Heme enzymes that bind and reduce O2 are susceptible to poisoning by NO. The high reactivity and affinity of NO for ferrous heme produces stable ferrous-NO complexes, which in theory should preclude O2 binding and turnover. However, NO inhibition is often competitive with respect to O2 and rapidly reversible, thus providing cellular and organismal survival advantages. This kinetic paradox has prompted a search for mechanisms for reversal and hence resistance. Here, I critically review proposed resistance mechanisms for NO dioxygenase (NOD) and cytochrome c oxidase (CcO), which substantiate reduction or oxidation of the tightly bound NO but nevertheless fail to provide kinetically viable solutions. A ferrous heme intermediate is clearly not available during rapid steady-state turnover. Reversible inhibition can be attributed to NO competing with O2 in transient low-affinity interactions with either the ferric heme in NOD or the ferric heme-cupric center in CcO. Toward resolution, I review the underlying principles and evidence for kinetic control of ferric heme reduction via an O2-triggered ferric heme spin crossover and an electronically-forced motion of the heme and structurally-linked protein side chains that elicit electron transfer and activate O2 in the flavohemoglobin-type NOD. For CcO, kinetics, structures, and density functional theory point to the existence of an analogous O2 and reduced oxygen intermediate-controlled electron-transfer gate with a linked proton pump function. A catalytic cycle and mechanism for CcO is finally at hand that links each of the four O2-reducing electrons to each of the four pumped protons in time and space. A novel proton-conducting tunnel and channel, electron path, and pump mechanism, most notably first hypothesized by Mårten Wikström in 1977 and pursued since, are laid out for further scrutiny. In both models, low-energy spin-orbit couplings or ‘spintronic’ interactions with O2 and NO or copper trigger the electronic motions within heme that activate electron transfer to O2, and the exergonic reactions of transient reactive oxygen intermediates ultimately drive all enzyme, electron, and proton motions.

Nitric-oxide dioxygenases (NODs) activate and combine O2 with NO to form nitrate. A variety of oxygen-binding hemoglobins with associated partner reductases or electron donors function as enzymatic NODs. Kinetic and structural investigations of the archetypal two-domain microbial flavohemoglobin-NOD have illuminated an allosteric mechanism that employs selective tunnels for O2 and NO, gates for NO and nitrate, transient O2 association with ferric heme, and an O2 and NO-triggered, ferric heme spin crossover-driven, motion-controlled, and dipole-regulated electron-transfer switch. The proposed mechanism facilitates radical-radical coupling of ferric-superoxide with NO to form nitrate while preventing suicidal ferrous-NO formation. Diverse globins display the structural and functional motifs necessary for a similar allosteric NOD mechanism. In silico docking simulations reveal monomeric erythrocyte hemoglobin alpha-chain and beta-chain intrinsically matched and tightly coupled with NADH-cytochrome b5 oxidoreductase and NADPH-cytochrome P450 oxidoreductase, respectively, forming membrane-bound flavohemoglobin-like mammalian NODs. The neuroprotective neuroglobin manifests a potential NOD role in a close-fitting ternary complex with membrane-bound NADH-cytochrome b5 oxidoreductase and cytochrome b5. Cytoglobin interfaces weakly with cytochrome b5 for O2 and NO-regulated electron-transfer and coupled NOD activity. The mechanistic model also provides insight into the evolution of O2 binding cooperativity in hemoglobin and a basis for the discovery of allosteric NOD inhibitors.

Kinetic and structural investigations of the flavohemoglobin-type NO dioxygenase have suggested critical roles for transient Fe(III)O2 complex formation and O2-forced movements affecting hydride transfer to the FAD cofactor and electron-transfer to the Fe(III)O2 complex. Stark-effect theory together with structural models and dipole and internal electrostatic field determinations provided a semi-quantitative spectroscopic method for investigating the proposed Fe(III)O2 complex and O2-forced movements. Deoxygenation of the enzyme causes Stark effects on the ferric heme Soret and charge-transfer bands revealing the Fe(III)O2 complex. Deoxygenation also elicits Stark effects on the FAD that expose forces and motions that create a more restricted NADH access to FAD for hydride transfer and switch electron-transfer off. Glucose also forces the enzyme toward an off state. Amino acid substitutions at the B10, E7, E11, G8, D5, and F7 positions influence the Stark effects of O2 on resting heme spin states and FAD consistent with the proposed roles of the side chains in the enzyme mechanism. Deoxygenation of ferric myoglobin and hemoglobin A also induces Stark effects on the hemes suggesting a common 'oxy-met' state. The ferric myoglobin and hemoglobin heme spectra are also glucose-responsive. A conserved glucose or glucose-6-phosphate binding site is found bridging the BC-corner and G-helix in fla-vohemoglobin and myoglobin suggesting novel allosteric effector roles for glucose or glucose-6-phosphate in the NO dioxygenase and O2 storage functions. The results support the proposed roles of a ferric O2 intermediate and protein motions in regulating electron-transfer during NO dioxygenase turnover.

