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Published in final edited form as: Bioessays. 2023 Jun 5;45(8):e2300055. doi: 10.1002/bies.202300055

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

Dennis J Stuehr 1, Pranjal Biswas 1, Yue Dai 1, Arnab Ghosh 1, Sidra Islam 1, Dhanya Thamaraparambil Jayaram 1
PMCID: PMC10478511  NIHMSID: NIHMS1902744  PMID: 37276366

Abstract

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 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.

Keywords: nitric oxide, iron, heme, anemia, evolution, physiology

Graphical Abstract

graphic file with name nihms-1902744-f0001.jpg

We hypothesize that tissues exist naturally in a heme deficient condition that limits the heme content and functions of heme proteins. The nitric oxide (NO) that is made in our tissues can control cell heme availability in a bimodal way, and so regulate heme protein functions in health and disease.

Introduction and Hypothesis

Iron protoporphyrin IX (heme) is a special iron-containing molecule with key roles in biological electron transfer, enzyme catalysis, cell signaling, and in the storage, transfer, or detection of O2 or NO [52][64]. In eukaryotes the initial and final steps of heme biosynthesis occur inside the mitochondria, and heme is the major iron-containing molecule in mammals, representing about 70–80% of the total iron (Fig. 1) [72]. Most of the heme in mammals (about 90%) resides in the hemoglobin contained in red blood cells, about 5% is in muscle myoglobin, and the remaining heme (5% of total) is distributed into diverse heme proteins that exist and function outside the circulation in our tissues and cells (Fig. 1) [72]. Research is revealing that other than in the red blood cells, the level of heme availability is kept in deficit, and this causes heme proteins that are expressed in cells and tissues outside the circulation to not be fully heme-saturated, such that their heme-free or apo forms typically represent 30 to 75% of the total (Table 1 and references therein). We hypothesize that this heme deficit that exists in tissues and cells outside of the circulation is a natural and deliberate biological condition. Moreover, based on recent findings we hypothesize that nitric oxide (NO), a natural signaling molecule that is generated in the body [33], operates in this circumstance as a master regulator of heme distribution to broadly and dynamically control the heme contents and the functions of heme proteins in health and disease.

Figure 1. Relative iron and heme distribution in mammals.

Figure 1.

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See text for details.

Table 1.

Heme proteins that have incomplete heme saturation when they are expressed in cells or tissues outside the circulation

Heme protein System References
Soluble guanylyl cyclase Cells and tissues [25], [31] and refs. therein
Trp dioxygenase Cells and tissues [6], [7], [32]
Indoleamine dioxygenase Cells [57], [25] and refs. therein
Hemoglobin α, β, γ Cells and tissues [31], [70]
Myoglobin Cells and tissues [31,70]
Cytochrome P450 Cells and tissues [58]
Myeloperoxidase Cells [31]
NADPH oxidase 5 Cells [71]
Ascorbate peroxidasea Cells [72]
Horseradish peroxidasea Cells [72]
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a

plant enzymes were expressed in mammalian cells

Outside of the erythroid system, cells and tissues are heme anemic

We have coined the term “heme anemic” to define a biological condition where the availability of heme is too low to fully saturate the heme binding sites of heme proteins that are expressed in cells or tissues. It is important to note that the heme-anemic condition exists outside the circulatory system, and thus is not present in red blood cells or in their immediate erythroid progenitors. The heme anemic condition that we describe here is also distinct from the well-known medical anemias that are associated with a low number of red cells that arise due either to low iron availability, poor red blood cell production, excessive lysis of red blood cells, or physical loss of blood from the circulation.

The concept that a widespread heme anemic condition exists outside of the circulation is new. It gradually arose from studies conducted on heme proteins that are expressed in healthy cells and tissues, which were found to never be fully heme-replete and instead existed where a significant proportion of their total (ca. 30–75%) was present in the heme-free or apo form. This phenomenon has been amply demonstrated for specific heme proteins like soluble guanylate cyclase (sGC), which is expressed in many different tissues and whose functional form requires that it contain a bound heme to sense NO, which stimulates it to generate cGMP in signal transduction cascades [57]. Besides sGC, a heme deficiency has also been demonstrated for other heme proteins when they are expressed outside the circulation including myoglobin, hemoglobin α, β, and γ, a variety of peroxidases, and indoleamine and Trp dioxygenases (Table 1). The first report of this phenomenon may have been in 1951 for the liver heme protein Trp dioxygenase (TDO), which was shown to be only 30–40 % heme saturated in the liver of healthy rats [5][39]. Why the partial heme saturation phenomenon was largely ignored over the years in the heme protein community is an interesting question. We suspect it relates to inherent biases of human nature: the apo-heme proteins are colorless and therefore easy to ignore compared to the deep red pigment of heme that was the focus of numerous spectroscopic studies on the heme proteins. In practice, apo-heme proteins were considered a nuisance and were typically thought to arise from heme loss during the protein purification process, and so were either ignored or in some cases a reconstitution was attempted with exogenous heme, for example see [34]. Nevertheless, several reports and sources now suggest that a heme deficit related to the heme anemic condition is common and impacts many different heme proteins (Table 1), and thus is a general state of being independent of heme protein identity. Given that the heme anemic condition and consequent poor heme saturation stands to have a broad fundamental impact on heme proteins in biology, it needs to be better studied regarding its prevalence and basis in health and disease.

