Hemoglobin: A Nitric-Oxide Dioxygenase

Members of the hemoglobin superfamily efficiently catalyze nitric-oxide dioxygenation, and when paired with native electron donors, function as NO dioxygenases (NODs). Indeed, the NOD function has emerged as a more common and ancient function than the well-known role in O2 transport-storage. Novel hemoglobins possessing a NOD function continue to be discovered in diverse life forms. Unique hemoglobin structures evolved, in part, for catalysis with different electron donors. The mechanism of NOD catalysis by representative single domain hemoglobins and multidomain flavohemoglobin occurs through a multistep mechanism involving O2 migration to the heme pocket, O2 binding-reduction, NO migration, radical-radical coupling, O-atom rearrangement, nitrate release, and heme iron re-reduction. Unraveling the physiological functions of multiple NODs with varying expression in organisms and the complexity of NO as both a poison and signaling molecule remain grand challenges for the NO field. NOD knockout organisms and cells expressing recombinant NODs are helping to advance our understanding of NO actions in microbial infection, plant senescence, cancer, mitochondrial function, iron metabolism, and tissue O2 homeostasis. NOD inhibitors are being pursued for therapeutic applications as antibiotics and antitumor agents. Transgenic NOD-expressing plants, fish, algae, and microbes are being developed for agriculture, aquaculture, and industry.


Background and Introduction
Nitric-oxide dioxygenases (NODs) are enzymes that efficiently convert NO and O 2 to nitrate (1). Most, if not all, NODs are hemoglobins (Hbs), and most, if not all, Hbs have the capacity to function as NODs. Hb-NODs appear to be widely distributed in nature. In fact, the NOD function appears more common and ancient than the classic O 2 transport-storage function, or any other function, within the Hb superfamily [1]. Nevertheless, textbook familiarity with the O 2 transport-storage function continues to blind investigators to the enzymatic functions of various members of the Hb superfamily. For example, the genome of the nematode Caenorhabditis elegans encodes >33 candidate Hbs [2,3], many of which are thought to store or transport O 2 . Multiple globins are also normally expressed in nonerythroid vertebrate cells and tissues [4,5], and globin expression is deranged in cancer cells [6][7][8][9]. When asking the question "what are all these oxygen-binding heme proteins doing?" [10], investigators are now obliged to thoroughly consider enzyme functions, and in particular a NOD function NODs are one of the most recent additions to the growing family of free radical and peroxide scavenging enzymes that includes the family of peroxidases [11] and peroxiredoxins (alkyl hydroperoxide reductases) [12], catalases [13], superoxide dismutases (SODs) [14,15], superoxide reductases [16], and NO reductases (NORs) [17]. ere is also preliminary evidence for a NO dismutase in certain methanotrophs [18].
Seminal concepts within the family of peroxide and free radical scavenging enzymes can be traced to Oskar Loew's pioneering description of "catalase", a heme-containing enzyme with the "power of catalyzing hydrogen peroxide" [13,19]. In 1900, Loew wrote "ere seems to exist no plant and no animal which is without that enzyme [catalase]". He also presciently wrote "One of the functions of this enzyme appears to be to prevent any accumulation of hydrogen peroxide which might be formed as a by-product in the series of energetic oxidations that characterize the cellular respiration process. Hydrogen peroxide is a poison for the living protoplasm, hence the activity of catalase is of vital importance. " Loew also made the early and important distinction between the substrate (H 2 O 2 ) and enzyme in the catalytic reaction at a time when enzymes were only vaguely understood. While peroxidations had been described as early as 1855, the enzymatic nature and biological function of peroxidases would only be investigated much later [11,20,21]. e idea of dangerous toxic free radicals formed as byproducts of an aerobic metabolism blossomed following the discovery of superoxide dismutase(s) and the demonstrations of superoxide radical actions in biological systems by Irwin Fridovich and his students [14,15,22]. Incalculable efforts have been expended to achieve our current understanding of the poisonous and damaging reactions, sources of the radicals and oxidants, as well as the full repertoire of detoxifying enzymes and their roles in physiology and pathophysiology. In addition, novel roles for radicals and oxidants in cell signaling and scavengers in signal modulation were appreciated by the end of the 20th century [23], but are still not fully comprehended.
Indeed, paradigms established by the pioneers of antioxidant and free radical-scavenging enzymes guided the discovery of NOD and continue to instruct investigations. Experiments have also been facilitated by a wealth of knowledge, as well as questions waiting for solution, in the areas of Hb structure, distribution, expression, functions, and evolutionary origins [24,25]. On the other hand, the discovery of NO metabolizing enzymes followed rapidly on the heels of discoveries of the biological production and functions of NO, thus leaving much to discern and discover in a rapidly evolving �eld. Substantial effort is now being aimed at understanding the biological functions and enzymatic mechanisms of various NODs and the development of NODbased technologies including research tools, recombinant plants and other organisms, and therapeutic drugs including antibiotics. Novel Hbs continue to be examined for a possible NOD function. In addition, novel non-Hb type NODs and NOD mimetics are under investigation [26].

Purpose and Scope
While several reactions of NO with erythrocyte Hb and muscle myoglobin (Mb) including the tight-binding of NO to the ferrous heme, the NO (Stickstoffoxyd, Saltpeterstoffgas)mediated oxidation of the oxy-heme, and the formation of NO from nitrite have been under investigation for many decades [27][28][29][30][31][32][33], our investigations published in 1998 [34,35] provided our �rst glimpse of the ∼2 billion-year-old evolutionary link between NO and Hb vis-à-vis the NOD function of E. coli �avoHb. e observations offered a distinct and common enzymatic function for the primitive lowabundance Hbs �rst formally formulated and investigated by David Keilin around 1945 [36, 37] Riggs [39,52], Michiko Nakano [53], Robert Poyton [54], Daniel Goldberg [55][56][57], Keiji Shikama [58][59][60][61], Malcolm Potts [62], Robert Hill [63,64] and many others clearing a wide path to the common NOD function. However, while appearing attractive to some, the well-known tight-binding of NO to ferrous Hbs [ =∼ 10 pM] made the NOD enzyme hypothesis appear rather dubious in the eyes of experts. Despite this and other challenges, data supporting a primal NOD mechanism-function for diverse members of the ancient Hb superfamily has expanded appreciably in the last 14 years. While the facts that lay obscure or dormant in the pre-1998 literature, and the �ndings of the �rst ∼7 years have been dutifully and thoroughly reviewed in our 2005 and 2006 publications [65,66] and elsewhere, my intent here is not only to describe important advances in the area of Hb/NOD research, but also to pose critical questions and discuss new concepts that will help move investigations forward and into new areas. is, of course, has demanded critiques of what I consider lingering misconceptions that continue to plague the �eld, stymie progress, and lead investigators off the path. It is hoped that the reader �nds this paper rigorous, not trite, too pedantic, tame, or polemical. e reader is also directed to more than a dozen recent reviews presenting in-depth perspectives on various aspects of the topic [1,. I have attempted to highlight these reviews in pertinent sections of my paper. A distillation and synthesis of the available evidence is crucial since some investigators continue to ponder upon a "common but still poorly de�ned function of globins" [88]. Here, I thoroughly scrutinize the merits of a common NOD function in the light of the results of more than a decade of research.