The substrates O-2 and NO cooperatively activate the NO dioxygenase function of Escherichia coli flavohemoglobin. Steady-state and transient kinetic measurements support a structure-based mechanistic model in which O-2 and NO movements and conserved amino acids at the E11, G8, E2, E7, B10, and F7 positions within the globin domain control activation. In the cooperative and allosteric mechanism, O-2 migrates to the catalytic heme site via a long hydrophobic tunnel and displaces LeuE11 away from the ferric iron, which forces open a short tunnel to the catalytic site gated by the ValG8/ IleE15 pair and LeuE11. NO permeates this tunnel and leverages upon the gating side chains triggering the CD loop to furl, which moves the E and F-helices and switches an electron transfer gate formed by LysF7, GlnE7, and water. This allows FADH(2) to reduce the ferric iron, which forms the stable ferric-superoxide-TyrB10/GlnE7 complex. This complex reacts with internalized NO with a bimolecular rate constant of 1010 M-1 s(-1) forming nitrate, which migrates to the CD loop and unfurls the spring-like structure. To restart the cycle, LeuE11 toggles back to the ferric iron. Actuating electron transfer with O-2 and NO movements averts irreversible NO poisoning and reductive inactivation of the enzyme. Together, structure snapshots and kinetic constants provide glimpses of intermediate conformational states, time scales for motion, and associated energies.

Background: Sulfite, an intermediate in sulfur metabolism, can be oxidized to the potentially toxic sulfur trioxide anion radical (STAR).Results: Diverse globins efficiently reduced STAR in vitro, and flavohemoglobin protected yeast from STAR toxicity.Conclusion: Globins can function as STAR scavengers.Significance: The data suggest roles for diverse globins in protecting cells against sulfite stress.Ferrous myoglobin was oxidized by sulfur trioxide anion radical (STAR) during the free radical chain oxidation of sulfite. Oxidation was inhibited by the STAR scavenger GSH and by the heme ligand CO. Bimolecular rate constants for the reaction of STAR with several ferrous globins and biomolecules were determined by kinetic competition. Reaction rate constants for myoglobin, hemoglobin, neuroglobin, and flavohemoglobin are large at 38, 120, 2,600, and >= 7,500 x 10(6) m(-1) s(-1), respectively, and correlate with redox potentials. Measured rate constants for O-2, GSH, ascorbate, and NAD(P)H are also large at -100, 10, 130, and 30 x 10(6) M-1 s(-1), respectively, but nevertheless allow for favorable competition by globins and a capacity for STAR scavenging in vivo. Saccharomyces cerevisiae lacking sulfite oxidase and deleted of flavohemoglobin showed an O-2-dependent growth impairment with nonfermentable substrates that was exacerbated by sulfide, a precursor to mitochondrial sulfite formation. Higher O-2 exposures inactivated the superoxide-sensitive mitochondrial aconitase in cells, and hypoxia elicited both aconitase and NADP(+)-isocitrate dehydrogenase activity losses. Roles for STAR-derived peroxysulfate radical, superoxide radical, and sulfo-NAD(P) in the mechanism of STAR toxicity and flavohemoglobin protection in yeast are suggested.

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.

Cytoglobin (Cygb) was investigated for its capacity to function as a NO dioxygenase (NOD) in vitro and in hepatocytes. Ascorbate and cytochrome b5 were found to support a high NOD activity. Cygb-NOD activity shows respective Km values for ascorbate, cytochrome b5, NO, and O2 of 0.25 mm, 0.3 μm, 40 nm, and ∼20 μm and achieves a kcat of 0.5 s−1. Ascorbate and cytochrome b5 reduce the oxidized Cygb-NOD intermediate with apparent second order rate constants of 1000 m−1 s−1 and 3 × 106 m−1 s−1, respectively. In rat hepatocytes engineered to express human Cygb, Cygb-NOD activity shows a similar kcat of 1.2 s−1, a Km(NO) of 40 nm, and a kcat/Km(NO) (k′NOD) value of 3 × 107 m−1 s−1, demonstrating the efficiency of catalysis. NO inhibits the activity at [NO]/[O2] ratios >1:500 and limits catalytic turnover. The activity is competitively inhibited by CO, is slowly inactivated by cyanide, and is distinct from the microsomal NOD activity. Cygb-NOD provides protection to the NO-sensitive aconitase. The results define the NOD function of Cygb and demonstrate roles for ascorbate and cytochrome b5 as reductants.

