Nitrite Reductase Reaction Research Articles

171 - Nitrite improves Zebrafish Cardiac Regeneration Potentially by Cytoglobin 1

Treatment with nitrite (NO2‒) has been shown to have a beneficial effect on ischemia/reperfusion injury via the production of nitric oxide (NO) and modulation of reactive oxygen/nitrogen species (ROS/RNS). Nitrite can be converted to NO through the action of heme globins via a nitrite reductase reaction. The role of NO2‒ and globins has not yet been investigated in the context of heart regeneration. Zebrafish can fully regenerate their hearts following ventricular amputation and were used a model to study the effects of hypoxia and NO2‒ treatment on heart regeneration. We hypothesize that treatment with hypoxia/ NO2‒ will improve cardiac regeneration through increased production of NO or signaling ROS/RNS. We have found that NO2‒ /hypoxia treatment improves the rate of cardiomyocyte proliferation and improves neovascularization compared to hypoxia alone five days post amputation (dpa). Preceding these events, we observe an increase in thrombocytes (1 dpa) and neutrophils (3 dpa) migration to the injured area and a decrease in the blood clot size, suggesting that NO2‒ treatment is able to modulate the immune response to improve healing following injury. Zebrafish Cytoglobin 1 (Cygb1) is a globin that may mediate the cardiac regenerative response to NO2‒. Our data show that Cygb1 is expressed in the epicardium (a tissue that is known to drive the regeneration process) and its expression is increased following cardiac injury. Cygb1 may be mediating this response via its fast nitrite reductase rate (28.6 ± 3.1 M (-1) s(-1)) in its deoxy state or oxy-Cygb1 may react with NO2- to produce RNS, modulating the immune response via ROS/RNS signaling. Given the extremely low Cygb1 oxygen P50 (0.277 torr), we hypothesize that it is the latter, even in the context of the hypoxic heart. Our current research aims to identify the mechanism by which nitrite treatment modulates the immune response in the healing heart and determine if Cygb1 expression is necessary and/or sufficient for cardiac regeneration.

Generating S-Nitrosothiols from Hemoglobin: MECHANISMS, CONFORMATIONAL DEPENDENCE, AND PHYSIOLOGICAL RELEVANCE

In vitro, ferrous deoxy-hemes in hemoglobin (Hb) react with nitrite to generate nitric oxide (NO) through a nitrite reductase reaction. In vivo studies indicate Hb with nitrite can be a source of NO bioactivity. The nitrite reductase reaction does not appear to account fully for this activity because free NO is short lived especially within the red blood cell. Thus, the exporting of NO bioactivity both out of the RBC and over a large distance requires an additional mechanism. A nitrite anhydrase (NA) reaction in which N2O3, a potent S-nitrosating agent, is produced through the reaction of NO with ferric heme-bound nitrite has been proposed (Basu, S., Grubina, R., Huang, J., Conradie, J., Huang, Z., Jeffers, A., Jiang, A., He, X., Azarov, I., Seibert, R., Mehta, A., Patel, R., King, S. B., Hogg, N., Ghosh, A., Gladwin, M. T., and Kim-Shapiro, D. B. (2007) Nat. Chem. Biol. 3, 785-794) as a possible mechanism. Legitimate concerns, including physiological relevance and the nature of the mechanism, have been raised concerning the NA reaction. This study addresses these concerns demonstrating NO and nitrite with ferric hemes under near physiological conditions yield an intermediate having the properties of the purported NA heme-bound N2O3 intermediate. The results indicate that ferric heme sites, traditionally viewed as a source of potential toxicity, can be functionally significant, especially for partially oxygenated/partially met-R state Hb that arises from the NO dioxygenation reaction. In the presence of low levels of nitrite and either NO or a suitable reductant such as L-cysteine, these ferric heme sites can function as a generator for the formation of S-nitrosothiols such as S-nitrosoglutathione and, as such, should be considered as a source of RBC-derived and exportable bioactive NO.

A model for the nitric oxide producing nitrite reductase activity of hemoglobin as a function of oxygen saturation

The production of nitric oxide by the nitrite reductase activity of hemoglobin has been proposed to play a major role in hypoxic vasodilation. The bimolecular reaction rate constant for nitric oxide formation is a complex function of hemoglobin oxygenation stemming from the intrinsic allosteric character of hemoglobin, resulting in an unsymmetrical inverted U shape profile of activity versus oxygen saturation. We present an analysis of the hemoglobin nitrite reductase activity based on the Monod Wyman Changeux (MWC) allosteric model and derive a set of equations that enabled us to express the rate constant of bimolecular reaction of nitrite with hemoglobin as a function of hemoglobin saturation and use this expression to explore the factors controlling the shape of the nitrite reductase activity versus hemoglobin saturation curve. From the value of the maximum reductase activity, we derive equations to calculate microscopic nitrite reductase reaction rate constants for the R and T quaternary states. We have also developed two methods to parameterize the MWC model based on the Hill equation, with its parameters, h and P50, and the knowledge that these two descriptions of the binding curve coincide in the region of the curve where h is defined. This has allowed the calculation of the hemoglobin nitrite reductase activity rate profiles for the human hemoglobin and for bovine hemoglobin. The properties of these rate profiles are discussed.

