain docking model, we assessed the importance of cross-domain charge interactions in the FMN to heme electron transfer...

Significance Maturation of functional adult (α2β2) or fetal (α2γ2) hemoglobin (Hb) tetramers requires that a heme cofactor be incorporated into each globin. During erythropoiesis, Hb-α maturation is aided by the alpha Hb-stabilizing protein (AHSP), but what enables the maturation and heme insertion of the other globins is unknown. We found that cha...

Cigarette smoking can cause fatal pulmonary injury involving extensive structural and functional damage of the lung through a disease called emphysema followed by extensive vascular remodeling, that restricts pulmonary circulation to cause pulmonary hypertension and right ventricular dystrophy of the heart. We demonstrate that oxidant(s) present in...

Endothelial nitric oxide synthase (eNOS) is a membrane-anchored enzyme. To highlight the potential role and effect of membrane phospholipids on the structure and activity of eNOS, we have incorporated the recombinant oxygenase subunit of eNOS into lipid nanodiscs. Two different size distribution modes were detected by multi-angle dynamic light scat...

The signaling molecule nitric oxide (NO) is synthesized in animals by structurally related NO synthases (NOSs), which contain NADPH/FAD- and FMN-binding domains. During catalysis, NADPH-derived electrons transfer into FAD and then distribute into the FMN domain for further transfer to internal or external heme groups. Conformational freedom of the...

Cigarette smoking causes emphysema, a fatal disease involving extensive structural and functional damage of the lung followed by pulmonary hypertension that restricts pulmonary circulation and causes right ventricular dysfunction of the heart. Using a guinea-pig model and human lung cells we show that oxidant(s) present in tobacco smoke not only ca...

The NO synthases (NOS) catalyze a two-step oxidation of L-Arginine (Arg) to generate NO. In the first step, O2 activation involves one electron being provided to the heme by an enzyme-bound 6R-tetrahydro-L-biopterin cofactor (H4B), which then forms a H4B radical that must be reduced back to H4B in order for NOS to continue catalysis. Although an NA...

The activity of endothelial NO synthase (eNOS) is triggered by calmodulin (CaM) binding and is often further regulated by phosphorylation at several positions in the enzyme. Phosphorylation at Ser(1179) occurs in response to diverse physiologic stimuli and increases the NO synthesis and cytochrome c reductase activities of eNOS, thereby enhancing i...

Significance Tobacco smoking causes emphysema, a fatal disease involving extensive structural damage of the lung. Besides directly oxidizing lung proteins, tobacco smoke activates Rtp801, a proinflammatory cellular factor that induces overproduction of NO by inducible NOS and consequent lung protein nitration and damage. Such oxidized or nitrated l...

NADPH-Cytochrome P450 oxidoreductase (CYPOR) transfers electrons from NADPH to cytochromes P450 via its FAD and FMN. To understand the biochemical and structural basis of electron transfer from FMN-hydroquinone to its partners, three deletion-mutants in a conserved loop near the FMN were characterized. Comparison of oxidized and reduced wild type a...

The impairment of vasodilator NO production is well accepted as a typical marker of endothelial dysfunction in vascular diseases, including in the pathophysiology of Pulmonary Arterial Hypertension (PAH), but the molecular mechanisms accounting for loss of NO production are unknown. We hypothesized that low NO production by pulmonary arterial endot...

Significance Asthmatics depend on β-agonist bronchodilator drugs, but a majority develop resistance to these drugs in their lifetime, and new ways to bronchodilate are needed. We show that brochodilation can be triggered in normal human and asthmatic mouse airways through an alternative signaling pathway, using new pharmacologic agents that directl...

Significance: Soluble guanylate cyclase (sGC) is an intracellular enzyme that plays a primary role in sensing nitric oxide (NO) and transducing its multiple signaling effects in mammals. Recent Advances: The chaperone heat shock protein 90 (hsp90) associates with signaling proteins in cells, including sGC, where it helps to drive heme insertion in...

Nitric oxide (NO) signaling regulates various physiological processes in both animals and plants. In animals, NO synthesis is mainly catalyzed by NO synthase (NOS) enzymes. Although NOS-like activities that are sensitive to mammalian NOS inhibitors have been detected in plant extracts, few bona fide plant NOS enzymes have been identified. We search...