A variety of hemoglobins, including several microbial flavohemoglobins, enzymaticatly dioxygenate the free radical nitric oxide ((center dot)NO) to form nitrate. Many of these (center dot)NO dioxygenases have been shown to control (center dot)NO toxicity and signaling. Furthermore, (center dot)NO dioxygenation appears to be an ancient and intrinsic function for members of the hemoglobin superfamily found in Archaea, eukaryotes, and bacteria. Yet for many hemoglobins, a function remains to be elucidated. Methods for the assay and characterization of the ONO dioxygenase (EC 1.14.12.17) activity and function of flavohemoglobins are described. The methods may also be applied to the discovery and design of inhibitors for use as antibiotics or as modulators of (center dot)NO signaling.

Structurally diverse members of the ancient hemoglobin (Hb) superfamily show NO dioxygenase (NOD) activity (EC 1.14.12.17), suggesting a common and primal function for Hbs and myoglobins (Mbs). NO reacts rapidly with the oxy complexes of Hbs, Mbs, flavoHb, truncated Hb, legume Hb, and neuroglobin, generating stoichiometric ferric heme and nitrate, and incorporating both O-atoms of the bound O2. Associated flavin-containing reductases, cytochrome b5, or ascorbate reduce ferric heme to allow O2 rebinding and catalytic turnover. Hb functions as a true enzyme by controlling O2 binding and electrochemistry, by guiding NO diffusion and the dioxygenation reaction, and by shielding reactive FeIII−O2•,FeIII−OONO,FeIV=O, and NO2 intermediates from solvent water and biomolecules. The activity protects against NO toxicity and modulates NO signaling in a variety of life forms. NODs and NOD inhibitors are finding applications in medicine, agriculture, and biotechnology.

ABSTRACT Flavohemoglobins metabolize nitric oxide (NO) to nitrate and protect bacteria and fungi from NO-mediated damage, growth inhibition, and killing by NO-releasing immune cells. Antimicrobial imidazoles were tested for their ability to coordinate flavohemoglobin and inhibit its NO dioxygenase (NOD) function. Miconazole, econazole, clotrimazole, and ketoconazole inhibited the NOD activity of Escherichia coli flavohemoglobin with apparent Ki values of 80, 550, 1,300, and 5,000 nM, respectively. Saccharomyces cerevisiae, Candida albicans, and Alcaligenes eutrophus enzymes exhibited similar sensitivities to imidazoles. Imidazoles coordinated the heme iron atom, impaired ferric heme reduction, produced uncompetitive inhibition with respect to O2 and NO, and inhibited NO metabolism by yeasts and bacteria. Nevertheless, these imidazoles were not sufficiently selective to fully mimic the NO-dependent growth stasis seen with NOD-deficient mutants. The results demonstrate a mechanism for NOD inhibition by imidazoles and suggest a target for imidazole engineering.

Nitric oxide (NO) serves critical signaling, energetic, and toxic functions throughout the biosphere. NO steady-state levels and functions are controlled in part by NO metabolism or degradation. Dioxygen-dependent NO dioxygenases (EC 1.14.12.17) and dioxygen-independent NO reductases (EC 1.7.99.7) are being identified as major routes for NO metabolism in various life forms. Here we describe the use of the Clark-type NO electrode, mechanistic inhibitors, and nitrate/nitrite assays to measure, characterize, and identify major NO metabolic pathways and enzymes in bacteria, fungi, plants, mammalian cells, and organelles. The methods may prove to be particularly useful for mechanistic investigations and the development of inhibitors, inducers, and other novel NO-modulating therapeutics.

ABSTRACT The yeast Candida albicans is an opportunistic pathogen that threatens patients with compromised immune systems. Immune cell defenses against C. albicans are complex but typically involve the production of reactive oxygen species and nitrogen radicals such as nitric oxide (NO) that damage the yeast or inhibit its growth. Whether Candida defends itself against NO and the molecules responsible for this defense have yet to be determined. The defense against NO in various bacteria and the yeast Saccharomyces cerevisiae involves an NO-scavenging flavohemoglobin. The C. albicans genome contains three genes encoding flavohemoglobin-related proteins, CaYHB1 , CaYHB4 , and CaYHB5 . To assess their roles in NO metabolism, we constructed strains lacking each of these genes and demonstrated that just one, CaYHB1 , is responsible for NO consumption and detoxification. In C. albicans , NO metabolic activity and CaYHB1 mRNA levels are rapidly induced by NO and NO-generating agents. Loss of CaYHB1 increases the sensitivity of C. albicans to NO-mediated growth inhibition. In mice, infections with Candida strains lacking CaYHB1 still resulted in lethality, but virulence was decreased compared to that in wild-type strains. Thus, C. albicans possesses a rapid, specific, and highly inducible NO defense mechanism involving one of three putative flavohemoglobin genes.