Evolution and Distribution of Hbs
Several recent phylogenomic studies and reviews have catalogued the broad distribution and lineages of Hbs of various subtypes in the three life kingdoms [1,[89][90][91][92][93][94]. e amazing array of globins expressed even within individual life forms [3,42,74,95] including humans [96,97] raise myriad questions that will occupy investigators for many years. Most strikingly, the genomes of most organisms encode multiple Hbs with unknown function. Moreover, relatively few Hbs have been scrutinized for a NOD function. Which subtypes function as NODs? If a life form cannot express a Hb, how does the organism metabolize NO? Why do organisms express multiple Hbs and various Hb sub-types ? What primary structure features reliably predict a NOD function and allow annotation of Hb genes? What is the signi�cance of these structural features to function? What are all the possible functions of Hb ? And the biggest question-How did Hb function evolve?
Answers to these many questions have been slow to emerge, but there are fundamental concepts guiding our understanding of Hb functions and evolution. Clearly, numerous proteins possess multiple reactivities and functions [98,99]; a property that has been colorfully referred to as "moonlighting" [100] or "catalytic promiscuity" [101].
Indeed, those reactivities serve as a framework for protein [globin] evolution. As astutely pointed out by Shikama and coworkers [61,102], "Whatever the possible roles of such primitive or ancient globins may be [or might have been], the reversible binding of molecular oxygen to iron [II] must be the primary event to manifest their physiological functions in vivo." e fairly unique electronic structure of the ferric hemesuperoxo bond in Hbs [103] suggests an even more unique chemical-biological niche for the globins. us, while a focus on the evolution of globin structural differences suggests great functional variation [24,25,104,105], a focus on the potentially limited reactivity of the conserved ferric hemesuperoxo electronic structure suggests a common function.
As Max Perutz eloquently stated "Evolution is a brilliant chemist" [106]. Proteins evolve by eliminating old traits and creating new traits from a single protein scaffold. In the case of Hbs, the old traits of �avoHb-NODs include high O 2 affinities, high autooxidation rates, and internal electron transfer rates that are suited for rapid NO dioxygenation catalysis. New traits include the low O 2 affinities, low autooxidation rates, external electron donors, lower NO dioxygenation rate constants [65], and a single globin domain that is adapted for O 2 storage-transport [106]. What is more difficult to fathom and demonstrate is a major O 2 -independent function for Hb that predates the ∼2 billion year old, contemporary, and common NOD function [1]. Albeit, NO reductase [69,107] and electron transfer [108] functions have been proposed for �avoHb and neuroglobin (Ngb), respectively. e discoveries of an estimated 3.8 billion-year-old primordial protoglobin in a "strictly" anaerobic methanogen Methanosarcina acetivorans [109,110] and the single domain "thermoglobin" in Aquifex aeolicus (AE000678) [52,111] provide relic experimental systems to further explore the functional origins of the globin family.
For many single domain globins (SDGs), an annotation as a NOD has always been less clear. It would appear that all single domain oxy-Hbs catalyze the NO dioxygenation reaction with reported in vitro bimolecular rate constants ranging from 10 6 to >10 9 s −1 M −1 [65,66,[120][121][122][123][124][125]. e Arabidopsis thalania GLB1 and GLB2 (trHbs) convert NO to nitrate in vivo as evidenced by increased NO emissions and decreased tissue nitrate concentrations in globin-de�cient mutants [126,127]. But few SDGs have been shown to couple with a native redox partner for efficient enzymatic NO consumption [128][129][130][131][132]. Moreover, when demonstrations of weak or nonspeci�c redox coupling have been observed to support turnover in vitro, or in heterologous organisms, there has been an understandable reluctance to declare that sufficient evidence for a NOD function [133]. Clearly, full proof of a NOD function requires the demonstration of catalytic 2 -dependent NO metabolism within the native organism. �urthermore, a speci�c electron donor needs to be identi�ed for demonstrations of efficient catalysis in vitro. To date, only the mammalian Cygb-NOD has been investigated both in cells [134,135] and reconstituted with a native electron donor (ascorbate or cytochrome b 5 ) in vitro [135,136]. In addition, many SDGs have shown evidence of a protective enzymatic NOD function including muscle Mb [137], Synechococcus truncated Hb [138], Synechocystis cyanoglobin [133], and the dual function nitrate reductase-fused trHb in raphidophytes [139]. However, one cannot declare a NOD function a priori. A NOD function requires a demonstration of enzymatic turnover. Protective effects of SDGs could also be attributed to nonenzymatic, rapid, and competing NO oxidation (dioxygenation) and nitrosylation reactions of Hbs similar to those of erythrocyte Hb and muscle Mb [31,32,140]. is is a small distinction, but a potentially important one.

Myriad NO Functions:
Bioenergetic Intermediate, N Source, Toxin, and Signal Molecule To understand the physiological functions of NODs (Hbs), knowledge of the burgeoning �eld of NO biology is required. NO is fairly ubiquitous and serves important roles in bioenergetic transformations, immunity, and signaling in diverse life forms. Quantitative knowledge of the sources of NO, target reactions of NO, and localized steady-state NO levels is also required to discern the competing and overlapping roles of NO. Moreover, within speci�c cells and tissues, these factors vary in complex ways [198,199]. Nevertheless, instructive generalizations can be made. In microbes, algae, and plants, NO serves as an important role as a bioenergetic intermediate and nitrogen source. e process of anaerobic or microaerobic denitri�cation, particularly in soil microorganisms, generates NO as an obligate intermediate in the energy-yielding reductive dissimilation of NO 3 − to N 2 [17,200]. Nitrate reductases generate nitrite, and nitrite reductases generate NO for reduction by proton-motive force generating membrane-bound NO reductases and N 2 O reductases. FlavoHb (NOD) can recycle the denitri�cation intermediate NO to NO 3 − and is required for optimal microaerobic denitri�cation, and presumably energy production, by fungi [200] and bacteria [48]. NO is also an important intermediate in the energy-yielding pathway of microaerobic ammonia oxidation or nitri�cation [201,202]. Alternatively, NO can be generated through non-speci�c reduction of nitrite by nitrate reductases found abundantly in plants, algae, and microbes [203][204][205]. Neighboring soil microorganisms and plants can assimilate N through the Hb-catalyzed dioxygenation of diffusible NO to NO 3 − . In addition, NO 3 − generated via catalytic NO dioxygenation can provide an electron sink for fermentative energy production by plants [206], bacteria, and fungi [52,200].
NO is also an important intermediate in the Earth's nitrogen cycle [207,208]. Anthropogenic sources, such as N-fertilization, NO pollution, and waste generation, may contribute to excess formation of NO and the greenhouse gas N 2 O [207]. Rhizobial NODs serve an important role in converting NO to NO 3 − in plants and in doing so avert N 2 O formation by rhizobia and soil microbe NO reductases [87]. Indeed, Pseudomonas stutzeri engineered for �avoHb overproduction emit less N 2 O during microaerobic den-itri�cation [209]. Photoilluminated leaf chloroplast nitrite reductase is also an important source of atmospheric N 2 O presumably via the reduction of assimilated NO 2 − to NO [210], and NO to NO − followed by the combination of 2 NO − to form N 2 O. In this case, a chloroplast NOD (trHb) may serve as an important geochemical function by decreasing NO levels and N 2 O formation.
NO acts as a toxin and signal molecule throughout the biosphere. In addition to the numerous sources of NO described above, NO is produced by nitric-oxide synthases (NOSs) and various nitrite-reducing activities in many different organisms including humans [211]. Removal of NO by catalytic NO dioxygenation can thus serve to prevent NO toxicity or attenuate NO signaling. Understanding NO toxicity and signaling in the biosphere requires an understanding of the many reactions of NO as well as its concentrations. I have listed only a few of the important biochemical reactions of NO and the relevant biological functions or consequences in Table 1. e representative list of reactions clari�es why NO is an outstanding natural antibiotic and antitumor agent and why NO metabolism by NODs or NORs provide a direct path for cellular resistance. e toxicology of NO has been discussed in greater detail in an excellent recent review by Toledo Jr. and Augusto [189]. Not too surprisingly, many of the toxic reactions of NO have been exploited by Nature for NO signaling functions. ese bifunctional toxic reactions include ferrous heme nitrosylation (e.g., soluble guanylate cyclase activation), iron-sulfur center disruption (NsrR and IRE-BP) [193], mononuclear iron-binding (e.g., prolyl hydroxylase,

NOD Functions for FlavoHbs and Hbs in Microbial Pathogenesis
NO is produced as a natural antibiotic and antitumor agent in the innate immune response of animals and plants.
Microbes and tumor cells have the capacity to resist NO toxicity [228]. Microarray analyses of mRNA [152,183,213,214,220,[229][230][231][232][233][234][235][236][237][238] and in silico reconstruction of transcription networks [152,233,[239][240][241] in microbes and mammalian cells have revealed myriad adaptive changes potentially protecting against NO toxicity during microbial pathogenesis and in�ammation. NODs, NORs [17,151,242], Fe-S cluster [243], and DNA repair enzymes [243], heme biosynthetic pathways [113], NO-resistant metabolic pathways (e.g., glucose metabolism and respiratory oxidases) [159-161, 240, 241], nitrite metabolism [213], and components of iron uptake systems form important and common elements of the nitrosative stress defense. Remarkably, in plants [141], �ibrio �scheri [162], and the fungal pathogen Candida albicans [238], NO poisoning of respiration is apparently averted by the induction of NO-resistant alternative oxidase. Mycobacteria survive the hazardous environment of macrohages with GlbN (trHbN), GlbO (trHbO), and heat shock protein GroEL2 induction with GlbO providing greater protection than GlbN within macrophages [244]. Some aerobic organisms do not utilize a �avoHb-NOD, but apparently utilize sole NORs for NO metabolism [232], but these are the exception rather than the rule. e accumulated evidence supports the hypothesis that NO metabolism and detoxi�cation by NODs and�or NO�s form the �rst� and most critical� line of defense against NO toxicity in microbes. However, results reviewed in the pre-2005 literature revealed only a modest advantage of the inducible �avoHbs and Hbs for microbes in the chosen infection models [65]. Tail vein injections of NOD-de�cient Candida albicans showed limited effects on mouse survival [95,238,245], and those effects appeared NOS-independent [238]. Inhalation of Cryptococcus neoformans in mice showed similar modest extensions of mouse survival times with a �avoHb de�ciency [246]. e effects of Hmp� (�avoHb) deletions in Erwinia chrysanthemi infections of Saintpaulia ionantha (African violet) plants were more impressive, but interpretations were subject to potential effects of deletions of neighboring pectate lyase virulence genes [115,247]. Nevertheless, Boccara et al. convincingly argued an important role for HmpX in the pathogenesis of E. chrysanthemi. Not only does HmpX (NOD) protect against NO toxicity, it also impacts the NO-regulated hypersensitive response required for plant immunity [115,224,248]. Clearly, more and better models of microbial infection are required to discover the full involvement of �avoHbs and Hbs in microbial pathogenesis, albeit the prospects appear limitless. For example, mucosal infections by C. albicans are far more common and applicable than blood stream infections. Most experimental designs also ignore the effects of the metabolic state of microbes upon subsequent infectivity [249]. For example, naïve and metabolically depleted E. coli are more susceptible to NO toxicity and bacteriostasis than actively growing bacteria [35]. More impressive protective roles for the �avoHb-NOD in rodent models of Salmonella typhimurium [250], Staphylococcus aureus [214], uropathogenic E. coli [251], Yersinia pestis [217], Vibrio cholerae [252] infections and in squid models of Vibrio �scheri symbiosis [218] have been subsequently reported. Interestingly, in the intestinal model of V. cholerae infection, �avoHb (Hmp) was more critical for virulence than the NO defense regulator. Stern and coworkers suggest that low constitutive levels of �avoHb protect V. cholerae from NO-dependent elimination [252]. In the case of the plant fungal necrotroph Botrytis cinerea, �avoHb-NOD was not found to be a virulence factor [222].