The puzzle of the heme anemic condition

If one accepts that a heme anemic condition is natural, it leads to the question, why is this so? In animals, heme homeostasis is a well-regulated and dynamic process, and cells have several means to govern their heme biosynthesis and their steady state heme availability, for example by regulating their mitochondrial heme production, heme destruction by heme oxygenase enzymes, and import or export of heme by cell membrane transporters [13][16][17][40][59]. Indeed, experimental manipulation of these or related parameters can alter the heme levels of intracellular heme proteins such as sGC or TDO [15], and intentionally increasing cell mitochondrial heme biosynthesis can significantly increase the heme saturation levels of several hemeproteins in cells [6][66]. Thus, despite having means to better saturate their heme proteins, cells fail to do so. We suggest that the following possible reasons could justify the heme anemic condition:

  1. It provides an additional way for cells to regulate their heme protein functions independent of any changes in heme protein gene expression. Regulating heme protein function via control of cell heme allocation is straightforward, but it is presently unclear how reversible the process might be, which may depend on the heme affinity of the individual heme protein. The dynamics of heme acquisition by and heme loss from heme proteins in living cells may differ from their behaviors when in purified form, and will require further study.

  2. It allows heme to serve as a signaling or regulatory molecule within cells [45]. There are several heme-responsive transcription factors and kinases whose functions are proportional to their bound heme contents. Heme anemia allows these types of proteins to respond to changes in cellular heme availability, and so if cell heme availability rose to levels that would support full heme saturation, such regulation could not exist.

  3. It reflects a practical compromise that balances the beneficial effects and uses of heme versus the toxic effects of heme that can arise when it is in excess [35]. Heme contains a redox active iron that is prone to generating reactive oxygen species, so perhaps keeping cellular heme levels at a level below some threshold of redox reactivity is preferable to a fuller heme saturation of heme proteins.

  4. It reflects a survival adaption that limits the ability of eukaryotic heme to serve as an iron source for invasive microbes [55]. Iron is often a limiting factor in biology and microbes have adapted multiple strategies to obtain iron from host organisms. Because heme is the most abundant form of iron in mammals, perhaps maintaining a heme anemic condition and a lower heme saturation level reflects an evolutionary compromise that discourages iron-driven microbial growth inside our non-erythroid cells.

These and other possible reasons for maintaining a heme anemic condition have interesting and fundamental biological implications that should be investigated.

NO is a regulator of heme allocation in biology

The biophysical properties of NO enable it to influence heme allocation and function in biology. In particular, its high binding affinity toward transition metal ions, metalloproteins, and heme proteins is well regarded [23]. Any heme protein whose heme iron has an open coordination site will typically bind NO at pM to high nM affinities unless there is steric hinderance within the protein. Thus, if NO is present, it will likely find heme. In fact, in NO synthase enzymes, which are heme proteins that generate NO from L-Arg, all of the newly formed NO molecules bind to the enzyme’s heme iron multiple times before they can exit the enzyme’s active site [46][61]. This makes the heme-NO binding event a fundamental feature of their catalysis and it impacts their behavior in important ways. For example, it helps to explain why NO synthases evolved to utilize tetrahydrobiopterin as an electron donor and it explains the odd relationship that these enzymes display between their steady state activities and the O2 concentration in cells and tissues across the physiologic O2 range [61].

Once NO escapes from NO synthase it can impact other heme proteins in different ways and according to different time frames. As occurs in NO synthases, the released NO can impact by binding to the heme iron, and such heme-NO binding events typically inhibit or enable function depending on the heme protein in question [8][38]. The NO can also lead to post-translational modifications within the heme protein, typically at Cys or Tyr groups, which may in turn alter the structure or function of the protein, and NO-based modifications can ultimately lead to changes in heme protein stability or gene expression in cells.

Our understanding of how NO impacts the maturation of heme proteins has gone full circle. NO was first shown to negatively impact heme protein maturation by preventing cellular heme from being incorporated into the immature apo-heme proteins. Newly synthesized heme proteins are typically heme-free and thus must incorporate heme as a required post translational step to mature into their functional forms. This process typically requires heme delivery by GAPDH and participation by cell chaperone Hsp90, which is usually bound to the apo-heme proteins [60]. A negative impact of NO on heme protein maturation was first reported in 1996 as evidenced by NO blocking heme incorporation into the NO synthase enzyme itself as it was being expressed in cells and began generating NO [2]. Subsequently, NO was found to inhibit cell heme incorporation into a variety of heme proteins including hemoglobin α and β, catalase, and cytochrome P450’s [12][68]. It took another fourteen years before NO was appreciated to also exert a positive effect on cell heme allocation, with the demonstration that NO exposure initially could stimulate maturation of sGC by promoting cell heme allocation into its beta subunit [26]. In retrospect, one reason that the positive effect of NO was initially missed is that the NO concentrations used in the earlier studies had been too high. Indeed, recent studies are revealing that NO has a concentration-dependent, hormetic effect on cell heme allocation. In all cases examined so far, there is a relatively narrow window of very low NO concentration that stimulates cell heme allocation, and as the NO concentration goes above this range it increasingly has a negative impact that ultimately overcomes the positive effect and even can lead to a net loss of heme from the heme proteins [7][15][27]. In all cases tested, the NO-driven heme allocations require the participation of GAPDH and Hsp90, indicating that the NO-driven mechanism involves the same cellular machinery as does normal heme allocation. One mechanism (among several) for NO-driven cell heme allocation that is consistent with the results to date is shown in Fig. 2, using sGC as an example heme protein. Remarkably, studies so far are revealing that the beneficial concentration window of NO is the same no matter what cell type or heme protein is studied, implying that NO stimulates cell heme allocation by some common mechanism. Moreover, the biphasic NO response that cells exhibit regarding their heme allocation, in which lower concentrations are beneficial while higher concentrations have a negative impact, reflect in general a hormesis that is observed for many other biological NO responses [9] . Overall, these concepts have fundamental implications for biology and medicine that should be further investigated.