Consumption of Hydrogen Ions in Rapid-Equilibrium Enzyme Kinetics

Enzyme-catalyzed reductase reactions in particular are characterized by large changes in the binding of hydrogen ions Δ(r)N(H). This is a thermodynamic property of the reaction that is catalyzed. For example, in the ferredoxin-nitrite reductase reaction, there is an increase of eight in the binding of hydrogen ions for every molecule of nitrite reduced to ammonia H(2)O. If these hydrogen ions are consumed in the rate-determining reaction, the limiting velocity is proportional to [H(+)](8). This would make it practically impossible to determine the kinetic parameters. This article shows that when n hydrogen ions are consumed in reactions preceding the rate-determining reaction the limiting velocity is not proportional to [H(+)](n) and may only vary with pH according to the pK's of the enzyme-substrate complex that produces products. Rapid-equilibrium rate equations for ordered A + B → products are derived for two mechanisms in which a single hydrogen ion is consumed prior to the rate-determining reaction. Rate equations are tested by calculating velocities for the minimum number of velocity measurements required to estimate the kinetic parameters and using these velocities to estimate the kinetic parameters.

Production Of Bioactive NO From The Reaction Of Met-nitrite Hemoglobin With NO: Use Of Glassy Matices And Sol-gels

There is growing evidence that nitrite-mediated reactions of Hb can generate bioactive forms of nitric oxide. The nitrite reductase reaction in which deoxy Hb reacts with nitrite has been studied from this perspective; however, this reaction generates NO under conditions where it can be easily scavenged. In contrast the proposed reaction of NO with nitrite bound to ferric heme derivatives to generate N2O3 is a much more promising mechanism. This follows because N2O3 can rapidly react with thiol containing peptides such as glutathione to produce S-NO derivatives. These species are likely to be the relatively long lived bioactive forms of NO that produce positive physiological effects in the vascular endothelium as occurs for blood substitutes derived from PEGylated Hb. We present results obtained using Hb samples encased in either glassy matrices or sol-gels that directly support this model. In particular there is spectroscopic evidence for the formation of a ferrous intermediate resulting from the direct reaction of NO with met-nitrite derivatives that precedes the formation of either ferrous NO or ferrous CO derivatives. This intermediate is attributable to a ferrous N2O3 complex.

Unraveling the Reactions of Nitric Oxide, Nitrite, and Hemoglobin in Physiology and Therapeutics

The ability of oxyhemoglobin to inhibit nitric oxide (NO)-dependent activation of soluble guanylate cyclase and vasodilation provided some of the earliest experimental evidence that NO was the endothelium-derived relaxing factor (EDRF). The chemical behavior of this dioxygenation reaction, producing nearly diffusion limited and irreversible NO scavenging, presents a major paradox in vascular biology: The proximity of large amounts of oxyhemoglobin (10 mmol/L) to the endothelium should severely limit paracrine NO diffusion from endothelium to smooth muscle. However, several physical factors are now known to mitigate NO scavenging by red blood cell encapsulated hemoglobin. These include diffusional boundaries around the erythrocyte and a red blood cell free zone along the endothelium in laminar flowing blood, which reduce reaction rates between NO and red cell hemoglobin by 100- to 600-fold. Beyond these mechanisms that reduce NO scavenging by hemoglobin within the red cell, 2 additional mechanisms have been proposed suggesting that NO can be stored in the red blood cell either as nitrite or as an S-nitrosothiol (S-nitroso-hemoglobin). The latter controversial hypothesis contends that NO is stabilized, transported, and delivered by intra-molecular NO group transfers between the heme iron and beta-93 cysteine to form S-nitroso-hemoglobin (SNO-Hb), followed by hypoxia-dependent delivery of the S-nitrosothiol in a process that links regional oxygen deficits with S-nitrosothiol-mediated vasodilation. Although this model has generated a field of research examining the potential endocrine properties of intravascular NO molecules, including S-nitrosothiols, nitrite, and nitrated lipids, a number of mechanistic elements of the theory have been challenged. Recent data from several groups suggest that the nitrite anion (NO2-) may represent the major intravascular NO storage molecule whose transduction to NO is made possible through an allosterically controlled nitrite reductase reaction with the heme moiety of hemoglobin. As subsequently understood, the hypoxic generation of NO from nitrite is likely to prove important in many aspects of physiology, pathophysiology, and therapeutics.