Critical Roles for Hb-NODs in Preserving Bioenergetics
Hbs serve two most vital roles in the production of energy via oxidative-phosphorylation. When oxygenated, Hbs can prevent NO poisoning of the citric acid cycle enzyme aconitase, heme-copper terminal oxidases, and respiration by catalyzing NO dioxygenation [35,163]. NO poisoning of terminal oxidases and respiration is particulary problematic during hypoxia because of the strong competition between NO and O 2 [∼1 : 1000] for binding the binuclear heme a 3 /Cu site and the noncompetitive binding of NO to the oxidized Cu site [159,160,164]. If sufficiently abundant, and having a low affinity for O 2 binding, Hbs can also store and release O 2 to sustain respiration during transient episodes of hypoxia. ese two functions, or NOD activity alone, can explain the strong protection nerve Hbs afford nerve excitatory activity during episodes of anoxia/hypoxia [253,254], muscle Mb provides the heart during hypoxia [137,255], Cygb affords �broblast respiration [134], �avoHb affords E. coli respiration [163], Vitreoscilla Hb affords either E. coli, plants or zebra�sh (Danio rerio) engineered for biotechnological applications [78,[256][257][258], nonsymbiotic Hb expression has on maize ATP levels and energy charge during hypoxia [63,74], and legume Hb affords Rhodobacter japonicum respiration [43] and soybean nodule bacteroid N 2 �xation [125,259]. e inducible Sinorhizobium meliloti �avoHb plays a similar role in protecting alfalfa bacteroid N 2 �xation and fostering symbiosis [183,260]. ese strong dual effects on organism energetics (and survival) may have constituted the earliest, most powerful, selective force and circumstance for Hb to evolve from an efficient low-level expression NO dioxygenase to a highly expressed 2 storage-transport protein. Ironically, the existence of Antarctic ice�sh lacking in Mb and Hb also supports the notion that these proteins evolved for dual functions. However, in this case, the loss O 2 transport-storage and NOD functions are apparently compensated for by a NOaugmented tissue vascularization, luminal diameters of blood vessels, and mitochondrial densities [261]. From a different perspective, the product of the NOD reaction, nitrate, can increase glycolytic ATP production by microbes and plants during the transition from the hypoxic to anoxic state by serving as an oxidant reservoir for NADH oxidation via nitrate reductases and nitrite reductases [52,206].
�. Be�on� NO Deto�i�cation� �ignaling e ability of Hb to consume NO in the mammalian vasculature [262,263] and Mb to decompose NO in muscle [137,255,264] has been an important issue since the conception of the role of NO in controlling blood �ow. Indeed, the abundance of red blood cell Hb and myocyte Mb prompted the consideration of mechanisms that would be able to preserve NO in the vasculature or muscle tissues [265] in the face of certain destruction. ese mechanisms include NO diffusion barriers [266][267][268], Hb/Mb-mediated NO sequestration and release [140] and, more recently, Mb or Hb-mediated NO formation from NO-derived nitrite [269]. e rapid reactions of NO with oxy and deoxy Hb and Mb also led to the early view that these reactions would impair their O 2 transport-storage functions [31,270]. However, the role of Hbs in modulating spatial and temporal NO signaling is subtle and slowly becoming apparent. In tissues, Hb, Mb, Ngb and Cygb may act as catalytic NO sinks that together with NOS dynamically and spatially determine steady-state NO levels, soluble guanylate cyclase (sGC) activation, and myriad signaling actions [198,199,255,262,271,272] including regulating cerebral blood �ow, synaptic efficiency and neurotransmitter release [273].
Griffiths and Garthwaite [271] formulated a "clamp" model for a better understanding of the consequences of NO consumption on NO steady-state levels and NO signaling functions in mammalian tissues. In this model, the sink (i.e., NOD) translates different rates of NO formation with a tissue volume into proportional steady-state NO concentrations and clamp [NO]. e NOD thus serves to "amplitude-code" NO signals. e NO inactivation rate also governs the rates of rise and fall of NO concentrations as NO sources switch on and off. In target cells, activation of sGC then causes cGMP to accumulate rapidly to levels that are graded with the prevailing NO concentration. A high NO inactivation rate endows the NO signal with temporal meaning. In addition, NO sinks add a spatial dimension for NO signals and provide additional mechanisms for regulating NO signaling such as via cellular O 2 concentrations [142,274,275]. Furthermore, physiological inactivation mechanisms for signaling molecules generally have properties that are tuned to those of the receptors. In other words, a NOD needs to modulate NO steady-state levels at the 0.1-10 nM concentration levels that modulate sGC activity in vitro [191,192]. Using quantitative real-time recording and modeling of neuronal NO signals via Scienti�ca 7 sGC activation in phosphodiesterase-de�cient cells, �ood et al. [276] recently extended the estimates of steady-state [NO] in neuronal tissues to 0.25-3 nM with rates of NO generation estimated at 0.036 to 0.360 M NO s −1 .
Chen and Popel [277,278] have mathematically modeled steady-state NO production rates in vascular and perivascular tissues and estimated similar values ranging from 0.017 to 1.5 M NO s −1 that are dependent upon concentrations of the NOS isoforms and tissue O 2 concentrations. Endothelial NOS (NOS3) expressed at lower concentrations (0.045 M) produces lower NO �uxes whereas neuronal NOS (NOS1) is expressed at higher levels (0.3-0.9 M) and produces larger NO �uxes. In these studies, estimates of NO steadystate concentrations in the perivascular tissue ranged from 0.3 to 51 nM and were O 2 dependent. ese values for [NO] are more than an order of magnitude lower than previous estimates. Importantly, the resulting steady-state NO concentrations are well within the range required for sGC activation (0.1-10 nM) [191,192,272]. e authors of these investigations made a number of simplifying assumptions including a rate of tissue NO consumption that was linearly proportional to NO [275]. However, the liver parenchymal cell activity used for the estimation of " " may be low since mammalian cells express NO consumption activity levels ranging from 2 to 20 nmol NO min  [142], thus further complicating estimates of steady-state NO levels.
Modeling of NO levels in tissues can provide quantitative and qualitative insights into the roles of potential NO consumption pathways. For example, for superoxide radical to act as a bimolecular pathway for NO removal at rates of 0.17 to 1.7 M NO s −1 , steady-state O 2 − levels of 140 pM to 1.4 nM would be required. ese concentrations are well beyond the ∼8 pM O 2 − estimated within O 2 − -generating mitochondria [279] and far greater than the level expected in the cytosol. On the other hand, for a Hb-NOD, like Cygb-NOD with a maximal turnover rate of ∼1.2 s −1 [135], to act as the sole catalyst, a tissue Cygb concentration of 0.14 to 1.4 M would be required. ese concentrations are within range of the globin concentrations typically seen in nonerythroid cells [4,[280][281][282] and plants [74,283]   recently provided evidence and arguments supporting a role for Cygb-NOD in controlling NO levels and vasorelaxation. Similar roles for Mb expressed in smooth muscle [4,137] and oxidase-generated O 2 − [284] have been previously suggested. Various Hb-NODs are excellent candidates for the long sought dynamic sensor-regulator controlling tissue 2 delivery and 2 . e plots in Figure 1 illustrate the potential for Cygb, or other globins, to act as O 2 -dependent modulators of NO steady-state levels within the physiologically relevant parameters. Caution is warranted since this simple model assumes that the globin is the major catalyst. Cells may express multiple NO dioxygenation catalysts [135].
Cytosolic globins have the potential to in�uence NO signaling and cell phenotypes through NO scavenging; however, enzymatic NO scavenging by cytosolic mammalian globins has not been demonstrated in most cells to date (e.g., see [285]). Cygb is the exception [134,135]. In mammals, and/or -chains of the red blood cell HbA 0 have been 8 Scienti�ca detected in many different cells including hepatocytes [286], neurons and glial cells [287][288][289][290][291], macrophages [285], alveolar type II epithelial lung cells [282,292], and mesangial kidney cells [293] suggesting additional Hb functions. Hb is enriched in pyramidal hippocampus and parietal grey matter neurons of Alzheimer's patients [294]. Other studies have demonstrated the expression of HbA 0 -and -chain mRNA in non-erythroid cells. Visceral metastases of breast carcinoma express elevated Hb -chain [295]. Cygb and Ngb are expressed in a variety of animal cells and tissues including neurons [296]. In cancer, tumor growth is suppressed by expressing E. coli �avoHb-NOD in brain gliomas [297], overexpressing Cygb in head and neck squamous cell carcinomas [298,299] or, paradoxically, by decreasing Mb in breast cancer cells [7]. Globins, acting as NODs, may either decrease the NO signal eliciting a metastatic cell phenotype or increase NO detoxi�cation and the resistance of tumor cell targets to endogenously generated NO (see Table 1). Removal of NO would decrease NO-elicited hypoxic tissue vascularization, mitochondrial biogenesis [300,301], and the Hif-1 -orchestrated gene array expression important for bolstering hypoxic metabolism and the metastatic phenotype. e prolyl hydroxylase controlling Hif-1 stability and function is sensitive to inhibition by NO [170]. NO induces and stabilizes Hif-1 and bolsters the hypoxic adaptation of cells [302]. Moreover, Hif-1 upregulates Mb and Ngb expression [7,9], thus providing a potential feedback loop for [NO] homeostasis. In this model, increased NO removal would decrease prolyl hydroxylase inhibition, destabilize Hif-1 , and attenuate globin expression and NO removal. Globin-regulated NO signaling may also be important for the normal functioning of neurons. Expression of HbA 0 in dopaminergic neurons is linked with pathways involved in O 2 homeostasis, oxidative stress, iron metabolism, NO synthesis, and oxidative phosphorylation [287]. Hif-1 expression was decreased in HbA 0 expressing neurons and targets of Hif-1 were altered [287]. Ngb de�ciency in mouse brain exacerbates the Hif-1 -regulated response to hypoxia [303] further suggesting effects of the neuronal Ngb on [NO] and NO on Hif-1 stability.
ere is also evidence for (�avo)Hbs modulating NO signaling pathways in fungi and bacteria. FlavoHb expression affects Dictyostelium discoideum development [304], and Aspergillus nidulans sexual development and mycotoxin production [305]. NO scavenging by �avoHb attenuates the expression of the nitrosative stress response (e.g., norVW) [151], affects the swarming behavior of E. coli [306], and maintains squid-Vibrio �scheri [218] and Medicago truncatula-Sinorhizobium meliloti [260] symbioses. R. Hill recently published an up-to-date and thorough review discussing the many known and potential roles for Hbs in modulating NO signaling processes in plants [82]. For example, the Arabidopsis Hb, GLB1, modulates salicylate, ethylene, and jasmonic acid responses to Pseudomonas syringae and Botrytis cinerea infections [307]. Lowering GLB1 levels and increasing NO levels elevates the defense against pathogens via multiple hormones. GLB1 expression also decreases ethylene-induced upward leaf movements in response to root hypoxia (hyponasty) [126].  [65]. At that time, a role for the NO-sensitive regulator NorR in the context of the transcriptional regulation of norVW in E. coli [151,194,311] and norBC in Ralstonia eutropha [312] was known. e mechanism of NO-sensing by NorR was subsequently elucidated by D' Autréaux et al. [313]. Roles for the ferric uptake regulator (Fur), methionine repressor (MetR), superoxide response regulator (SoxRS), fumarate nitrate reductase regulator (Fnr) in E. coli, or Salmonella hmp (�avoHb) transcription had also been investigated and reported [69,[314][315][316], but those regulators did not satisfactorily explain NO induction [229,[317][318][319]. e advent of genomics and systematic and comparative operon analyses [239] greatly accelerated the identi�cation NO-sensing regulators and the characterization of globin regulation in various bacteria. Stephen Spiro recently reviewed the literature on NO sensor regulators in the larger context of gas sensors in bacterial nitrogen metabolism [80]. Efforts to identify NO-sensing regulators in representative yeast and fungi have progressed more slowly. Upregulation of globin expression in plants, algae, and animals in response to NO and hypoxia have also been described and is consistent with a NOD function. Here, I brie�y review the NO and hypoxia sensing regulators of globin expression in the context of the putative NOD function.