Figure 2. Model of NO-driven cellular heme allocation.

Figure 2.

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The example depicts one way that NO may drive heme allocation in cells, in this case to promote assembly of its receptor heme protein soluble guanylyl cyclase (sGC heterodimer). Cell-generated NO from NO synthase (left) diffuses into a neighboring cell (right) to promote heme allocation into an apo-sGCβ subunit in complex with chaperone hsp90. One way the NO might promote transfer of heme (red parallelogram) from GAPDH is by binding to the heme iron (Fe) (center, red arrow). The incorporation of heme-NO into the apo-sGCβ subunit prompts it to dissociate its GAPDH and hsp90 partners and bind an sGCα subunit (green) to generate a functional sGC heterodimer whose cGMP production can drive numerous biological responses.

Is there a widespread impact of NO on heme allocation and heme protein function?

The realization that NO has a sensitive hormetic effect on cell heme allocation, and that the heme anemic condition is likely to be a natural phenomenon, creates a circumstance where NO could broadly regulate heme protein functions by positively or negatively regulating heme availability. This concept is diagrammed in Fig. 3A. The curve depicts how the heme content and functions of cellular heme proteins rise and fall in relationship to the NO concentration. The area to the left of the peak maximum represents the heme anemic condition that is present in cells and tissues and causes heme proteins to be incompletely heme saturated. As NO concentrations rise, the heme content and functions of the hemeproteins increase due to NO stimulating cell heme allocation into the subpopulation of apo-heme proteins that are present in the cells. However, when NO concentrations go beyond an optimum level, the beneficial effect of NO is gradually lost and instead transitions to having a negative impact on cell heme allocation. In mammals, we envision that the lower levels of NO that benefit heme allocation might arise from the activities of the two constitutive NO synthase enzymes, commonly named endothelial and neuronal NO synthases, while the higher levels of NO that inhibit heme allocation may arise from the inducible NO synthase, whose expression is induced by immune stimulation, and typically generates more NO due to it being constitutively active [3] . The different impacts of the NO synthase isoforms on cell heme allocation as described above have been borne out in cell culture experiments [7][15].

Figure 3. Relationships between NO, heme protein heme content, function, and health and disease.

Figure 3.

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Panel A depicts how the heme content and function of cellular heme proteins changes with NO concentration due to a hormetic effect of NO on cell heme allocation. A heme anemic condition is present and associated with a suboptimal NO concentration range, in which cellular heme proteins are not fully heme-replete and thus have suboptimal function. Importantly, this heme anemic condition appears to be the natural state. An increase in NO can improve heme allocation and heme protein function, but beyond an optimal concentration the NO gradually loses its ability to promote cellular heme allocation. Panel B depicts four points on the curve (designated 1–4) as examples of where human or animal populations could exist. Some set points likely populate within a “healthy” range (points 1 and 2), while some set points may populate below or above the healthy range (points 3 and 4) and may be associated with or enable disease. See text for details.

Several fascinating questions arise within this context:

Where do humans or other mammals sit on this spectrum of NO production, heme allocation, and heme protein function? Is there a healthy range? Given the evidence for a widespread heme anemic condition, it follows that mammals are likely situated somewhere to the left of the optimal NO exposure-heme allocation position on the graph (Fig. 3B, point 1), even in healthy conditions. In this position, heme availability and heme saturation levels can be either increased or decreased by an increase or decrease in NO exposure, respectively. As noted previously, being naturally positioned below the optimal set point for heme saturation may reflect a practical or evolutionary compromise to balance heme availability with other aspects of its biology.

It is also possible that human populations occupy different positions on the spectrum (Fig. 3B, points 1 versus 2) depending on factors like age, genetics, and environment. For example, it is known that certain human populations who have settled at altitudes above 4,000 meters (for example, Tibetans) have greater levels of NO production than do lowland human populations, and it has been speculated that this may enable enhanced survival at altitude [4]. Perhaps their heme saturation levels and heme protein functions are enhanced by their higher NO production (Fig. 3B, point 2), and this provides one basis for enhanced survival? Indeed, diminished NO bioactivity and sGC heme protein function have sometimes been associated with maternal hypertension and preeclampsia [41][65], which are conditions exacerbated at high altitude. Is it possible that greater NO production in the highlander population enables a better level of heme saturation, particularly in their NO synthase and sGC enzymes, that in turn provides a survival advantage to childbearing women and newborns?

Similarly, there may be positions on the spectrum that are associated with a greater prevalence for disease. For example, pulmonary hypertension disease is associated with a lower-than-normal NO generation and sGC function [18][30]. Perhaps the lower NO production exacerbates the heme anemic condition in this population (Fig. 3B, point 3), resulting in poorer sGC function leading to hypertension? As an example, airway cells isolated from the lungs of severe asthmatic donors were found to have a greater heme deficit as indicated by much lower heme levels in their sGC [27]. Because this phenotype persisted in the airway cells even upon their passage in culture, it suggests they may have an underlying genetic basis for a more severe heme anemic condition. It is also possible that, due to the hormetic nature of the NO response, human populations could be positioned on the other side of the spectrum and thereby have increased prevalence toward disease (Fig. 3B, point 4). For example, many asthmatics generate higher than normal levels of NO in their airways and the level of exhaled NO is used as a clinical biomarker [49]. Perhaps the airway NO reaches a level that no longer aids and instead inhibits proper heme allocation into airway heme proteins with known anti-inflammatory roles like catalase or indoleamine dioxygenase [28][31][36][44], and in this way it exacerbates airway inflammation and disease?