Nitric oxide synthases (NOSs) catalyze a two-step oxidation of L-arginine to form nitric oxide (NO) and L-citrulline. NOS contains a N-terminal oxygenase domain (NOSoxy) that is the site of NO synthesis, and a C-terminal reductase domain (NOSred) that binds nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and...

Significance Electron transfer is a fundamental process in biology that can be coupled to catalysis within redox enzymes through a careful control of protein conformational movements. Using single-molecule fluorescence resonance energy transfer (FRET) spectroscopy, we find that calmodulin binding to neuronal NO synthase reductase domain (nNOSr) bot...

Heat shock protein 90 (hsp90) drives heme insertion into the β1 subunit of soluble guanylate cyclase (sGCβ1), which enables it to associate with a partner sGCα1 subunit and mature into a nitric oxide (NO)-responsive, active form. We utilized fluorescence polarization measurements and hydrogen-deuterium exchange mass spectrometry to define molecular...

Objectives Large Vessel Vasculitides (LVV) are a group of autoimmune diseases characterized by injury and anatomic modifications of large vessels including aorta and its branch vessels. Disease etiology is unknown. Using samples from aorta root, ascending aorta and aorta arch surgical specimens, we sought to identify antigen targets within affected...

Background Calmodulin (CaM) plays an important role in Ca2+-dependent signal transduction. Ca2+ binding to CaM triggers a conformational change, forming a hydrophobic patch that is important for target protein recognition. CaM regulates a Ca2+-dependent inactivation process in store-operated Ca2+ entry, by interacting Orai1. To understand the relat...

The ubiquitin ligase CHIP plays an important role in cytosolic protein quality control by ubiquitinating proteins chaperoned by Hsp70/Hsc70 and Hsp90, thereby targeting such substrate proteins for degradation. We present a 2.91 Å resolution structure of the tetratricopeptide repeat (TPR) domain of CHIP in complex with the α-helical lid subdomain an...

Catalase is a tetrameric heme-containing enzyme with essential antioxidant functions in biology. Multiple factors including nitric oxide (NO) have been shown to attenuate its activity. However, the possible impact of NO in relation to the maturation of active catalase, including its heme acquisition and tetramer formation, has not been investigated...

Nitric oxide synthases (NOS) are haem-thiolate enzymes that catalyse the conversion of L-Arginine (L-Arg) into NO and citrulline. Inducible NOS (iNOS) is responsible for delivery of NO in response to stressors during inflammation. The catalytic performance of iNOS is proposed to rely mainly on the haem midpoint potential and the ability of the subs...

Multi-domain enzymes often rely on large conformational motions to function. However, the conformational setpoints, rates of domain motions, and relationships between these parameters and catalytic activity is not well understood. To address this, we determined and compared the conformational setpoints and the rates of conformational switching betw...

During winter hibernation, brown bears (Ursus arctos) lie in dens for half a year without eating while their basal metabolism is largely suppressed. To understand underlying mechanisms of metabolic depression in hibernation, we measured type and content of blood metabolites of two ubiquitous inhibitors of mitochondrial respiration, hydrogen sulfide...

The chaperone heat shock protein 90 (hsp90) associates with signaling proteins in cells including soluble guanylate cyclase (sGC). Hsp90 associates with the heme-free (apo) sGC-β1 subunit and helps to drive heme insertion as required for maturation of sGC to its nitric oxide (NO)-responsive active form. Here we found that NO caused apo-sGC-β1 to ra...

Multi‐domain enzymes rely on conformational motions to function. However, the conformational setpoints, rates of domain motions, and how they might combine to determine catalytic activity, are not well understood. To address this, we measured and compared conformational setpoints and rates of FMN domain motion in four members of the di‐flavin NADPH...

Islet transplantation is an alternative to pancreas transplantation to cure type 1 diabetes, but both require chronic immunosuppression, which is often accompanied by deleterious side effects. The purpose of this study was to explore prolongation of islet allograft survival by cotransplantation with myeloid-derived suppressor cells (MDSCs) without...

Tetrahydrobiopterin (BH4) is a required cofactor for the synthesis of NO by NOS. Bioavailability of BH4 is a critical factor in regulating the balance between NO and superoxide production by endothelial NOS (eNOS coupling). Crystal structures of the mouse inducible NOS oxygenase domain reveal a homologous BH4-binding site located in the dimer inter...