Rate Of Nitric Oxide Consumption Research Articles

Nitric Oxide Does Not Inhibit but Is Metabolized by the Cytochrome bcc-aa3 Supercomplex.

Nitric oxide (NO) is a well-known active site ligand and inhibitor of respiratory terminal oxidases. Here, we investigated the interaction of NO with a purified chimeric bcc-aa3 supercomplex composed of Mycobacterium tuberculosis cytochrome bcc and Mycobacterium smegmatis aa3-type terminal oxidase. Strikingly, we found that the enzyme in turnover with O2 and reductants is resistant to inhibition by the ligand, being able to metabolize NO at 25 °C with an apparent turnover number as high as ≈303 mol NO (mol enzyme)−1 min−1 at 30 µM NO. The rate of NO consumption proved to be proportional to that of O2 consumption, with 2.65 ± 0.19 molecules of NO being consumed per O2 molecule by the mycobacterial bcc-aa3. The enzyme was found to metabolize the ligand even under anaerobic reducing conditions with a turnover number of 2.8 ± 0.5 mol NO (mol enzyme)−1 min−1 at 25 °C and 8.4 µM NO. These results suggest a protective role of mycobacterial bcc-aa3 supercomplexes against NO stress.

Branched-chain amino acid supplementation promotes aerobic growth of Salmonella Typhimurium under nitrosative stress conditions.

Nitric oxide (NO) inactivates iron-sulfur enzymes in bacterial amino acid biosynthetic pathways, causing amino acid auxotrophy. We demonstrate that exogenous supplementation with branched-chain amino acids (BCAA) can restore the NO resistance of hmp mutant Salmonella Typhimurium lacking principal NO-metabolizing enzyme flavohemoglobin, and of mutants further lacking iron-sulfur enzymes dihydroxy-acid dehydratase (IlvD) and isopropylmalate isomerase (LeuCD) that are essential for BCAA biosynthesis, in an oxygen-dependent manner. BCAA supplementation did not affect the NO consumption rate of S. Typhimurium, suggesting the BCAA-promoted NO resistance independent of NO metabolism. BCAA supplementation also induced intracellular survival of ilvD and leuCD mutants at wild-type levels inside RAW 264.7 macrophages that produce constant amounts of NO regardless of varied supplemental BCAA concentrations. Our results suggest that the NO-induced BCAA auxotrophy of Salmonella, due to inactivation of iron-sulfur enzymes for BCAA biosynthesis, could be rescued by bacterial taking up exogenous BCAA available in oxic environments.

Abstract 16720: Cytoglobin Regulates Cardiovascular Function and Vessel Tone by Controlling the Rate of Vascular Nitric Oxide Metabolism

Cytoglobin (Cygb) can effectively metabolize nitric oxide (NO), a potent vasodilator, in the presence of oxygen and reductants. Cygb in the vascular wall may affect cardiovascular functions by changing the rate of NO metabolism. In this study, we directly tested the vascular role of Cygb using Cygb knockout (Cygb-/-) mice. The mean blood pressure of Cygb-/- and C57BL/6 wild type (WT) mice was 65.3 ± 1.9 mmHg and 93.7 ± 1.5 mmHg, respectively (n=10). Using echocardiography, we observed that cardiac output (CO) was increased in Cygb-/- mice compared to WT with values of 29.8 ± 3.9 vs 17.7 ± 0.9 ml/min. The systemic vascular resistance (SVR) of Cygb-/- mice was decreased by ~60% vs that of WT mice (Fig. 1). Further, the inner diameter (id) of aorta of Cygb-/- mice was dilated compared to WT with values of 2.2 ± 0.1 mm vs 1.5 ± 0.05 mm (n=5), respectively. After treatment with the NO synthase inhibitor L-NAME, no difference in the aortic id remained between Cygb-/- (1.55 ± 0.03 mm) and WT (1.49 ± 0.02 mm) mice, indicating that the NO pathway is responsible for the difference in vascular inner diameters and tone. Myograph experiments show that the aortic vasodilation response of Cygb-/- mice is much more sensitive to acetylcholine (Ach) or the NO donor nitroprusside (SNP) (EC50 shifts from 13 nM and 2.9 nM (WT mice) to 0.33 nM and 0.16 nM (Cygb-/-) for Ach and SNP, respectively). Using NO electrodes to measure the rate of NO consumption by SMCs and quantitative imunoblotting to estimate Cygb content in RSMCs-AR and Cygb knockdown RSMCs, we observed that 90% of NO consumption by RSMCs-AR is caused by the intracellular Cygb. Our results indicate that Cygb deficiency in the vascular wall of Cygb-/- mice greatly reduces the rate of NO metabolism and increases vascular NO concentration, resulting in vasodilation, increase in vessel lumen diameter, and decrease in SVR. These results demonstrate that Cygb regulates cardiac function and vessel tone by controlling the rate of vascular NO metabolism.