Finally, we raise a most fundamental question: Has biological heme allocation evolved to depend on low amounts of NO? Humans and higher animals have sites of relatively high NO production, including in the vasculature, skin, sinuses, and upper airways [10][24][42][54]. Does proper heme allocation depend on some tonic level of NO production within the organism? The remarkable sensitivity that heme allocation displays towards NO suggests that this could be possible. Indeed, a recent study showed that when the expression of two of the three NO synthase isoforms were knocked out by genetic manipulation in mice, the mice had a significantly greater heme deficit as indicated by much lower levels of heme in the sGC expressed in their airway and in the hemoglobin and myoglobin in their heart tissue [27]. This suggests that a tonic level of NO generation by NO synthase enzymes is relied upon to facilitate proper heme allocation in mammals. Could we alter our NO production to achieve a level of heme allocation that is more optimal for health? There are fascinating possibilities: Physical exercise increases the expression of NO synthases and increases NO bioavailability [19][48]. Might an NO-induced increase in heme availability and heme protein function help explain the health benefits of exercise? A similar mechanism could possibly explain the health benefits of a high nitrate diet [43][51][58][69]. Humming is also known to stimulate significant NO release from the sinuses [42][70], might this extra NO increase heme availability and function in the body and thus contribute to the health effects of humming or chanting [20]? The connections between NO, heme bioavailability, and heme protein function can be logically extended to most other animal species, and might even involve the background NO that is naturally present in the environment. This all deserves further study.

Approaches to test the hypotheses

There are ways to gauge the severity of the heme anemic condition and to investigate its scope in biology. The easiest and oldest method is to measure the heme-dependent functional activity of a given heme protein of interest in a biological sample in the absence or presence of added heme. This has been used for example to indicate the percentage heme saturation in TDO, IDO, sGC, and cytochromes P450 in homogenates of various cells or tissues [5][7][14][34][39][47], but its usefulness depends on the ability of the given heme protein of interest to be amenable to in vitro heme reconstitution. Another way would be to determine the heme levels in the heme proteins by mass spectrometry [67]. For sGC, there is an additional way to judge its heme level which involves comparing its cGMP production activity in response to agonists that either can only activate the heme-containing sGC or can only activate the heme-free sGC [56]. Because such sGC agonists are commercially available and sGC activity is broadly expressed in many tissues and cells, this approach could possibly serve as one common way to qualitatively compare the heme tone in different tissue or cell samples. For example, measures of the sGC agonist response profile have potentially shown that a more severe heme-anemic condition exists in airway smooth muscle cells isolated from severe asthmatic donors than from non-asthmatics [25], and may also exist in tissues from mice with genetic knockout of NO synthase isoforms [27]. However, it remains to be seen if sGC heme levels as indicated in this way can serve as a general marker of heme level in other heme proteins in the same tissues or cells. However, initial results concerning this are promising [27].

To test the hypothesis that NO controls heme allocation, this can easily be done in cell culture and has already been demonstrated, as discussed above. In whole animal systems, the NO level can be manipulated up and down in a number of ways, including by knockout of the NO synthases [27][32], adding nitrate or nitrite to the diet [11], injecting NO carriers like S-nitrosated Hb into the bloodstream [50], adding NO gas at ppm levels into the breathing air [29], or by injecting immunostimulants like bacterial lipopolysaccharide to induce whole animal NO synthase expression [5]. Some of these manipulations are already known to change the NO levels and heme contents of heme proteins in animals in predictable ways that support the hypothesis [5][27]. In humans, although it is restrictive to do direct experiments, it may be possible to utilize the sGC-based pharmacologic response as noted above to study systemic effects of NO on the level of heme in sGC and thus potentially report on the heme-anemic condition in general. For example, by measuring forearm blood flow or skin conductance [37]in response to the different sGC agonists, one could investigate the impact of a high-nitrate diet [11] or of breathing a medically-approved level of NO-supplemented air [29]. Possible correlations could be investigated between the sGC agonist response and the level of NO in exhaled air from the lungs [22], the level of S-nitrosated Hb or heme-nitrosyl Hb present in the blood [53], and/or the overall NO production as measured by nitrate levels in blood and urine samples (for example in highland versus lowland populations) [21]. If results obtained with the sGC agonists proved interesting, perhaps the heme content of other heme proteins could also be assessed from a tissue or cell sample obtained in some minimally invasive way.

Conclusion

A heme anemic condition that naturally exists outside of the circulation limits heme availability and thereby limits the heme contents and functions of heme proteins expressed in tissues and cells. Within this context, the natural endogenous signaling molecule, NO, was recently found to up- or down-regulate cellular heme availability in a sensitive and concentration-dependent manner. Together, these findings reveal how NO, by controlling cellular heme allocation, may play a fundamental role in regulating the functions of diverse heme proteins in biology that impact many aspects of human health and disease.

Funding

Supported by National Institute of Health Grants HL081064 and GM130624 to DJS and HL150049 to AG

Data availability statement

Data sharing not applicable to this article as no data sets were generated or analyzed during the current study.