Reactions of spinach nitrite reductase with its substrate, nitrite, and a putative intermediate, hydroxylamine.

Plant nitrite reductase (NiR) catalyzes the reduction of nitrite (NO(2)(-)) to ammonia, using reduced ferredoxin as the electron donor. NiR contains a [4Fe-4S] cluster and an Fe-siroheme, which is the nitrite binding site. In the enzyme's as-isolated form ([4Fe-4S](2+)/Fe(3+)), resonance Raman spectroscopy indicated that the siroheme is in the high-spin ferric hexacoordinated state with a weak sixth axial ligand. Kinetic and spectroscopic experiments showed that the reaction of NiR with NO(2)(-) results in an unexpectedly EPR-silent complex formed in a single step with a rate constant of 0.45 +/- 0.01 s(-)(1). This binding rate is slow compared to that expected from the NiR turnover rates reported in the literature, suggesting that binding of NO(2)(-) to the as-isolated form of NiR is not the predominant type of substrate binding during enzyme turnover. Resonance Raman spectroscopic characterization of this complex indicated that (i) the siroheme iron is low-spin hexacoordinated ferric, (ii) the ligand coordination is unusually heterogeneous, and (iii) the ligand is not nitric oxide, most likely NO(2)(-). The reaction of oxidized NiR with hydroxylamine (NH(2)OH), a putative intermediate, results in a ferrous siroheme-NO complex that is spectroscopically identical to the one observed during NiR turnover. Resonance Raman and absorption spectroscopy data show that the reaction of oxidized NiR ([4Fe-4S](2+)/Fe(3+)) with hydroxylamine is binding-limited, while the NH(2)OH conversion to nitric oxide is much faster.

Copper complexes of a new tridentate imidazole-containing ligand: spectroscopy, structures and nitrite reductase reactivity: The molecular structures of [Cu(biap)(NO 2) 2] and [Cu(biap)Br 2

The ligand N, N-bis(2-ethyl-5-methyl-imidazol-4-ylmethyl)-aminopropane (abbreviated as biap) forms several copper(II) coordination compounds with general formula [Cu(biap)X 2], with X −=Cl −, Br −, NO 3 − , NO 2 − and NCS −. In all these compounds the ligand biap acts as a tridentate ligand. Single crystals of [Cu(biap)(NO 2) 2] and [Cu(biap)Br 2] were used for X-ray structure determinations. All compounds were characterized by IR, ligand field and EPR spectroscopy, and cyclic voltammetry. The geometry of copper(II) in the (pseudo-) halide complexes is five-coordinate, whereas copper(II) is distorted octahedrally coordinated in the nitrate and nitrite complexes, whereby one of the anions binds didentate chelating and the other one monodentate. The affinity of nitrite for the [Cu(biap)] 2+ cation was studied by determining the binding constant of the anion to the perchlorate salt of the complex in methanol solution. The data show that nitrite exhibits high affinity for the complex and its behavior is comparable with that of azide. The reduction of nitrite, to produce nitric oxide, by the reduced complex [Cu(biap)] + has been studied anaerobically. Kinetic experiments showed that the reaction exhibits saturation behavior with respect to nitrite concentration. The kinetic data indicate that [Cu(biap)] + has a moderate efficiency in the nitrite reductase reaction.

Application of nitrite reductase from Alcaligenes faecalis S-6 for nitrite measurement

The enzymatic reaction of nitrite reductase (NIR) from Alcaligenes faecalis S-6 was applied to the measurement of nitrite. NIR was immobilized on the surface of a gold electrode using filter paper and a dialysis membrane, and used as a working electrode in a three-electrode system. Amperometric methods were applied using NIR and the electron mediator 1-methoxy PMS (1-methoxy-5-methylphenazinium methylsulfate). The decrease in catholic current showed a correlation to nitrite concentration over the range 0–1 mg/l. Measurements using a batch-flow type system gave a lower detection limit of 0·01 mg/l. This is sufficient for the detection of nitrite in natural waters.

Aerobic dissimilatory reduction of nitrite by cells of Paracoccus denitrificans: the role of nitric oxide

With anaerobically grown cells of Paracoccus denitrificans it was previously found (Kučera, I. and Dadák, V. (1983) Biochem. Biophys. Res. Commun. 117, 252–258) that, in the presence of an uncoupler, nitrite as terminal acceptor was preferred to oxygen, the consumption of which was simultaneously inhibited. In the present study it is shown that besides an increased inhibition of terminal oxidases brought about by NO 2 − anion another potent inhibitor originating in the course of nitrite reductase reaction affects the division of electron flow between oxygen and nitrite. The inhibitor, the creation of which is accompanied by the aerobic nitrite reduction, is formed, even in the absence of an uncoupler, only as a result of a slight inhibition of oxygen respiration exhibited by hydroxylamine addition. From the comparison of the inhibitory effect of the intermediate on aerobically grown cells and membrane vesicles derived from them, it was proved that at neutral pH this substance does not carry an electric charge. A complex absorbing at 563 nm was formed due to the interaction of the inhibitor (generated from nitrite in the presence of uncoupler) with ferricytochrome c from bovine heart. From these findings we were led to conclude that it was most probably nitric oxide (NO).