Differences in oxygen‐dependent nitric oxide metabolism by cytoglobin and myoglobin account for their differing functional roles

The endogenous vasodilator nitric oxide (NO) is metabolized in tissues in an oxygen-dependent manner. In skeletal and cardiac muscle, high concentrations of myoglobin (Mb) function as a potent NO scavenger. However, the Mb concentration is very low in vascular smooth muscle, where low concentrations of cytoglobin (Cygb) may play a major role in metabolizing NO. Questions remain regarding how low concentrations of Cygb and Mb differ in terms of NO metabolism, and the basis for their different cellular roles and functions. In this study, electrode techniques were used to perform comparative measurements of the kinetics of NO consumption by Mb and Cygb. UV/Vis spectroscopic methods and computer simulations were performed to study the reaction of Mb and Cygb with ascorbate (Asc) and the underlying mechanism. It was observed that the initial rate of Cygb(3+) reduction by Asc was 415-fold greater than that of Mb(3+). In the low [O2] range (0-50 μM), the Cygb-mediated NO consumption rate is ~ 500 times more sensitive to changes in O2 concentration than that of Mb. The reduction of Cygb(3+) by Asc follows a reversible kinetic model, but that of Mb(3+) is irreversible. A reaction mechanism for Cygb(3+) reduction by Asc is proposed, and the reaction equilibrium constants are determined. Our results suggest that the rapid reduction of Cygb by cellular reductants enables Cygb to efficiently regulate NO metabolism in the vascular wall in an oxygen-dependent manner, but the slow rate of Mb reduction does not show this oxygen dependence.

Characterization of the Function of Cytoglobin as an Oxygen-Dependent Regulator of Nitric Oxide Concentration

The endogenous vasodilator nitric oxide (NO) is metabolized in tissues in an O(2)-dependent manner. This regulates NO levels in the vascular wall; however, the underlying molecular basis of this O(2)-dependent NO consumption remains unclear. While cytoglobin (Cygb) was discovered a decade ago, its physiological function remains uncertain. Cygb is expressed in the vascular wall and can consume NO in an O(2)-dependent manner. Therefore, we characterize the process of the O(2)-dependent consumption of NO by Cygb in the presence of the cellular reductants and reducing systems ascorbate (Asc) and cytochrome P(450) reductase (CPR), measure rate constants of Cygb reduction by Asc and CPR, and propose a reaction mechanism and derive a related kinetic model for this O(2)-dependent NO consumption involving Cygb(Fe(3+)) as the main intermediate reduced back to ferrous Cygb by cellular reductants. This kinetic model expresses the relationship between the rate of O(2)-dependent consumption of NO by Cygb and rate constants of the molecular reactions involved. The predicted rate of O(2)-dependent consumption of NO by Cygb is consistent with experimental results supporting the validity of the kinetic model. Simulations based on this kinetic model suggest that the high efficiency of Cygb in regulating the NO consumption rate is due to the rapid reduction of Cygb by cellular reductants, which greatly increases the rate of consumption of NO at higher O(2) concentrations, and binding of NO to Cygb, which reduces the rate of consumption of NO at lower O(2) concentrations. Thus, the coexistence of Cygb with efficient reductants in tissues allows Cygb to function as an O(2)-dependent regulator of NO decay.