References

  • [1].Abraham NG, Quan S, Mieyal PA, Yang L, Burke-Wolin T, Mingone CJ, … Wolin MS (2002). Modulation of cGMP by human HO-1 retrovirus gene transfer in pulmonary microvessel endothelial cells. Am J Physiol Lung Cell Mol Physiol, 283(5), L1117–1124. doi: 10.1152/ajplung.00365.2001 [DOI] [PubMed] [Google Scholar]
  • [2].Albakri QA, & Stuehr DJ (1996). Intracellular assembly of inducible NO synthase is limited by nitric oxide-mediated changes in heme insertion and availability. J Biol Chem, 271(10), 5414–5421. doi: 10.1074/jbc.271.10.5414 [DOI] [PubMed] [Google Scholar]
  • [3].Alderton WK, Cooper CE, & Knowles RG (2001). Nitric oxide synthases: structure, function and inhibition. Biochem J, 357(Pt 3), 593–615. doi: 10.1042/0264-6021:3570593 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [4].Beall CM, Laskowski D, & Erzurum SC (2012). Nitric oxide in adaptation to altitude. Free Radic Biol Med, 52(7), 1123–1134. doi: 10.1016/j.freeradbiomed.2011.12.028 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [5].Bissell DM, & Hammaker LE (1977). Effect of endotoxin on tryptophan pyrrolase and delta-aminolaevulinate synthase: evidence for an endogenous regulatory haem fraction in rat liver. Biochem J, 166(2), 301–304. doi: 10.1042/bj1660301 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [6].Biswas P, Dai Y, & Stuehr DJ (2022). Indoleamine dioxygenase and tryptophan dioxygenase activities are regulated through GAPDH- and Hsp90-dependent control of their heme levels. Free Radic Biol Med, 180, 179–190. doi: 10.1016/j.freeradbiomed.2022.01.008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [7].Biswas P, & Stuehr DJ (2023). Indoleamine Dioxygenase and Tryptophan Dioxygenase Activities are Regulated through Control of Cell Heme Allocation by Nitric Oxide. J Biol Chem, 104753. doi: 10.1016/j.jbc.2023.104753 [DOI] [PMC free article] [PubMed]
  • [8].Boon EM, & Marletta MA (2005). Ligand specificity of H-NOX domains: from sGC to bacterial NO sensors. J Inorg Biochem, 99(4), 892–902. doi: 10.1016/j.jinorgbio.2004.12.016 [DOI] [PubMed] [Google Scholar]
  • [9].Calabrese EJ, Agathokleous E, Dhawan G, Kapoor R, Dhawan V, Manes PK, & Calabrese V (2023). Nitric oxide and hormesis. Nitric Oxide, 133, 1–17. doi: 10.1016/j.niox.2023.02.001 [DOI] [PubMed] [Google Scholar]
  • [10].Cals-Grierson MM, & Ormerod AD (2004). Nitric oxide function in the skin. Nitric Oxide, 10(4), 179–193. doi: 10.1016/j.niox.2004.04.005 [DOI] [PubMed] [Google Scholar]
  • [11].Carlstrom M, Moretti CH, Weitzberg E, & Lundberg JO (2020). Microbiota, diet and the generation of reactive nitrogen compounds. Free Radic Biol Med, 161, 321–325. doi: 10.1016/j.freeradbiomed.2020.10.025 [DOI] [PubMed] [Google Scholar]
  • [12].Chakravarti R, Gupta K, Majors A, Ruple L, Aronica M, & Stuehr DJ (2015). Novel insights in mammalian catalase heme maturation: effect of NO and thioredoxin-1. Free Radic Biol Med, 82, 105–113. doi: 10.1016/j.freeradbiomed.2015.01.030 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [13].Chiabrando D, Vinchi F, Fiorito V, Mercurio S, & Tolosano E (2014). Heme in pathophysiology: a matter of scavenging, metabolism and trafficking across cell membranes. Front Pharmacol, 5, 61. doi: 10.3389/fphar.2014.00061 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [14].Correia MA, Sinclair PR, & De Matteis F (2011). Cytochrome P450 regulation: the interplay between its heme and apoprotein moieties in synthesis, assembly, repair, and disposal. Drug Metab Rev, 43(1), 1–26. doi: 10.3109/03602532.2010.515222 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [15].Dai Y, Faul EM, Ghosh A, & Stuehr DJ (2022). NO rapidly mobilizes cellular heme to trigger assembly of its own receptor. Proc Natl Acad Sci U S A, 119(4). doi: 10.1073/pnas.2115774119 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [16].Desuzinges-Mandon E, Arnaud O, Martinez L, Huche F, Di Pietro A, & Falson P (2010). ABCG2 transports and transfers heme to albumin through its large extracellular loop. J Biol Chem, 285(43), 33123–33133. doi: 10.1074/jbc.M110.139170 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [17].Doty RT, Sanchez-Bonilla M, Keel SB, & Abkowitz JL (2013). FLVCR1a But Not FLVCR1b Is Required For Effective Erythropoiesis In Adult Mice. Blood, 122(21), 308–308. doi: 10.1182/blood.V122.21.308.308 [DOI] [Google Scholar]
  • [18].Dunham-Snary KJ, Wu D, Sykes EA, Thakrar A, Parlow LRG, Mewburn JD, … Archer SL (2017). Hypoxic Pulmonary Vasoconstriction: From Molecular Mechanisms to Medicine. Chest, 151(1), 181–192. doi: 10.1016/j.chest.2016.09.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [19].Dyakova EY, Kapilevich LV, Shylko VG, Popov SV, & Anfinogenova Y (2015). Physical exercise associated with NO production: signaling pathways and significance in health and disease. Front Cell Dev Biol, 3, 19. doi: 10.3389/fcell.2015.00019 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [20].Eby GA (2006). Strong humming for one hour daily to terminate chronic rhinosinusitis in four days: a case report and hypothesis for action by stimulation of endogenous nasal nitric oxide production. Med Hypotheses, 66(4), 851–854. doi: 10.1016/j.mehy.2005.11.035 [DOI] [PubMed] [Google Scholar]