Regulation of Globin Expression and NOD Function
10.1. Bacteria. NO-regulated NORs and NODs cooperate in the NO defense in bacteria. However, within any single organism, NOD and NOR appear, for the most part, to be under the control of different NO sensor regulators. On the other hand, homologous regulators control either NOR or NOD in different organisms. For example, the sigma-54 dependent transcription activator NorR regulates NOR expression in Ralstonia eutropha [312] and E. coli [151,194,311] in response to nanomolar NO, but NorR regulates NOD (hmp) expression in Vibrio cholera [239,252]. Similarly, in Pseudomonas aeruginosa, rpoN, a sigma-54 homologue, and FhpR, a NorR homologue, regulate p (�avoHb) in response to NO [320]. rough a veritable tour de force, S. Spiro and coworkers demonstrated that NorR utilizes an EPR-detectable mononuclear iron center embedded in a PAS-GAF domain to bind and sense NO and to activate transcription of norVW in E. coli [313]. It is this iron center that can also react with peroxide to impair the NO response [321].
Many bacteria have long been known to upregulate (�avo)Hb expression in response to hypoxia [46,50,53,326]. e common regulator appears to be Fnr, and the signal is O 2 , not NO [309]. Induction of a NOD in response to hypoxia makes sense since O 2 is a cosubstrate for the NOD reaction and since NO is more toxic to respiration at a low pO 2 .

Yeast/Fungi.
Progress has also been made in understanding NO regulation of �avoHbs in fungi and yeast. In Saccharomyces cerevisiae, the Fzf1p regulator is required for NO induction of YHB transcription [236]. In Candida albicans, CTA4 a zinc-�nger protein is required for induction of YHB1 transcription [245], and Cwt1p acts as a repressor of a nitrosative stress regulon that includes YHB1 [241]. NO upregulates �avoHb (NOD) expression by Aspergillus oryzae [216], the plant pathogen Botrytis cinerea [222] and other fungi [65] presumably via similar mechanisms. e full details of NO sensing and transcription activation remain to be elucidated.
One of the more interesting discoveries has been that the Fusarium graminearum virus-D�21 downregulates �avoHb mRNA and protein expression by the pathogenic plant fungus Fusarium graminearum along with virulence [327]. A role for the �avoHb in virulence is suggested.

Photosynthetic Organisms.
Numerous examples of NO and hypoxia upregulating Hbs in plants have been described and reviewed [74,81,82,328]. e fused nitrate reductase-trHb (NR2-2/2HbN) is induced by NO in the microalgal species Heterosigma akashiwo [139]. Arabidopsis class 1 nsHb is induced during growth with nitrate [329]. Two nonsymbiotic trHb genes are strongly induced by nitrate, nitrite, and NO in cultured rice cells [330]. Cotton and wheat nsHbs are induced by NO [224] or NO donors [331]. Only one of the �ve Hb genes, LjHb1 encoding a nsHb, is induced by NO and hypoxia in Lotus japonicum [308,332]. Another nsHb (LjHb2) is induced by sucrose, abscisic acid, and osmotic stress. Clearly, not every Hb with a capacity for a NOD function is induced by NO or hypoxia. For example, the cyanobacterium Synechococcus sp. GlbN provides resistance to NO, but is apparently not inducible by NO, nitrate, or nitrite [138].
10.5. Animals/Humans. Numerous animal Hbs are reportedly induced by hypoxia, but apparently none by NO. e Hb -chain is induced in macrophages by interferon and lipopolysaccharide [285] which also elicit NO synthesis. However, the induced Hb -chain failed to increase NO consumption by macrophages [285]. HbA 0 chains are expressed in rat ischaemic rat neurons [333] and are upregulated in response to hemorrhage [291], hemin, erythropoeitin [289], and oxygen-glucose deprivation [333]. e chains are not always coordinately regulated; oxygen-glucose deprivation increases rat neuronal -chain mRNA 1.9-fold, but decreases Hb -chain mRNA 3-fold [333]. HbA 0 is also reportedly present in hippocampal and parietal grey matter neurons of Alzheimer's patients [294], a hypoxic and in�ammatory condition that may induce globin expression. Hb -and -chains are also expressed in alveolar type II lung epithelial cells in response to hypoxia [282,292] and Hif-2 or Hif-1 control transcription [334]. Ngb is induced by hypoxia [335] and is under control of the master transcriptional regulator of the hypoxic response, Hif-1 [336][337][338]. Hypoxia also induces �sh Mb in nonmuscle cells including liver, gill, and brain [5]. Numerous other studies have shown elevated expression of mRNAs for Hb -and -chains, Mb, Cygb, and Ngb in non-erythroid cells and especially hypoxic tumors [7,9]. What remains unclear is whether these animal globins have a signi�cant capacity for NO dioxygenation and modulation of NO functions and how this relates to pathophysiology. For example, Ngb-de�ciency in mice leads to changes in Hif-1regulated pathways in response to hypoxia [303]. Is this due to de�cient NO metabolism and NO stabilization of Hif-1 via prolyl hydroxylase inhibition? Is NO, like O 2 , a signal that normally controls Hif-1 ?
While knowledge of the many factors and mechanisms controlling transcription or translation of globins in various life forms is revealing and oen supportive of a NOD function, a quantitative knowledge of the NO and O 2 concentration dependencies of the responses is critical for understanding Hb function in NO homeostasis control, detoxi�cation, and�or signal modulation. e overriding question is: What level of NO is normal and how do cells maintain and respond to changes in the concentration of NO via globin synthesis regulation?

Hb Structure(s) and the NOD Mechanism
a mechanism for nitrate egress, (7) a mechanism for univalent reduction.
Here, I will focus on new knowledge, key concepts, and current questions about Hb chemistry that are directly related to the NO dioxygenation reaction, NOD catalysis, and a NOD function. New investigators of Hb-NODs should consult the seminal literature [34,49,52,[339][340][341] and prior reviews describing (�avo)Hb structure-function-evolution [1, 65-67, 71, 72, 102, 342] for background and additional information. Mowat et al. provide an excellent recent overview of the �avoHb-NOD structure-function [72].  [65,344]. For many Hbs, the (NO) value determined by laser photolysis and stopped-�ow is ∼10 pM. Yet, steady-state assays of NOD activity reveal much weaker inhibition by NO [52,135,341,344] thus suggesting the existence of mechanisms for decreasing NO binding during catalysis (4)

High O 2 Affinity.
e most attractive explanation for this phenomenon is the allosteric modulation of an NO tunnel or gate by O 2 binding. In the M. tuberculosis trHbN, O 2 and NO access the ferrous heme through two different gated tunnels [345]. O 2 binding opens a long tunnel for NO and increases NO access [346][347][348]. Tunnel switching occurs on the picosecond to nanosecond time scale and is compatible with a role in NOD catalysis [349]. A novel role for PheE15 in gating O 2 and NO migration through channels has been suggested in which the distal H-bonding TyrB10 and GlnE11 act as the triggers [348,350,351]. Similar tunnels or gates may control NO access in other Hbs. Bis-histidyl ligation and structural plasticity may serve a similar role in controlling ligand access in the NOD function of plant nsHb [84,352].
Other explanations for diminished NO inhibition include NO reduction by the �avoHbs [341], yet, rates of NO reduction by some �avoHbs appear too low [52]. Interestingly, Liu et al. were able to mathematically model the Cygb-NOD activity with the available transient kinetic rate constants for NO and O 2 binding and NO dioxygenation [136] suggesting the absence of mechanisms for decreasing NO binding in some Hbs.