Nitric oxide emissions from black soil, northeastern China: A laboratory study revealing significantly lower rates than hitherto reported

Nitric oxide (NO) is an important component of biogeochemical cycling of nitrogen, produced via biologically mediated processes of nitrification and denitrification in soils. The production and consumption processes of NO in black soils are not fully understood. We established how moisture and temperature affect NO dynamics for black soil samples of maize land in the temperate zone of northeastern China. The optimum soil moisture for the maximum NO production and emission was determined to be 41% water-filled pore space (WFPS), based on laboratory experiments and modeling. For a given moisture, NO fluxes increased exponentially with soil temperature at any given soil moisture. The optimum soil moisture for the maximum NO emission was constant and independent of soil temperature. The NO consumption rate constant (k) in the studied soil (range 9.31 × 10−6–15.1 × 10−6 m3 kg−1 s−1) was in the middle of the range of similar k values published to date. The maximum NO emission potential for black soils at 25 °C and 15 °C were about 18.6 and 9.0 ng N m−2 s−1, respectively. Based on laboratory results and field monitoring data of soil water content and soil temperature, the average NO fluxes from black soils in the region were estimated to be 10.7 ng N m−2 s−1 for an entire plant growth period. NO emissions likely occur principally in July, associated with optimum soil moisture. The present study suggests that NO fluxes from black soil are much lower than the previous reports from cropland in southern parts of China.

Oxygen regulates the effective diffusion distance of nitric oxide in the aortic wall

Endothelium-derived nitric oxide (NO) is critical in maintaining vascular tone. Accumulating evidence shows that NO bioavailability is regulated by oxygen concentration. However, it is unclear to what extent the oxygen concentration regulates NO bioavailability in the vascular wall. In this study, a recently developed experimental setup was used to measure the NO diffusion flux across the aortic wall at various oxygen concentrations. It was observed that for a constant NO concentration at the endothelial surface, the measured NO diffusion flux out of the adventitial surface at [O 2] = 0 μM is around fivefold greater than at [O 2] = 150 μM, indicating that NO is consumed in the aortic wall in an oxygen-dependent manner. Analysis of experimental data shows that the rate of NO consumption in the aortic wall is first order with respect to [NO] and first order with respect to [O 2], and the rate constant k 1 was determined as (4.0 ± 0.3) × 10 3 M −1 s −1. Computer simulations demonstrate that NO concentration distribution significantly changes with oxygen concentration and the effective NO diffusion distance at low oxygen level ([O 2] ≤ 25 μM) is significantly longer than that at high oxygen level ([O 2] = 200 μM). These results suggest that oxygen-dependent NO consumption may play an important role in dilating blood vessels during hypoxia by increasing the effective NO diffusion distance.

Cytochrome P450 oxidoreductase participates in nitric oxide consumption by rat brain

In low nanomolar concentrations, NO (nitric oxide) functions as a transmitter in brain and other tissues, whereas near-micromolar NO concentrations are associated with toxicity and cell death. Control of the NO concentration, therefore, is critical for proper brain function, but, although its synthesis pathway is well-characterized, the major route of breakdown of NO in brain is unclear. Previous observations indicate that brain cells actively consume NO at a high rate. The mechanism of this consumption was pursued in the present study. NO consumption by a preparation of central glial cells was abolished by cell lysis and recovered by addition of NADPH. NADPH-dependent consumption of NO localized to cell membranes and was inhibited by proteinase K, indicating the involvement of a membrane-bound protein. Purification of this activity yielded CYPOR (cytochrome P450 oxidoreductase). Antibodies against CYPOR inhibited NO consumption by brain membranes and the amount of CYPOR in several cell types correlated with their rate of NO consumption. NO was also consumed by purified CYPOR but this activity was found to depend on the presence of the vitamin E analogue Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), included in the buffer as a precaution against inadvertent NO consumption by lipid peroxidation. In contrast, NO consumption by brain membranes was independent of Trolox. Hence, it appears that, during the purification process, CYPOR becomes separated from a partner needed for NO consumption. Cytochrome P450 inhibitors inhibited NO consumption by brain membranes, making these proteins likely candidates.