  • [21].Erzurum SC, Ghosh S, Janocha AJ, Xu W, Bauer S, Bryan NS, … Beall CM (2007). Higher blood flow and circulating NO products offset high-altitude hypoxia among Tibetans. Proc Natl Acad Sci U S A, 104(45), 17593–17598. doi: 10.1073/pnas.0707462104 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [22].Escamilla-Gil JM, Fernandez-Nieto M, & Acevedo N (2022). Understanding the Cellular Sources of the Fractional Exhaled Nitric Oxide (FeNO) and Its Role as a Biomarker of Type 2 Inflammation in Asthma. Biomed Res Int, 2022, 5753524. doi: 10.1155/2022/5753524 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [23].Ford PC, Pereira JCM, & Miranda KM (2014). Mechanisms of Nitric Oxide Reactions Mediated by Biologically Relevant Metal Centers. In Mingos DMP (Ed.), Nitrosyl Complexes in Inorganic Chemistry, Biochemistry and Medicine II (pp. 99–135). Berlin, Heidelberg: Springer Berlin Heidelberg. [Google Scholar]
  • [24].Forstermann U, & Munzel T (2006). Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation, 113(13), 1708–1714. doi: 10.1161/CIRCULATIONAHA.105.602532 [DOI] [PubMed] [Google Scholar]
  • [25].Ghosh A, Koziol-White CJ, Jester WF Jr., Erzurum SC, Asosingh K, Panettieri RA Jr., & Stuehr DJ (2021). An inherent dysfunction in soluble guanylyl cyclase is present in the airway of severe asthmatics and is associated with aberrant redox enzyme expression and compromised NO-cGMP signaling. Redox Biol, 39, 101832. doi: 10.1016/j.redox.2020.101832 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [26].Ghosh A, Stasch JP, Papapetropoulos A, & Stuehr DJ (2014). Nitric oxide and heat shock protein 90 activate soluble guanylate cyclase by driving rapid change in its subunit interactions and heme content. J Biol Chem, 289(22), 15259–15271. doi: 10.1074/jbc.M114.559393 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [27].Ghosh A, Sumi MP, Tupta B, Okamoto T, Aulak K, Tsutsui M, … Stuehr DJ (2022). Low levels of nitric oxide promotes heme maturation into several hemeproteins and is also therapeutic. Redox Biol, 56, 102478. doi: 10.1016/j.redox.2022.102478 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [28].Ghosh S, Janocha AJ, Aronica MA, Swaidani S, Comhair SA, Xu W, … Erzurum SC (2006). Nitrotyrosine proteome survey in asthma identifies oxidative mechanism of catalase inactivation. J Immunol, 176(9), 5587–5597. doi: 10.4049/jimmunol.176.9.5587 [DOI] [PubMed] [Google Scholar]
  • [29].Gladwin MT, Schechter AN, Shelhamer JH, Pannell LK, Conway DA, Hrinczenko BW, … Ognibene FP (1999). Inhaled nitric oxide augments nitric oxide transport on sickle cell hemoglobin without affecting oxygen affinity. J Clin Invest, 104(7), 937–945. doi: 10.1172/JCI7637 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [30].Grzesk G, Witczynska A, Weglarz M, Wolowiec L, Nowaczyk J, Grzesk E, & Nowaczyk A (2023). Soluble Guanylyl Cyclase Activators-Promising Therapeutic Option in the Pharmacotherapy of Heart Failure and Pulmonary Hypertension. Molecules, 28(2). doi: 10.3390/molecules28020861 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [31].Hayashi T, Beck L, Rossetto C, Gong X, Takikawa O, Takabayashi K, … Raz E (2004). Inhibition of experimental asthma by indoleamine 2,3-dioxygenase. J Clin Invest, 114(2), 270–279. doi: 10.1172/JCI21275 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [32].Huang PL (2000). Mouse models of nitric oxide synthase deficiency. J Am Soc Nephrol, 11 Suppl 16, S120–123. [PubMed] [Google Scholar]
  • [33].Ignarro LJ (2019). Nitric oxide is not just blowing in the wind. Br J Pharmacol, 176(2), 131–134. doi: 10.1111/bph.14540 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [34].Ignarro LJ, Degnan JN, Baricos WH, Kadowitz PJ, & Wolin MS (1982). Activation of purified guanylate cyclase by nitric oxide requires heme. Comparison of heme-deficient, heme-reconstituted and heme-containing forms of soluble enzyme from bovine lung. Biochim Biophys Acta, 718(1), 49–59. doi: 10.1016/0304-4165(82)90008-3 [DOI] [PubMed] [Google Scholar]