The high affinity of Paracoccus denitrificans cells for nitrate as an electron acceptor. Analysis of possible mechanisms of nitrate and nitrite movement across the plasma membrane and the basis for inhibition by added nitrite of oxidase activity in permeabilised cells

(1) The apparent K m for nitrate of the electron-transport system in intact cells of Paracoccus denitrificans was less than 5 μM. In contrast the apparent K m for nitrate of inverted membrane vesicles oxidising NADH was greater than 50 μM. When azide, a competitive inhibitor, was present, the apparent K m observed with the vesicles was raised to 0.64 mM, consistent with values previously reported for purified preparations of the reductase. In membrane vesicles the nitrate reductase is probably not rate-limiting for NADH-nitrate oxido-reductase activity, and thus a lower limit for K m (NO 3 −) is obtained. It is suggested that the very low K m (NO 3 −) in intact cells must arise from either a transport process or a nitrate-specific pore that allows access of nitrate directly to the active site of its reductase from the periplasm. (2) The swelling of spheroplasts has been studied under both aerobic and anaerobic conditions to probe possible mechanisms of nitrate and nitrite transport across the plasma membrane of P. denitrificans. Nitrate reductase was inhibited by azide to prevent reduction of internal nitrate. No evidence for operation of either nitrate-nitrite antiport or proton-nitrate symport was obtained. (3) Measurements from the fluorescence intensity of 8-anilino-naphthalene-1-sulphonate of the rates of decay of diffusion potentials generated by addition of potassium salts to valinomycin-treated plasma membrane vesicles from P. denitrificans showed that the permeability of the membrane to anions is SCN − > NO 3 −, NO 2 −, pyruvate, acetate > CI − > SO 4 2−. In the presence of a protonophore the rate of decay of the diffusion potential was considerably enhanced with potassium acetate or potassium nitrite, but not with potassium salts of nitrate, chloride or pyruvate. This result indicates that HNO 2 and CH 3COOH can rapidly and passively diffuse across the cell membrane. This finding suggests that transport systems for nitrite are in general probably not required in bacteria. The failure of a protonophore to enhance the dissipation of the diffusion potential generated by potassium nitrate is evidence against the operation of a proton-nitrate symporter. (4) Low concentrations of added nitrite very strongly inhibit electron flow to oxygen in anaerobically grown cells, provided that they have been treated with Triton X-100 or an uncoupler. This inhibition is not observed with aerobically grown cells. It is concluded that the inhibitory species is a reaction product or an intermediate of the nitrite reductase reaction. The requirement for collapse of protonomotive force by uncoupler or permeabilising the plasma membrane suggests that any such species could be negatively charged. Nitroxyl anion (NO −) can be considered, as its conjugate acid is a postulated intermediate between nitrite and nitrous oxide; nitroxyl anion can bind to heme centres to give nitrosyl derivatives. (5) The basis for the ability of permeabilised, but not intact, cells of P. denitrificans to reduce oxygen and nitrate simultaneously is discussed.

On the purification of nitrite reductase from [formula omitted] and its reaction with nitrite under reducing conditions

Nitrite reductase (cytochrome cd ) from T. denitrificans has been crystallized in high yield in three simple and rapid steps. The spectral absorption ratio at 408 to 280 nm was 1.52. Light absorption spectra in the oxidized and reduced states were virtually identical to those of nitrite reductase from P. aeruginosa . EPR spectroscopy of nitrite reductase at 12° showed a low-spin ferric heme resonance with g-values at 2.52, 2.45 and 1.73 assigned to the d-heme. Reaction of nitrite reductase with nitrite in the presence of the reducing systems [(ascorbate + PMS) or sulfide] resulted in the formation of nitric oxide (confirmed by gas chromatography) which reacted with both c - and d -hemes of nitrite reductase yielding an EPR-detectable enzyme-NO complex with g-values at 2.07, 2.04 and 1.99 and a 14N hyperfine splitting constant of 22.5 gauss. The amount of nitric oxide produced enzymatically with sulfide as electron donor was only 5% of that found when ascorbate plus PMS served as reductant. To our knowledge the detection of the unique enzyme-NO complex is the first definitive EPR evidence for the mandatory liganding of nitric oxide with pure nitrite reductase during nitrite reduction.