Biogenic nitric oxide emission from saline sodic soils in a semiarid region, northeastern China: A laboratory study

It is well‐known that nitric oxide (NO) is an important component in nitrogen biogeochemical cycling produced through biological process of nitrification and denitrification in soils, but the production and the consumption processes of NO in sodic saline soil are less understood. Through a series of laboratory experiments focusing on NO biogenic emissions from four kinds of saline sodic soils of different land use in western Songnen Plain northestern China, we found that the optimum soil moisture for the maximum NO production and emission were 14.0%, 9.0%, 9.5%, and 18% water‐filled pore space (WFPS) for soil samples from natural pasture, man‐made pasture, paddy field of saline sodic soil mixed sandy soil, and paddy field of pure saline sodic soil, respectively. For a given moisture, NO fluxes increased exponentially with soil temperature at any given soil moisture. The optimum soil moisture for the maximum NO emission for a certain soil type, however, was constant and independent of soil temperature. The NO consumption processes for different land uses were similar in all studied saline sodic soils since the difference of NO consumption rate constant in these soils was small (ranged from 1.07 × 10−6 to 7.45 × 10−6 m3 kg−1 s−1). The NO emission potential for paddy field soils was about 1.2–2‐fold higher than pasture soils. On the basis of laboratory results and field monitoring data of soil water content and soil temperature, the average NO fluxes from these saline sodic soils in the region were estimated to be 1.3–4.9 ng m−2 s−1 for an entire plant growth period. NO fluxes for pastures mainly occurred in the dry season and were about threefold higher than that for paddy fields, which was strongly influenced by field management.

A Model of NO/O2 Transport in Capillary-perfused Tissue Containing an Arteriole and Venule Pair

The goal of this study was to investigate the complex co-transport of nitric oxide (NO) and oxygen (O2) in a paired arteriole-venule, surrounded by capillary-perfused tissue using a computer model. Blood flow was assumed to be steady in the arteriolar and venular lumens and to obey Darcy's law in the tissue. NO consumption rate was assumed to be constant in the core of the arteriolar and venular lumen and to decrease linearly to the endothelium. Average NO consumption rate by capillary blood in a unit tissue volume was assumed proportional to the blood flux across the volume. Our results predict that: (1) the capillary bed, which connects the arteriole and venule, facilitates the release of O2 from the vessel pair to the surrounding tissue; (2) decreasing the distance between arteriole and venule can result in a higher NO concentration in the venular wall than in the arteriolar wall; (3) in the absence of capillaries in the surrounding tissue, diffusion of NO from venule to arteriole contributes little to NO concentration in the arteriolar wall; and (4) when capillaries are added to the simulation, a significant increase of NO in the arteriolar wall is observed.

Direct and Indirect Roles of Cytochrome b in the Mediation of Superoxide Generation and NO Catabolism by Mitochondrial Succinate-Cytochrome c Reductase

Mitochondrial superoxide (O2*-) production is an important mediator of oxidative cellular injury. Succinate-cytochrome c reductase (SCR) of the electron transport chain has been implicated as an essential part of the mediation of O2*- generation and an alternative target of nitric oxide (NO) in the regulation of mitochondrial respiration. The Q cycle mechanism plays a central role in controlling both events. In the present work, O2*- generation by SCR was measured with the EPR spin-trapping technique using DEPMPO (5-diethoxylphosphoryl-5-methyl-1-pyrroline N-oxide) as the spin trap. In the presence of succinate, O2*- generation from SCR was detected as the spin adduct DEPMPO/*OOH. Inhibitors of the Q(o*-) site only marginally reduced (20-30%) this O2*- production, suggesting a secondary role of Q(o*-) in the mediation of O2*- generation. Addition of cyanide significantly decreased (approximately 70%) O2*- production, indicating the involvement of the heme component. UV-visible spectral analysis revealed that oxidation of ferrocytochrome b was accompanied by cytochrome c(1) reduction, and the reaction was mediated by the formation of an O2*- intermediate, indicating a direct role for cytochrome b in O2*- generation. In the presence of NO, DEPMPO/*OOH production was progressively diminished, implying that NO interacted with SCR or trapped the O2*-. The consumption of NO by SCR was investigated by electrochemical detection using an NO electrode. In the presence of succinate, SCR-mediated NO consumption was observed and inhibited by the addition of superoxide dismutase, suggesting the involvement of O2*-. Under the conditions of argon saturation, the NO consumption rate was not enhanced by succinate, suggesting a direct role for O2*- in the mediation of NO consumption. In the presence of succinate, oxidation of the ferrocytochrome b moiety of SCR was accelerated by the addition of NO, and was inhibited by argon saturation, indicating an indirect role for cytochrome b in the mediation of NO consumption.