  • [35].Immenschuh S, Vijayan V, Janciauskiene S, & Gueler F (2017). Heme as a Target for Therapeutic Interventions. Front Pharmacol, 8, 146. doi: 10.3389/fphar.2017.00146 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [36].Jia S, Guo P, Ge X, Wu H, Lu J, & Fan X (2018). Overexpression of indoleamine 2, 3-dioxygenase contributes to the repair of human airway epithelial cells inhibited by dexamethasone via affecting the MAPK/ERK signaling pathway. Exp Ther Med, 16(1), 282–290. doi: 10.3892/etm.2018.6163 [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  • [37].Kellogg DL Jr., Zhao JL, Wu Y, & Johnson JM (2011). Antagonism of soluble guanylyl cyclase attenuates cutaneous vasodilation during whole body heat stress and local warming in humans. J Appl Physiol (1985), 110(5), 1406–1413. doi: 10.1152/japplphysiol.00702.2010 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [38].Kim YM, Bergonia HA, Muller C, Pitt BR, Watkins WD, & Lancaster JR Jr. (1995). Loss and degradation of enzyme-bound heme induced by cellular nitric oxide synthesis. J Biol Chem, 270(11), 5710–5713. doi: 10.1074/jbc.270.11.5710 [DOI] [PubMed] [Google Scholar]
  • [39].Knox WE (1951). Two mechanisms which increase in vivo the liver tryptophan peroxidase activity: specific enzyme adaptation and stimulation of the pituitary adrenal system. Br J Exp Pathol, 32(5), 462–469. [PMC free article] [PubMed] [Google Scholar]
  • [40].Liu L, Dumbrepatil AB, Fleischhacker AS, Marsh ENG, & Ragsdale SW (2020). Heme oxygenase-2 is post-translationally regulated by heme occupancy in the catalytic site. J Biol Chem, 295(50), 17227–17240. doi: 10.1074/jbc.RA120.014919 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [41].Lopez-Jaramillo P, Arenas WD, Garcia RG, Rincon MY, & Lopez M (2008). The role of the L-arginine-nitric oxide pathway in preeclampsia. Ther Adv Cardiovasc Dis, 2(4), 261–275. doi: 10.1177/1753944708092277 [DOI] [PubMed] [Google Scholar]
  • [42].Lundberg JO, Farkas-Szallasi T, Weitzberg E, Rinder J, Lidholm J, Anggaard A, … Alving K (1995). High nitric oxide production in human paranasal sinuses. Nat Med, 1(4), 370–373. doi: 10.1038/nm0495-370 [DOI] [PubMed] [Google Scholar]
  • [43].Lundberg JO, Weitzberg E, & Gladwin MT (2008). The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics. Nat Rev Drug Discov, 7(2), 156–167. doi: 10.1038/nrd2466 [DOI] [PubMed] [Google Scholar]
  • [44].Mbongue JC, Nicholas DA, Torrez TW, Kim NS, Firek AF, & Langridge WH (2015). The Role of Indoleamine 2, 3-Dioxygenase in Immune Suppression and Autoimmunity. Vaccines (Basel), 3(3), 703–729. doi: 10.3390/vaccines3030703 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [45].Mense SM, & Zhang L (2006). Heme: a versatile signaling molecule controlling the activities of diverse regulators ranging from transcription factors to MAP kinases. Cell Res, 16(8), 681–692. doi: 10.1038/sj.cr.7310086 [DOI] [PubMed] [Google Scholar]
  • [46].Negrerie M, Berka V, Vos MH, Liebl U, Lambry JC, Tsai AL, & Martin JL (1999). Geminate recombination of nitric oxide to endothelial nitric-oxide synthase and mechanistic implications. J Biol Chem, 274(35), 24694–24702. doi: 10.1074/jbc.274.35.24694 [DOI] [PubMed] [Google Scholar]
  • [47].Nelp MT, Kates PA, Hunt JT, Newitt JA, Balog A, Maley D, … Groves JT (2018). Immune-modulating enzyme indoleamine 2,3-dioxygenase is effectively inhibited by targeting its apo-form. Proc Natl Acad Sci U S A, 115(13), 3249–3254. doi: 10.1073/pnas.1719190115 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [48].Nosarev AV, Smagliy LV, Anfinogenova Y, Popov SV, & Kapilevich LV (2014). Exercise and NO production: relevance and implications in the cardiopulmonary system. Front Cell Dev Biol, 2, 73. doi: 10.3389/fcell.2014.00073 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [49].Pianigiani T, Alderighi L, Meocci M, Messina M, Perea B, Luzzi S, … Cameli P (2023). Exploring the Interaction between Fractional Exhaled Nitric Oxide and Biologic Treatment in Severe Asthma: A Systematic Review. Antioxidants (Basel), 12(2). doi: 10.3390/antiox12020400 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [50].Premont RT, Reynolds JD, Zhang R, & Stamler JS (2021). Red Blood Cell-Mediated S-Nitrosohemoglobin-Dependent Vasodilation: Lessons Learned from a beta-Globin Cys93 Knock-In Mouse. Antioxid Redox Signal, 34(12), 936–961. doi: 10.1089/ars.2020.8153 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [51].Raat NJ, Noguchi AC, Liu VB, Raghavachari N, Liu D, Xu X, … Gladwin MT (2009). Dietary nitrate and nitrite modulate blood and organ nitrite and the cellular ischemic stress response. Free Radic Biol Med, 47(5), 510–517. doi: 10.1016/j.freeradbiomed.2009.05.015 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [52].Reddi AR, & Hamza I (2016). Heme Mobilization in Animals: A Metallolipid’s Journey. Acc Chem Res, 49(6), 1104–1110. doi: 10.1021/acs.accounts.5b00553 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [53].Reynolds JD, Posina K, Zhu L, Jenkins T, Matto F, Hausladen A, … Stamler JS (2023). Control of tissue oxygenation by S-nitrosohemoglobin in human subjects. Proc Natl Acad Sci U S A, 120(9), e2220769120. doi: 10.1073/pnas.2220769120 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [54].Ricciardolo FL (2003). Multiple roles of nitric oxide in the airways. Thorax, 58(2), 175–182. doi: 10.1136/thorax.58.2.175 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [55].Runyen-Janecky LJ (2013). Role and regulation of heme iron acquisition in gram-negative pathogens. Front Cell Infect Microbiol, 3, 55. doi: 10.3389/fcimb.2013.00055 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [56].Sandner P, Follmann M, Becker-Pelster E, Hahn MG, Meier C, Freitas C, … Stasch JP (2021). Soluble GC stimulators and activators: Past, present and future. Br J Pharmacol doi: 10.1111/bph.15698 [DOI] [PubMed]