Superoxide Radical-Like
e ferric iron forms unique bonding interactions with the bound 2 − important for NOD function. e heme-Fe 3+ (3 5 ) unpaired electron interacts with the unpaired * O 2 − electron through strong antiferromagnetic coupling in a unique endon orientation [103]. In the NO dioxygenation reaction, the unpaired NO * electron associated with the N-atom couples with the unpaired * O 2 − electron. One electron is formally transferred from NO to the half-�lled * (O 2 − ) orbital (reduction of superoxo), but not to the metal; thus the strong O 2 bond is broken and the Fe-O bond is strengthened. e reaction is analogous to the diffusion-limited reaction of NO and O 2 − in solution [355] except that the strong antiferromagnetic coupling of an end-on 2 − has the capacity to preferentially localize the unpaired * electron density to the O-atom proximal to the iron and to direct the NO reaction (vide infra).
e anti-ferromagnetic coupling is eliminated with the return of the electron from the bound O 2 − to Fe 3+ permitting �cienti�ca 11 O 2 dissociation. However, many Hbs that function as NODs (e.g., E. coli �avoHb) show relatively high autooxidation rates and O 2 − release. Toxic O 2 − release is more problematic at high oxy-Hb concentrations and is slow for abundant Hbs and Mbs functioning in O 2 transport-storage [356][357][358]. In contrast, the less abundant neuronal Ngb shows high autooxidation rates in vitro [359]. Curiously, this has led some investigators to discount a NOD function [360]. However, high autooxidation rates can be an artefact of in vitro conditions and should not be the sole basis for conclusions about function. For example, autooxidation of the Candida norvigensis �avoHb-NOD oxy-complex is very slow, but increases when the reductase domain cofactor FAD is absent or when the reductase domain is separated from the Hb domain [61,102].

Protected
e half-life of the �eeting peroxynitrite intermediate is expected to be in the microsecond range due to facile ironcatalyzed isomerization to ferric-nitrato, whereas the ferricnitrato species has a measured half-life of milliseconds [361,362]. e putative HbFe 3+ (ONOO − ) intermediate detected in reactions of oxy-Mb, -Ngb and -GlbO in the millisecond time range [122,123,[363][364][365] must now be assigned to the ferric-nitrato species. Oxy-globin model compounds, Fe(Por)(NH 3 )(O 2 ), also react with NO at 80-100 K forming only the low-spin ferric-nitrato complexes, thus implying that peroxynitrite intermediates, if formed, also undergo very facile isomerization [366]. In contrast, NO reacts with oxycoboglobin model compounds, Co(Por)(NH 3 )(O 2 ), forming a detectable peroxynitrite intermediate and nitrato species at low temperature [367]. More recently, Navati and Friedman [365] reported preliminary evidence for Fe 3+ (ONOO − ) formation during the reaction of NO with oxy-Mb and oxy-Hb in a special "dry" glassy matrix.
e ferric-peroxynitrite would form in the distal pocket of Hb-NODs which is well isolated from nucleophiles and solvent. Hb pockets are typically lined with a large number of hydrophobic residues with histidine, glutamine, and/or tyrosine residues forming hydrogen bonds to the O 2 − ligand. A protected pocket may explain how Hb functions as a high-�delity catalyst of NO dioxygenation whereas the indoleamine dioxygenase-like oxy Turbo Mb cannot [66].

O-Atom Isomerization or Rearrangement Mechanism.
HbFe Two mechanisms for formation of nitrate from the reaction of NO and HbFe 3+ (O 2 − ) (7) have been previously discussed [66]. Both provide a pathway for O-O bond breaking and O-atom isomerization or rearrangement in the peroxynitrite intermediate. e �rst that we envisioned was an iron-catalyzed mechanism in which ferric iron, acting as a Lewis acid, facilitates O-atom rearrangement [34]. We wrote "e heme-Fe 3+ may facilitate an oxygen bond rearrangement by participating in a iron-mediated oxygen bond shi analogous to the proton-mediated shi suggested for the nonenzymic mechanism for HOONO decomposition to NO 3 − . " e mechanism was never fully rationalized, developed, or argued in the literature, but it was assumed that the mechanism would bear similarity to the mechanism of peroxynitrite isomerization accelerated by acidic pH (pH < 2) [368,369]. e second, a ferryl mechanism involving peroxynitrite, O-O bond homolysis, and ferryl O-atom transfer was strongly supported by theory, overwhelmingly favored, and thoroughly argued and investigated [65,66,342,364]. However, both the doubt cast by recent [121,362] and earlier [368] investigations and the paucity of strong experimental support for the ferryl mechanism have demanded new insights and the scrutiny of alternative Oatom rearrangement mechanisms.
In 1954, Anbar and Taube [370] suggested a concerted internal O-atom rearrangement mechanism for the isomerization of peroxynitrous acid to nitrate to explain O-atom retention, and a similar mechanism, as already mentioned, was suggested as a possibility for the Hb reaction, albeit remaining poorly de�ned (see above) [65,66]. Tsai et al. [371] had argued against an internal rearrangement mechanism for peroxynitrite/peroxynitrous acid in 1996 "because contracting of the O-O-N bond angle produces a strong repulsion between the terminal peroxide oxygen and the two oxygens bound to nitrogen. " Discussions with Henry Taube and recent descriptions of the unique Fe 3+ O 2 − bonding in Hbs [103] provided new insights for a novel concerted internal O-atom rearrangement mechanism (see below). e new mechanism casts doubt on the relevance and validity of mechanistic inferences drawn from experiments demonstrating peroxynitrite isomerization catalyzed by ferric Mb [372][373][374][375] or metalloporphyrins [26,66,376]. Models of heme-peroxynitrite adducts envision the negatively charged terminal O-atom bonded to iron [376,377]. e two possible O-atom rearrangement mechanisms are illustrated in Figure 2  e support for a ferryl mechanism was previously reviewed [65,66,364] and recently critiqued [121,362,375,378]. From atomistic simulations of the truncated group I HbN, Mishra and Meuwly [121] concluded that the O-O bond scission energy barrier was too high and homolysis too slow thus suggesting an internal rearrangement mechanism. Su and Groves [378] have concluded that NO 2 is an unavoidable product of the ferryl mechanism, yet, there is no evidence for NO 2 formation or ferryl O-atom scrambling with water during (�avo)Hb-catalyzed NO dioxygenation [66]. One would expect NO 2 -mediated nitration damage to the �10 tyrosine residue in the distal pocket of �avoHb-NOD and a loss of NOD activity similar to the nitrations of more distant tyrosines observed in Mb and Hb [66,373,374,[379][380][381][382][383]. e release of reactive toxic intermediates during NO oxidation by oxy-Hb is the antithesis of a NOD function, and the evidence for that is meager [33]. What then are the arguments and evidence suggesting a concerted Lewis acid mechanism (Figure 2, (1)) ? First, the reaction mechanism demands the application of density functional theory and further scrutiny. Past theoretical investigations of peroxynitrous acid isomerization [384] have not considered the effects of hydrogen ion interactions with peroxynitrous acid O-atoms. Nor has O-atom retention during peroxynitrous acid isomerization [370,385] been investigated as a function of pH. Clearly, hydrogen ions increase the rate of the reaction [368,369] well beyond the peroxynitrous acid pK of 6.5-7 [386], but this phenomenon has never been clari�ed. A proton, or Lewis acid, interacting with the peroxide O-atom proximal to N would decrease the strong repulsion between the terminal peroxide oxygen and the two oxygens bound to nitrogen. In an analogous reaction, hydrogen ions catalyze Oatom transfer from H 2 O 2 to I − and other two-electron donors [387][388][389][390]. e only apparent catalytic mechanism is through O-atom protonation, which withdraws electrons from the proximal O-atom, increases O or OH + reactivity with I − and which causes peroxide bond heterolysis forming water and IO − or IOH [389]. A Lewis acid (e.g., Fe 3+ ) would be expected to be able to catalyze a similar O-atom transfer. ere are also reasons why a mechanism involving NO attack of the O-atom proximal to iron in Hbs is attractive. e proximal O-atom is more accessible to NO. Moreover, it is the most probable location for the anti-ferromagnetically coupled unpaired * Fe 3+ (O 2 − ) electron [103] that couples with the unpaired NO electron. Hughes and Nicklin had suggested a heterolytic mechanism for peroxynitrous acid isomerization in 1968 [368], but the potential role of hydronium or hydrogen ions in catalyzing the reaction was not considered.