A Model of NO/O2Transport in Capillary-perfused Tissue Containing an Arteriole and Venule Pair.

The goal of this study was to investigate the complex co-transport of nitric oxide (NO) and oxygen (O2) in a paired arteriole-venule, surrounded by capillary-perfused tissue using a computer model. Blood flow was assumed to be steady in the arteriole and venular lumens and to obey Darcy's law in the capillary-perfused tissue. NO consumption rate in the arteriolar and venular lumen was assumed to be constant in the core of the arteriolar and venular lumen and to decrease linearly to the endothelium. Average NO consumption rate by capillary blood in a unit tissue volume was assumed proportional to the blood flux across the volume. Our preliminary results predict that: 1) The capillary bed, which connects the arteriole and venule, facilitates the release of O2 from the vessel pair to the surrounding tissue; 2) Decreasing the distance between arteriole and venule can result in a higher NO concentration in the venular wall than in the arteriolar wall; 3) In the absence of capillaries in the surrounding tissue, diffusion of NO from venule to arteriole contributes little to NO contents in the arteriole; and 4) when capillaries are added to the simulation, a significant increase in arteriole NO content is observed.

Biphasic modulation of vascular nitric oxide catabolism by oxygen.

Endothelium-derived nitric oxide (NO) plays an important role in the regulation of vascular tone. Lack of NO bioavailability can result in cardiovascular disease. NO bioavailability is determined by its rates of generation and catabolism; however, it is not known how the NO catabolism rate is regulated in the vascular wall under normoxic, hypoxic, and anaerobic conditions. To investigate NO catabolism under different oxygen concentrations, studies of NO and O2 consumption by the isolated rat aorta were performed using electrochemical sensors. Under normoxic conditions, the rate of NO consumption in solution was enhanced in the presence of the rat aorta. Under hypoxic conditions, NO consumption decreased in parallel with the O2 concentration. Like the inhibition of mitochondrial respiration by NO, the inhibitory effects of NO on aortic O2 consumption increased as O2 concentration decreased. Under anaerobic conditions, however, a paradoxical reacceleration of NO consumption occurred. This increased anaerobic NO consumption was inhibited by the cytochrome c oxidase inhibitor NaCN but not by the free iron chelator deferoxamine, the flavoprotein inhibitor diphenylene iodonium (10 microM), or superoxide dismutase (200 U/ml). The effect of O2 on the NO consumption could be reproduced by purified cytochrome c oxidase (CcO), implying that CcO is involved in aortic NO catabolism. This reduced NO catabolism at low O2 tensions supports the maintenance of effective NO levels in the vascular wall, reducing the resistance of blood vessels. The increased anaerobic NO catabolism may be important for removing excess NO accumulation in ischemic tissues.

Biogenic NO emissions from forest and pasture soils: Relating laboratory studies to field measurements

During September and October 1999, dynamic chamber measurements were carried out to determine nitric oxide (NO) fluxes from a primary forest soil and an old pasture in the Brazilian Amazon basin as part of the project “European Studies of Trace Gases and Atmospheric Chemistry as a Contribution to the Large‐Scale Biosphere‐Atmosphere Experiment in Amazonia” (LBA‐EUSTACH). In addition, soil samples were collected from these two sites, and laboratory experiments were conducted to determine the NO production and consumption rate constants as functions of soil temperature and soil moisture. These laboratory results were converted into NO fluxes using a simple algorithm, which required additional information on the gas diffusion in soil, the soil bulk density, and the field conditions (soil temperature and soil moisture). Over the entire measurement period, the calculated and measured NO fluxes agreed well both for the forest (6.9 ± 2.9 and 5.0 ± 4.6 ng m−2 s−1, respectively) and for the pasture (0.67 ± 0.09 and 0.65 ± 0.37 ng m−2 s−1, respectively). Forest to pasture conversion decreased NO production and gas diffusion and resulted in smaller NO fluxes from pasture than forest soil.