  • [57].Shah RC, Sanker S, Wood KC, Durgin BG, & Straub AC (2018). Redox regulation of soluble guanylyl cyclase. Nitric Oxide, 76, 97–104. doi: 10.1016/j.niox.2018.03.013 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [58].Stanaway L, Rutherfurd-Markwick K, Page R, & Ali A (2017). Performance and Health Benefits of Dietary Nitrate Supplementation in Older Adults: A Systematic Review. Nutrients, 9(11). doi: 10.3390/nu9111171 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [59].Stojanovski BM, Hunter GA, Na I, Uversky VN, Jiang RHY, & Ferreira GC (2019). 5-Aminolevulinate synthase catalysis: The catcher in heme biosynthesis. Mol Genet Metab, 128(3), 178–189. doi: 10.1016/j.ymgme.2019.06.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [60].Stuehr DJ, Dai Y, Biswas P, Sweeny EA, & Ghosh A (2022). New roles for GAPDH, Hsp90, and NO in regulating heme allocation and hemeprotein function in mammals. Biol Chem, 403(11–12), 1005–1015. doi: 10.1515/hsz-2022-0197 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [61].Stuehr DJ, Santolini J, Wang ZQ, Wei CC, & Adak S (2004). Update on mechanism and catalytic regulation in the NO synthases. J Biol Chem, 279(35), 36167–36170. doi: 10.1074/jbc.R400017200 [DOI] [PubMed] [Google Scholar]
  • [62].Sumi MP, Tupta B, & Ghosh A (2022). Nitric Oxide Trickle Drives Heme into Hemoglobin and Muscle Myoglobin. Cells, 11(18). doi: 10.3390/cells11182838 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [63].Sweeny EA, Hunt AP, Batka AE, Schlanger S, Lehnert N, & Stuehr DJ (2021). Nitric oxide and heme-NO stimulate superoxide production by NADPH oxidase 5. Free Radic Biol Med, 172, 252–263. doi: 10.1016/j.freeradbiomed.2021.06.008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [64].Swenson SA, Moore CM, Marcero JR, Medlock AE, Reddi AR, & Khalimonchuk O (2020). From Synthesis to Utilization: The Ins and Outs of Mitochondrial Heme. Cells, 9(3). doi: 10.3390/cells9030579 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [65].Teran E, Chedraui P, Vivero S, Villena F, Duchicela F, & Nacevilla L (2009). Plasma and placental nitric oxide levels in women with and without pre-eclampsia living at different altitudes. Int J Gynaecol Obstet, 104(2), 140–142. doi: 10.1016/j.ijgo.2008.09.010 [DOI] [PubMed] [Google Scholar]
  • [66].Tupta B, Stuehr E, Sumi MP, Sweeny EA, Smith B, Stuehr DJ, & Ghosh A (2022). GAPDH is involved in the heme-maturation of myoglobin and hemoglobin. FASEB J, 36(2), e22099. doi: 10.1096/fj.202101237RR [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [67].Upmacis RK, Hajjar DP, Chait BT, & Mirza UA (1997). Direct Observation of Nitrosylated Heme in Myoglobin and Hemoglobin by Electrospray Ionization Mass Spectrometry. Journal of the American Chemical Society, 119(43), 10424–10429. doi: 10.1021/ja964249l [DOI] [Google Scholar]
  • [68].Waheed SM, Ghosh A, Chakravarti R, Biswas A, Haque MM, Panda K, & Stuehr DJ (2010). Nitric oxide blocks cellular heme insertion into a broad range of heme proteins. Free Radic Biol Med, 48(11), 1548–1558. doi: 10.1016/j.freeradbiomed.2010.02.038 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [69].Webb AJ, Patel N, Loukogeorgakis S, Okorie M, Aboud Z, Misra S, … Ahluwalia A (2008). Acute blood pressure lowering, vasoprotective, and antiplatelet properties of dietary nitrate via bioconversion to nitrite. Hypertension, 51(3), 784–790. doi: 10.1161/HYPERTENSIONAHA.107.103523 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [70].Weitzberg E, & Lundberg JO (2002). Humming greatly increases nasal nitric oxide. Am J Respir Crit Care Med, 166(2), 144–145. doi: 10.1164/rccm.200202-138BC [DOI] [PubMed] [Google Scholar]
  • [71].Yuan X, Rietzschel N, Kwon H, Walter Nuno AB, Hanna DA, Phillips JD, … Hamza I (2016). Regulation of intracellular heme trafficking revealed by subcellular reporters. Proc Natl Acad Sci U S A, 113(35), E5144–5152. doi: 10.1073/pnas.1609865113 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [72].Zhang AS, & Enns CA (2009). Iron homeostasis: recently identified proteins provide insight into novel control mechanisms. J Biol Chem, 284(2), 711–715. doi: 10.1074/jbc.R800017200 [DOI] [PMC free article] [PubMed] [Google Scholar]

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