Mechanisms for Nitrate Egress. Martí et al. simulated
nitrate release from Mycobacterium tuberculosis trHbN [391]. e molecular dynamic simulations suggest that formation of the ferric-nitrato species causes a structural distortion of the pocket cavity walls forming pores for water entry. Water hydration weakens the bond between the heme iron atom and nitrate exits in ∼5 ns via a unique pathway differing from O 2 and NO tunnels. A role for rE2 in assisting nitrate egress was proposed. e role of plant nsHb pocket plasticity in nitrate removal has also been discussed [84]. e Mycobacterium leprae GlbO ferric-nitrato intermediate showed a 10-100-fold longer half-life than other globins [122] suggesting a slow nitrate egress mechanism.

Mechanisms for Univalent Reduction.
Each NO dioxygenation reaction consumes a single electron that must be supplied by an electron donor for catalytic turnover. e ferric heme is reduced by an electron (8a) for the O 2 binding reaction (3). Importantly, Hbs require mechanisms to prevent the transfer of a second electron to the higher potential ferric superoxo complex. A second electron would generate ferricperoxide (8b), and a third electron would generate ferryl. Reduction of the putative ferric-peroxynitrite intermediate could release peroxynitrite or generate hydroxyl radical (8c). ese reactive species would be expected to cause damage to the heme and/or protein.
Native electron donors are known for only a handful of Hb-NODs, but distinct patterns are emerging. e ultimate electron donors for the �avoHb-NOD [65] and dual function raphidophyte trHb-nitrate reductase [139] are �avincontaining reductases linked by a multidomain structure. e truncated cyanoglobin (GlbN) and ferredoxin reductase genes are also linked in a bi-cistronic operon controlled by the NO sensor-regulator NsrR in Legionella pneumophila [239]. Together, the data suggest a common role for ferredoxin reductases and other FNR-like proteins as electron donors for trHbs and SDGs either linked in multi-domain structures or as separate proteins. e role of the of pre-A sequence for truncated HbN-NOD activity in Mycobacteria may be to facilitate interactions with a speci�c ferredoxin reductase [392]. Ferredoxin:NADP + oxidoreductase from E. coli was systematically tested for its capacity to support NOD activity of various Hbs in vitro [133], however, in those reactions, "NOD activity" is impossible to discern from reactions of O 2 − . High concentrations of SOD are required to prevent reactions of NO with the O 2 − invariably released by reductases [344]. Red blood cell Hb is reduced by cytochrome b 5 , and cytochrome b 5 can support the Cygb-NOD activity in vitro [135]. Ascorbate also supports the Cygb-NOD activity at concentrations found within Cygb expressing �broblasts and neurons [135,136], and a putative binding site for ascorbate has been identi�ed [135]. An accessory role for NADPH:cytochrome P450 oxidoreductase in CygbFe 3+ reduction has also been suggested [136]. e barley nsHb can be reduced by an ascorbate-dependent monodehydroascorbate reductase [132]. A mass spectrometry approach has been utilized to identify the Ngb and Mb interactome [393]. e method may also reveal novel electron donor candidates.
Hb structures suggest two mechanisms for controlling univalent electron transfer. Bis-histidyl ligation in hexacoordinate nsHbs, Cygb, and Ngb provides structural plasticity [84] and a mechanism for controlling electron donor binding and electron transfer. For example, movements of the E-helix and ArgE10 with changes in O 2 binding may modulate ascorbate binding and electron transfer in Cygb [135]. In this case, bis-histidyl ligation to the ferric iron would be expected to induce an ascorbate binding-site, and bishistidyl ligation to Fe 3+ (O 2 − ) to decrease ascorbate binding. A rotating water bridge may also provide a mechanism for controlling univalent electron transfer from reduced �avins. e �avoHb and SDG structures [49,65] and electron pathway analyses [394] suggest an important role for water molecules bridging the FAD and heme in mediating electron transfer. In the E. coli �avoHb-NOD structure [339], the bridging water molecule hydrogen-bonded and anchored by LysF7 ammonium group has the capacity to rotate to ON and OFF orientations (Figure 3). Strengthening of a short, strong hydrogen bond between water and the heme propionate, with O 2 binding and a pK shi of the heme propionate, provides a mechanism for controlling the bridging water orientation and regulating univalent electron transfer in Hb-NODs. e oxygen core repels electrons in the OFF orientation and would block electron tunneling. In support of the hypothesis, mutation of LysF7 dysregulates univalent electron transfer, oxidatively destroys the heme, and incapacitates the NOD function [395]. e many adaptations of the Hbs for the NOD function show that Mother Nature is indeed a brilliant chemist!

The FlavoHb Denitrosylase (O 2 Nitrosylase) Concept
Following the report of NO metabolism by the NO-inducible E. coli �avoHb and kinetic investigations supporting a NOD mechanism [34,52,341], Hausladen et al. argued for a major revision by suggesting a denitrosylase mechanism for NO conversion to nitrate [396,397]. NO Despite a dearth of either experimental or theoretical support for the �avoHb denitrosylase mechanism a�er more than 10 years, a handful of investigators persistently assign merit to the mechanism [67,69,70,215,250,399] or equate the two mechanisms [87,400] and in so doing obscure the true function of (�avo)Hbs and impede general progress. Moreover, proponents of the denitrosylase mechanism continue to reject the dioxygenase mechanism and nomenclature, but have received only a partial scienti�c rebuttal [65,344,397,401]. Hence, a refutation of the denitrosylase mechanism is demanded.
e following is a list of reasons why a denitrosylase mechanism is theoretically unfeasible and weakly supported by experiment. (1) NO reduction ( 0 = −0.8 V) [189,402] to the nitroxyl (NO − ) intermediate has a large energy barrier. max values for NO reduction by �avoHb are >2000-fold lower than the max values for NO dioxygenation [52]; (2) the reaction of Fe 3+ (NO − ) with O 2 is not kinetically favored [123,403]; (3) O 2 would be more rate-limiting for a denitrosylation at low physiological O 2 concentrations than it would for the NOD mechanism; (4) the mechanism requires O 2 to react with the N-atom liganded to the iron which does not provide an obvious reaction path for O-atom rearrangement; (5) O 2 binding and NO scavenging are affected by the �avoHb distal pocket structure (TyrB10), but NO binding is not [341]; (6) CO competitively inhibits NO metabolism with respect to O 2 , not NO, throughout the physiological [O 2 ] range [52,344]; (7) the NO metabolic activity of �avoHb is saturable by O 2 [52,341,344] with no evidence of an activity increase at low O 2 as suggested and reported [396,398]; (8) NO inhibits the NO metabolic activity of �avoHb at NO:O 2 ratios >1 : 100 and not as potently as predicted from NO affinity measurements [52,341,344]; (9) �avoHb-NOD activity is induced by NO providing an effective escape from the otherwise problematic NO inhibition at low [O 2 ] [151,404]; (10) NO reductases are also expressed at low O 2 concentrations thus substituting for lost �avoHb function due to NO inhibition [151,404]; (11) the denitrosylase proposal assumes that micromolar NO concentrations are required for NO toxicity and thus the NO detoxi�cation function of �avoHb. Presumably, under these high NO levels, a dioxygenase mechanism would be impossible. But, even a "tiny" 50 nM NO is toxic to aerobic E. coli [35] and �avoHb protects against this toxicity [35,151,404]. us, �avoHb-NOD can detoxify NO without signi�cant NO inhibition even at 5 M O 2 . Furthermore, NOD turnover under normoxia (∼ 400 NO s −1 heme −1 ) far exceeds the activity expected from the denitrosylase mechanism. NO reduction by �avoHb, a step in the denitrosylase mechanism, has never exceeded 0.2 NO s −1 heme −1 [65,107]. For the two mechanisms to be comparable in rate, the NOD mechanism would need to be inhibited by 99.95%. At 50 nM NO, the available [O 2 ] would need to be <5 nM for 99.95% NOD inhibition for a remotely plausible and equivalent NO scavenging function. (12) Finally, the initially troubling observation of �avoHb metabolizing NO and NADH at an NO : O 2 ratio of >1 : 6 [398] can be explained by the fact that unusually high levels of �avoHb (0.5-1 M) were present in the reactions such that the added NO (35 M) was so rapidly consumed as not ever to achieve >5 M NO! (see Figure 1(a), [398]). Moreover, the substantial NADH oxidase activity of �avoHb can account for NADH oxidation observed at these high concentrations of �avoHb (see Figure 1(d), [398]). ese unfavorable conditions for NOD catalysis may also explain the relatively high yield of nitrite formation and would even compromise the deduced O 2 : NO : NADH reaction stoichiometry [398]. e concentrations of �avoHb protein used in steady-state kinetic analyses are typically 5,000-fold lower and involve high turnover numbers [52,341,344].
Unresolved issues nevertheless remain. For example, why is the a�nity of �avo�b for �� ∼15-fold greater when determined by the �ash photolysis-rebinding method than when estimated from steady-state kinetic analysis? NO removal by the �avoHb NO reductase activity during catalysis is one possibility [341], but the activity may be insufficient to account for NO resistance. Similar questions arise for all other globins including the mammalian Cygb. e Cygb NO : O 2 affinity ratio of ∼125,000 : 1 immediately suggests a much greater inhibition by NO than that observed at NO : O 2 ratios of >1 : 500 [135]. Liu et al. [136] have recently used modeling simulations to argue that the steady-state kinetics measured for the Cygb-NOD activity are allowed by the high NO affinity ( = 8 pM), but the model required assumptions including NO affinity decreases with temperature. Less consideration has been given to structural dynamics and changes in NO access [346,348] as a common mechanism for limiting NO binding and inhibition of catalysis (see above).