Human intestinal Caco-2 cells metabolize and detoxify NO via a dioxygen- and NADPH-dependent, cyanide- and CO-sensitive pathway that yields nitrate. Enzymes catalyzing NO dioxygenation fractionate with membranes and are enriched in microsomes. Microsomal NO metabolism shows apparent KM values for NO, O2, and NADPH of 0.3, 9, and 2 μM, respectively, values similar to those determined for intact or digitonin-permeabilized cells. Similar to cellular NO metabolism, microsomal NO metabolism is superoxide-independent and sensitive to heme-enzyme inhibitors including CO, cyanide, imidazoles, quercetin, and allicin-enriched garlic extract. Selective inhibitors of several cytochrome P450s and heme oxygenase fail to inhibit the activity, indicating limited roles for a subset of microsomal heme enzymes in NO metabolism. Diphenyleneiodonium and cytochrome c(III) inhibit NO metabolism, suggesting a role for the NADPH–cytochrome P450 oxidoreductase (CYPOR). Involvement of CYPOR is demonstrated by the specific inhibition of the NO metabolic activity by inhibitory anti-CYPOR IgG. In toto, the results suggest roles for a microsomal CYPOR-coupled and heme-dependent NO dioxygenase in NO metabolism, detoxification, and signal attenuation in mammalian cells.

Nitric oxide (NO) induces NO-detoxifying enzymes in Escherichia coli suggesting sensitive mechanisms for coordinate control of NO defense genes in response to NO stress. Exposure of E. coli to sub-micromolar NO levels under anaerobic conditions rapidly induced transcription of the NO reductase (NOR) structural genes, norV and norW, as monitored by lac gene fusions. Disruption of rpoN (sigma(54)) impaired the NO-mediated induction of norV and norW transcription and NOR expression, whereas disruption of the upstream regulatory gene, norR, completely ablated NOR induction. NOR inducibility was restored to NorR null mutants by expressing NorR in trans. Furthermore, an internal deletion of the N-terminal domain of NorR activated NOR expression independent of NO exposure. Neither NorR nor sigma(54) was essential for NO-mediated induction of the NO dioxygenase (flavohemoglobin) encoded by hmp. However, elevated NOR activity inhibited NO dioxygenase induction, and, in the presence of dioxygen, NO dioxygenase inhibited norV induction by NO. The results demonstrate the role of NorR as a sigma(54)-dependent regulator of norVW expression. A role for the NorR N-terminal domain as a transducer or sensor for NO is suggested.

Recent data indicate that cystic fibrosis (CF) airway mucus is anaerobic. This suggests that Pseudomonas aeruginosa infection in CF reflects biofilm formation and persistence in an anaerobic environment. P. aeruginosa formed robust anaerobic biofilms, the viability of which requires rhl quorum sensing and nitric oxide (NO) reductase to modulate or prevent accumulation of toxic NO, a byproduct of anaerobic respiration. Proteomic analyses identified an outer membrane protein, OprF, that was upregulated approximately 40-fold under anaerobic versus aerobic conditions. Further, OprF exists in CF mucus, and CF patients raise antisera to OprF. An oprF mutant formed poor anaerobic biofilms, due, in part, to defects in anaerobic respiration. Thus, future investigations of CF pathogenesis and therapy should include a better understanding of anaerobic metabolism and biofilm development by P. aeruginosa.

Nitric-oxide dioxygenase (NOD) and reductase (NOR) activities of flavohemoglobin (flavoHb) have been suggested as mechanisms for NO metabolism and detoxification in a variety of microbes. Mechanisms of NO detoxification were tested in Escherichia coli using flavoHb-deficient mutants and overexpressors. flavoHb showed negligible anaerobic NOR activity and afforded no protection to the NO-sensitive aconitase or the growth of anoxic E. coli, whereas the NOD activity and the protection afforded with O(2) were substantial. A NO-inducible, O(2)-sensitive, and cyanide-resistant NOR activity efficiently metabolized NO and protected anaerobic cells from NO toxicity independent of the NOR activity of flavoHb. flavoHb possesses nitrosoglutathione and nitrite reductase activities that may account for the protection it affords against these agents. NO detoxification by flavoHb occurs most effectively via O(2)-dependent NO dioxygenation.

SOCIETY OF CRITICAL CARE MEDICINE 32ND CRITICAL CARE CONGRESS SAN ANTONIO, TEXAS, USA JANUARY 28-FEBRUARY 2, 2003: ORAL/SANDWICH PRESENTATIONS: Poster Presentation: Basic Science: Sepsis NO and Signal Transduction: PDF Only