Cytochrome bo3 from Escherichia coli: the binding and turnover of nitric oxide

The reaction of nitric oxide (NO) with fast and reduced cytochrome bo 3(cyt bo 3) from Escherichia coli has been investigated. The stoichiometry of NO binding to cyt bo 3 was determined using an NO electrode in the [NO] range 1–14 μM. Under reducing conditions, the initial decrease in [NO] following the addition of cyt bo 3 corresponded to binding of 1 NO molecule per cyt bo 3 functional unit. After this “rapid” NO binding phase, there was a slow, but significant rate of NO consumption (∼0.3 mol NO mol bo 3 −1 min −1), indicating that cyt bo 3 possesses a low level of NO reductase activity. The binding of NO to fast pulsed enzyme was also investigated. The results show that in the [NO] range used (1–14 μM) both fast and pulsed oxidised cyt bo 3 bind NO with a stoichiometry of 1:1 with an observed dissociation constant of K d =5.6±0.6 μM and that NO binding was inhibited by the presence of Cl −. The binding of nitrite to the binuclear centre causes spectral changes similar to those observed upon NO binding to fast cyt bo 3. These results are discussed in relation to the model proposed by Wilson and co-workers [FEBS Lett. 414 (1997) 281] where the binding of NO to Cu B II results in the formation of the nitrosonium (Cu B I–NO +) complex. NO + then reacts with OH −, a Cu B ligand, to form nitrite, which can bind at the binuclear centre. This work suggests for the first time that the binding of NO to oxidised cyt bo 3 does result in the reduction of Cu B.

Erythrocyte consumption of nitric oxide in presence and absence of plasma-based hemoglobin.

Experimental measurements have suggested a consumption rate of nitric oxide (NO) by red blood cells (RBCs) that is orders of magnitude smaller than that of an equivalent concentration of free hemoglobin in solution. This difference has been attributed to external diffusion limitations in the transport of NO from the plasma to the surface of the RBC or to resistance in the transport through the erythrocytic membrane. A detailed mathematical model is developed to quantify the resistance to NO transport around a single RBC and to predict the consumption rate in the presence and absence of extracellular hemoglobin. We provide a description for the NO consumption rate as a function of hematocrit, RBC radius, membrane permeability, and extracellular hemoglobin concentration. We predict a first-order rate constant for NO consumption in blood between 7.5 x 10(2) and 6.5 x 10(3) s(-1) at a hematocrit of 45% for membrane permeability values between 0.1 and 40 cm/s. Our results suggest that the difference in NO uptake by RBCs and free hemoglobin is smaller than previously reported and it is hematocrit dependent.

The heme-copper oxidases of Thermus thermophilus catalyze the reduction of nitric oxide: evolutionary implications.

We show that the heme-copper terminal oxidases of Thermus thermophilus (called ba(3) and caa(3)) are able to catalyze the reduction of nitric oxide (NO) to nitrous oxide (N(2)O) under reducing anaerobic conditions. The rate of NO consumption and N(2)O production were found to be linearly dependent on enzyme concentration, and activity was abolished by enzyme denaturation. Thus, contrary to the eukaryotic enzyme, both T. thermophilus oxidases display a NO reductase activity (3.0 +/- 0.7 mol NO/mol ba(3) x min and 32 +/- 8 mol NO/mol caa(3) x min at [NO] approximately 50 microM and 20 degrees C) that, though considerably lower than that of bona fide NO reductases (300-4,500 mol NO/mol enzyme x min), is definitely significant. We also show that for ba(3) oxidase, NO reduction is associated to oxidation of cytochrome b at a rate compatible with turnover, suggesting a mechanism consistent with the stoichiometry of the overall reaction. We propose that the NO reductase activity of T. thermophilus oxidases may depend on a peculiar Cu(B)(+) coordination, which may be revealed by the forthcoming three-dimensional structure. These findings support the hypothesis of a common phylogeny of aerobic respiration and bacterial denitrification, which was proposed on the basis of structural similarities between the Pseudomonas stutzeri NO reductase and the cbb(3) terminal oxidases. Our findings represent functional evidence in support of this hypothesis.