Why So Many Different Hb-Type NODs?
While there is certain danger of a fallacy of composition in suggesting a NOD function for all Hbs, the literature now shows that many different Hb structures in many different forms of life function as NODs. is realization raises important questions. If various Hbs function as NODs, Scienti�ca 15 why the structural diversity and the multiplicity of Hbs within organisms?
Cursory answers to these questions can be readily gleaned from the anti-oxidant and radical scavenging enzyme systems. For example, in order to scavenge H 2 O 2 , organisms express catalase, glutathione peroxidases (8 isoforms in humans), cytochrome c peroxidase, ascorbate peroxidase, and peroxiredoxins (6 isoforms in mammals) [12]. And, for O 2 − removal, multiple SOD isoforms are expressed (7 isoforms in Arabidopsis) [11,15,405]. ese enzyme isoforms show differential localization in subcellular compartments, expression during development and stresses, electron sources, catalytic requirements, posttranslational regulation, and subtle differences in physiological functions.
e analogy implies a similar ubiquity and richness of NO-and Hb-related functions in nature. Moreover, examples are emerging to support this rubric. Aspergillus �avoHb-NODs are di�erentially distributed to the cytosol and mitochondria because FHb2 bears a mitochondrial Nterminal signal sequence [112,216]. A class I truncated Hb localizes along chloroplasts thylakoid membranes in algae [406]. Other examples of Hbs with membrane localization signals or affinities include the M. tuberculosis HbO [407,408], VHb [409,410], the myristoylated Crab globin [411], and the myristoylated or palmitoylated �sh Glb� [88]. Membrane association may also facilitate reduction by membrane-bound reductases. Saccharomyces cerevisiae �avoHb localizes to mitochondria and cytosol without an apparent signal sequence [412]. e wheat nsHb interacts with, and apparently cofunctions with, photosystem I and II [331]. Many Hbs show tissue-speci�c and developmental regulation of expression [82,116,222,296,304,336,413]. Some Hb-NODs are induced by NO stress, and others are not [95,116,216,310]. Some �avoHb-NODs prefer NADH while others prefer NADPH [52,114,216], and expression appears to correlate with the availability of the electron source. e Cygb-NOD utilizes ascorbate as an electron donor in vitro [135,136] and is expressed in ascorbate-rich �broblasts and neurons [296]. Transcript network analysis strongly suggests a role for ferredoxin: NADP + oxidoreductase as the electron donor for cyanoglobin (GlbN) in Legionella pneumophila [239], and GlbN would be required to interact speci�cally with the reductase. Some globins are suited for NOD catalysis at relatively high micromolar NO concentrations and �uxes (e.g., �avoHbs) [52,341] whereas others show lower turnover rates and are more susceptible to NO inhibition (e.g., Cygb and Ngb) [135]. is is reasonable since some globins serve primary NO detoxi�cation functions while others subserve NO signaling functions. Some Hbs may function better at low or high temperatures [111,219] while others may function under dehydrating osmotic stresses [308]. Potential mechanisms for post-translational regulation are also emerging. Reeder et al. have reported that lipid binding to Cygb alters heme coordination and suggest that this provides a mechanism for lipids to alter or regulate Cygb function [414]. Protein phosphorylation and thiol-disul�de interchange have been suggested as mechanisms regulating Ngb function [415][416][417]. In addition, some globins (e.g., muscle Mb and legume Hb) may be suited to serve dual functions in O 2 transport-storage and NO metabolism [125,137,255,262]. ese many differences are achieved by many unique Hb structures while intrinsic 2 binding and NO dioxygenation activity appear to be preserved in all Hbs.

Are Hemoglobins Unique O 2 -Dependent
Catalysts for NO Metabolism?

Other Enzymatic Functions for Hemoglobins?
In addition to NO dioxygenation, several specialized enzymatic activities and functions have been proposed for various Hbs. Discerning a meaningful biological function from myriad fortuitous in vitro activities of Hbs [65] continues to be an important challenge. In all cases, investigators must always ask whether (1) other enzymes coexist in cells that could better serve the proposed function, (2) the structure evolved for the proposed function, and (3) the reaction is bene�cial for long-term survival of an organism. In evaluating proposed functions of Hbs, it is also important to consider the fundamental argument of Shikama and coworkers [61,102] that (4) "the reversible binding of molecular oxygen to iron(II) must be the primary event to manifest their physiological functions in vivo. " Of the following recently proposed enzymatic functions, surprisingly, only the heme oxygenase activity meets the last and most basic of these criteria.

NO Reductase.
Anaerobic NO reduction was �rst suggested to be an important biological function for the E. coli �avoHb by �im et al. [107]. e reaction (10) served to explain the anaerobic induction of �avoHb by NO [50] and the anaerobic growth protection �avoHb provided against nitrosothiols [55] 2 However, the proposed function has critical weaknesses. For example, other microbial �avoHbs show much lower [52], or negligible [48], NO reductase activity. e maximal turnover rate for NO reduction is >2000-fold lower than that measured for NOD activity, and is unstable (unpublished results). Furthermore, the �avoHb shows no NO reductase activity within anaerobic E. coli [404]. Moreover, there are more efficient NO reductases in organisms expressing �avoHb or other Hbs [17,151], and �avoHbs are capable of reducing nitrosothiols directly [52], thus explaining the protection observed. Yet, uncertainty, and confusion, over a NO reductase function clearly persists in the �eld of nitrosative and oxidative stress research [431].

Peroxynitrite
Isomerase. e commonly accepted NOD mechanism includes the efficient isomerization of a ferric heme-bound peroxynitrite intermediate to nitrate (7). Given the formation of toxic peroxynitrite in cells from the rapid reaction of NO and O 2 − , Herold and others have suggested that various Hbs and Mb may also function as scavengers of peroxynitrite [125,138,432]. However, large rate constants (>10 6 s −1 M −1 ) for peroxynitrite isomerization were only measured for distal E7 histidine mutants [372] suggesting that entry, binding or isomerization of the ONOO − anion in the heme pocket is normally hindered by E7 histidine. Exposure of Mb or Hb to exogenous peroxynitrite also nitrates and damages the protein [373,374,[379][380][381][382][383]. Hence, these Hb structures do not appear adapted for a peroxynitrite isomerase function. Secondly, peroxynitrite reacts with a variety of abundant biomolecules (e.g., CO 2 and glutathione) [189], and these biomolecules would compete with ferric Hb or Mb. Moreover, proposals of a peroxynitrite isomerase function ignore (1) the important function of SODs and NODs in preventing toxic peroxynitrite formation within cells and (2) reactions with the predominantly oxy Hb within cells [382,383].

Nitrite Reductase.
As early as 2003, Mark Gladwin and others began pursuing possible NO-generating nitrite reductase functions for the mammalian red blood cell Hb [433,434] and muscle Mb [269,435,436]. e reaction (11) produces NO from nitrite under aerobic and hypoxic conditions in vitro and in vivo and is stimulated by acidic conditions that occur in tissue ischaemia A vital role for the reaction in the hypoxic vasodilation of capillaries and homeostatic control of tissue O 2 delivery was proposed. In addition, the reputed nitrite reductase function has been extended to other members of the Hb superfamily. For example, dithionite-reduced Ngb catalyzes NO production from millimolar nitrite under anaerobic conditions with rate constants estimated at 0.12 to 5 s −1 M −1 [416,437]. Furthermore, Gladwin and co-workers have reported effects of distal histidine interactions, disul�de bond formation, and protein phosphorylation on the rate of the deoxy Ngb-nitrite reaction [415,416]. e reaction rate constant increased to physiologically relevant values (∼50 s −1 M −1 ) with the elimination or weakening of the distal histidine-heme interaction. Li et al. reported similar low levels of nitrite reducing activity (0.14 s −1 M −1 ) for Cygb and demonstrated a signi�cant role for the activity in soluble guanylate cyclase activation in cultured aortic smooth muscle cells with 10 M nitrite [438]. is is remarkable since the theoretical maximum rate of NO generation under these conditions with an upper [deoxy-Cygb] estimate of 1 M is 1.4 pM s −1 . Neighboring endothelial cells normally generate and release NO at a far higher rate of 17 to 1500 nM s −1 [277,278]. As pointed out by Sturms et al., "the modest (low micromolar) levels of nitrite typically found in mammalian tissues diminish the likelihood that nitrite reductase activity is a major function of these proteins" [439]. Rather, Sturms and Hargrove suggest that plant hexacoordinate Hbs may be more suited to nitrite reduction since nitrite levels are higher in plant tissues. ey have demonstrated anaerobic nitrite reduction by deoxy forms of rice Hb and Synechocystis Hb with respective bimolecular rate constants of 166 and 130 s −1 M −1 [439], but these Hbs are of relatively low abundance. Tiso et al. subsequently reported comparable bimolecular rate constants of 5 and 20 s −1 M −1 for the Arabidopsis thaliana nonsymbiotic Hbs [440].
It is important to note, however, that the reaction of nitrite with globins nitrosylates ferrous heme and cysteine sulydryls [437] and that the reaction of nitrite with globins in the presence of H 2 O 2 nitrates proteins [441,442]. Moreover, high nitrite concentrations under mildly acidic aerobic conditions form nitriheme, a damaged heme [443]. us, the anoxic NO 2 − reduction reaction is likely a non-speci�c and damaging reaction of Hbs. Furthermore, the NO-generating reactions of Hbs must always be evaluated with other enzyme systems that also act as non-speci�c nitrite reductases [211] including the plant nitrate reductase [204] and mitochondria [444].  [138], Cygb [448][449][450][451], Ngb [450,452], and several Arabidopsis Hbs [441]. As pointed out by Paul [453], when evaluating a peroxidase function, one needs to determine overall turnover rate, rates of elementary steps, and speci�city for electron donors and compare them with those reported for genuine peroxidases. A peroxidase function must also be demonstrated within the living organism in the normal background of peroxidases and catalase. Only in the case of the peroxidative dehalogenation of phenols by the polychaete Amphitite ornate Hb [454] has a peroxidase function been convincingly demonstrated.