The reaction of nitric oxide with ubiquinol: kinetic properties and biological significance

The reaction of nitric oxide ( NO) with ubiquinol-0 and ubiquinol-2, short-chain analogs of coenzyme Q, was examined in anaerobic and aerobic conditions in terms of formation of intermediates and stable molecular products. The chemical reactivity of ubiquinol-0 and ubiquinol-2 towards NO differed only quantitatively, the reactions of ubiquinol-2 being slightly faster than those of ubiquinol-0. The ubiquinol/ NO reaction entailed oxidation of ubiquinol to ubiquinone and reduction of NO to NO −, the latter identified by its reaction with metmyoglobin to form nitroxylmyoglobin and indi rectly by measurement of nitrous oxide (N 2O) by gas chromatography. Both the rate of ubiquinone accumulation and NO consumption were linearly dependent on ubiquinol and NO concentrations. The stoichiometry of NO consumed per either ubiquinone formed or ubiquinol oxidized was 1.86 Å 0.34. The reaction of NO with ubiquinols proceeded with intermediate formation of ubisemiquinones that were detected by direct EPR. The second order rate constants of the reactions of ubiquinol-0 and ubiquinol-2 with NO were 0.49 and 1.6 × 10 4 M −1s −1, respectively. Studies in aerobic conditions revealed that the reaction of NO with ubiquinols was associated with O 2 consumption. The formation of oxyradicals — identified by spin trapping EPR— during ubiquinol autoxidation was inhibited by NO, thus indicating that the O 2 consumption triggered by NO could not be directly accounted for in terms of oxyradical formation or H 2O 2 accumulation. It is suggested that oxyradical formation is inhibited by the rapid removal of superoxide anion by NO to yield peroxynitrite, which subsequently may be involved in the propagation of ubiquinol oxidation. The biological significance of the reaction of ubiquinols with NO is discussed in terms of the cellular O 2 gradients, the steady-state levels of ubiquinols and NO, and the distribution of ubiquinone (largely in its reduced form) in biological membranes with emphasis on the inner mitochondrial membrane.

Estimation of nitric oxide production and reaction rates in tissue by use of a mathematical model.

Nitric oxide (NO) produced by the vascular endothelium is an important biologic messenger that regulates vessel tone and permeability and inhibits platelet adhesion and aggregation. NO exerts its control of vessel tone by interacting with guanylyl cyclase in the vascular smooth muscle to initiate a series of reactions that lead to vessel dilation. Previous efforts to investigate this interaction by mathematical modeling of NO diffusion and reaction have been hampered by the lack of information on the production and degradation rate of NO. We use a mathematical model and previously published experimental data to estimate the rate of NO production, 6.8 x 10(-14) micromol . micron-2 . s-1; the NO diffusion coefficient, 3,300 micron2 s-1; and the NO consumption rate coefficient in the vascular smooth muscle, 0.01 s-1 (1st-order rate expression) or 0.05 microM-1 . s-1 (2nd-order rate expression). The modeling approach is discussed in detail. It provides a general framework for modeling the NO produced from the endothelium and for estimating relevant physical parameters.

Simultaneous measurement of nitric oxide production and consumption in soil using a simple static incubation system, and the effect of soil water content on the contribution of nitrification

Nitric oxide (NO) compensation concentrations ( m c), NO uptake rate constants ( k) and NO production rates ( P) were determined in four different soils at three different soil moisture contents using serum bottles as a static incubation system. Two different approaches were compared. In the first approach the release of NO was measured until a constant NO concentration (= m c) was reached indicating that the NO production rate equalled the NO consumption rate so that the net NO release became zero. In a second approach NO was added to the bottles and the consumption of NO was measured until the compensation concentration was reached. In both cases, k was determined by curve fitting and P was calculated as the product of k times m c. Both approaches resulted in similar values of m c. However, values of k and P were often different. If the soils produced only small amounts of NO, the increase of NO was usually too small to allow a reliable curve fitting for calculation of k and P. Thus, the determination of k and P was more reliable when using the NO consumption approach. Nitrification was specifically inhibited in the presence of 10 Pa acetylene. The inhibition experiments showed that nitrification was the main NO-producing process in all soils except in an acidic forest soil. Increase of soil moisture contents from 30 to 60% maximum water-holding capacity resulted in a decrease of the contribution of nitrification to NO production.