Lactate Dehydrogenase.
Gupta et al. [118] have recently reported the failure of Mycobacterium tuberculosis �avoHb [Rv0385] to meet the quali�cations of a NOD. e protein is hexa-coordinate and shows very low NOD activity [119] and little ability to protect against nitrosative stress imposed by acidi�ed nitrite when expressed in E. coli or M. smegmatis [118]. A function as a D-lactate speci�c dehydrogenase was suggested. e proposed mechanism is similar to that of the L-lactate oxidizing �avocytochrome b 2 [72] where lactate transfers electrons to the �avin, and the �avin transfers electrons to the heme. A rather low in vitro turnover number for D-lactate oxidation of 0.026 s −1 is estimated from the published data [118]. e possibility of post-translational modi�cations in�uencing activity was not considered. Interestingly, the putative M. tuberculosis �avoHb (Rv3571) was previously suggested to function in NO detoxi�cation [455], but the work has not been con�rmed or extended.  [460]. Several of the >33 C. elegans globins are fused within multi-domain proteins and likely possess O 2 -sensing regulatory functions [2,3]. Whether SDGs function in a similar capacity, but in non-covalent association with enzymes, is an important consideration when evaluating the function of Hbs. For example, oxidized ferric Ngb has been found act as a heterotrimeric G -protein guanine nucleotide dissociation inhibitor and has been proposed to regulate G -protein signaling [461,462]. Importantly, the facile NO dioxygenation reaction may also be relevant to the biological function of some multidomain sensor-regulators. For example, the O 2 binding Azotobacter vinelandii AvGReg, a globin-coupled sensor, reacts rapidly with NO and is thought to serve a NO detoxi�cation [463] or NO sensing function. In the case of the Mycobacterium tuberculosis PAS domain sensors DevS and DosT, NO rapidly reacts with the oxy forms to generate nitrate and the ferric heme form [196]. e putative NO dioxygenation reaction triggers the dormancy program through subsequent formation of the kinase activating ferrous-NO form. e results are intriguing and may explain the ability of NO to induce and maintain the antibiotic-resistant dormant state of the bacterium in tuberculosis [464,465]. A similar NO dioxygenation reaction of the FixL regulator may explain a major part of the NO transcriptional response of Sinorhizobium meliloti [183].

Hb-NOD Technologies
Progress in understanding the NO-scavenging NOD function and mechanism of various (�avo)Hbs continues to inspire medical, agricultural, and industrial inventions and is helping to unravel the complex biology of NO in a variety of organisms.
For example, human red blood cell Hb has been modi�ed and is being tested for use as an injectable long-lived NO and oxidant scavenger for treatment of septic shock [466]. Future designs that incorporate a catalytic Hb-NOD activity are anticipated. Alternatively, small molecule NOD mimetics [26] may �nd therapeutic applications once shielding of reactive intermediates in the NO dioxygenation reaction can be achieved.
Mechanistic inhibitors of NODs are being investigated for therapeutic use against microbial infections, hypertension, and malignant tumors [467][468][469]. Imidazoles bearing bulky hydrophobic groups such as the antifungal agent miconazole are particularly effective inhibitors of the �avoHbs [469]. X-ray crystal structures of miconazole and other imidazoles with R. eutropha �avoHb reveal key interactions with hydrophobic residues (e.g., Ile25) in the distal heme pocket [470]. ese interactions suggest strategies for the rational design of therapeutic inhibitors such as the addition of speci�c hydrophobic groups to the miconazole nucleus. Imidazoles may also be especially effective towards S. aureus and other microbes. Miconazole increases O 2 − /H 2 O 2 generation by �avoHb in S. aureus in addition to blocking the NOD function [471].
In agriculture, Monsanto Corp. is engineering corn, soybean, and other crop plants expressing E. coli, Erwinia chrysanthemi and yeast �avoHb-NODs for increased resistance to stresses generating NO, increased capacity for soil nitrogen (NO) sequestration in the form of nitrate, and improved growth and fruit production characteristics [472]. Using the patented technology, projected growth and fruit yields increase by ∼20% and ∼5%, respectively. Many others have engineered transgenic plants to express the Vitreoscilla Hb, or other SDGs with the capacity for catalytic NO dioxygenation [75,129,256]. e potential bene�cial effects for growth, resistance to hypoxia, nitrosative stress, and so forth as well as potential detrimental effects have been recently reviewed [75]. In aquaculture, hypoxia resistant �sh are being created through uniform expression of the Vitreoscilla Hb directed by the carp actin promoter [257].
Vitreoscilla Hb is now being frequently exploited to increase the industrial production of proteins and other molecules by bacteria and yeasts under hypoxic reactor conditions [473][474][475][476][477][478][479][480][481], and the primary bene�t appears to be increased microaerobic respiration vis-à-vis the NOD function [256,258]. Another potential application is the use of the recently discovered dual function algal raphidophyte NOD-nitrate reductase [139] in bioremediation reactors to scavenge NO produced in fossil fuel combustion and to supply algae with nitrate for increased production of biofuels [482].
More recently, the �avoHb-NOD has been touted as a molecular tool for determining the role of NO in biological processes [483]. As stressed by Forrester et al. [483], the strategy has clear advantages over pharmacological methods employing NOS inhibitors or gene knock-outs. Indeed, �avoHb and SDGs have already been used to discover and dissect the role of NO in glioma growth and cancer [297], fungal development and mycotoxin production [305], the plant hypersensitive response [115,224,484], symbiosis and nodule development [87,260], N 2 �xation [183,184], and plant senescence [485].

Summary and Outlook
In all �elds, we �nd change and permanence in thinking [20] and a maturation of obscure phenomena to fundamental theories and advances through the scienti�c process, as lucidly stated by Werner Heisenberg .
��or an understanding of the phenomena, the �rst condition is the introduction of adequate concepts; only with the help of the correct concepts can we really know what has been observed. When we enter a new �eld, very o�en new concepts are needed, and these new concepts usually come up in a rather unclear and undeveloped form. Later they are modi�ed, sometimes they are almost completely abandoned and are replaced by better concepts which then, �nally are clear and well-de�ned. � Only through the introduction of "correct concepts", controlled experiments, exacting scrutiny, careful retrospection, and the revision or refutation of faulty concepts can basic truths be established from obscure phenomena. is has been aptly illustrated by our progress in understanding the biology of Hb and NO. Sir Humphry Davy's investigations of the "respiration" of Stickstoffoxyd (NO/NO 2 ) by blood and tissues in 1800 produced the �rst relevant, yet unclear, phenomena including the remarkable oxidation of the red pigment of blood by the gas [30]. Since then, the redpigmented proteins Hb/Mb have been shown to carry O 2 in multicellular organisms, while the NO-metabolizing activity of Hb is more ancient, widespread and only beginning to become clear. An understanding of O 2 binding remains important for both functions, but the discovery of the NO dioxygenase function has introduced numerous new questions including some that challenge dogma and traditional views of Hb structure-function. In addition to continuing to ask which Hbs function as NODs, greater knowledge of enzyme mechanism and biological function are needed to understand the diverse Hb structures and their evolution. A better understanding of the structures governing the confounding competitive binding of O 2 and NO to the ferrous heme during catalysis, or rather lack thereof, is requisite. Catalysis also needs to be understood in terms of the relevant in vivo concentrations of NO and O 2 . Also, much remains to be discovered in the area of electron donors and electron transfer.
Knowledge of the NOD activity and function of various Hbs and �avoHbs is being applied to the development of NOD inhibitors for use as therapeutic antibiotics, antitumor agents, and vasomodulators. FlavoHbs are being widely used as tools to assess the involvement of NO in the physiology and pathophysiology of plants, animals, fungi, and bacteria. Plants are being genetically modi�ed for (�avo)Hb expression for increased NO resistance, nitrogen assimilation, growth, and crop yields. Hbs and �avoHbs are also being used to increase the productivity of bacteria, yeasts, and algae in biotechnological and environmental remediation applications. It is hoped that success in any of these or other applications provides tangible rewards for past and current efforts aimed at understanding the NOD function of Hbs.
Finally, I end with a tribute to the spirit of the discoverer of the "Sauerstoffsaugung" (Hb's O 2 -absorbing function) and pioneer of molecular biology, Friedrich Hünefeld [486], by echoing his motto 172 years later.
Whatever is still hidden reveals ages in the light!

Acknowledgments
Many colleagues contributed to the evolution of the ideas and knowledge presented in this paper. If the author was remiss in citation, it was due to the rapid expansion of the literature and breadth of the �eld. e author is especially thankful to Dr. Anne Gardner for many thoughtful discussions and experimental contributions. e author thanks his students, assistants, and collaborators listed on many of the publications for their contributions. e author gratefully acknowledges discussions with the late Professor Henry Taube that led him to formulate the internal O-atom rearrangement mechanism presented herein. is work was initiated with support from the American Heart Association Scientist Development Grant (9730193N), the Cincinnati Children's Hospital Trustee Grant, and the National Institutes of Health Grant (R01 GM65090).