A theoretical study of myoglobin working as a nitric oxide scavenger

The mechanism for the reaction between nitric oxide (NO) and O₂ bound to the heme iron of myoglobin (Mb), including the following isomerization to nitrate, has been investigated using hybrid density functional theory (B3LYP). Myoglobin working as a NO scavenger could be of importance, since NO reversibly inhibits the terminal enzyme in the respiration chain, cytochrome c oxidase. The concentration of NO in the cell will thus affect the respiration and thereby the synthesis of ATP. The calculations show that the reaction between NO and the heme-bound O₂ gives a peroxynitrite intermediate whose O–O bond undergoes a homolytic cleavage, forming a NO₂ radical and myoglobin in the oxo-ferryl state. The NO₂ radical then recombines with the oxo-ferryl, forming heme-bound nitrate. Nine different models have been used in the present study to examine the effect on the reaction both by the presence and the protonation state of the distal His64, and by the surroundings of the proximal His93. The barriers going from the oxy-Mb and nitric oxide reactant to the peroxynitrite intermediate and further to the oxo-ferryl and NO₂ radical are around 10 and 7 kcal/mol, respectively. Forming the product, nitrate bound to the heme iron has a barrier of less than ~7 kcal/mol. The overall reaction going from a free nitric oxide and oxy-Mb to the heme bound nitrate is exergonic by more than 30 kcal/mol.     

Implication of CO inactivation on myoglobin function

Myoglobin (Mb) has a purported role in facilitating O₂ diffusion in tissue, especially as cellular PO₂ drops or the respiration demand increases. Inhibiting Mb with CO under conditions that accentuate the facilitated diffusion role should then elicit a significant physiological response. In one set of experiments, the perfused myocardium received buffer with decreasing PO₂ (225, 129, and 64 mmHg). Intracellular PO₂ declined, as reflected in the ¹H NMR Val E11 signal of MbO₂ (67%, 32%, and 18%). The addition of 6% CO further reduced the available MbO₂ (11%, 9%, and 7%), as evidenced by the decline of the MbO₂ Val E11 signal intensity at –2.76 ppm. In a second set of experiments, electrical stimulation increased the heart rate (300, 450, and 540 beats/min) and correspondingly the O₂ consumption rate (MO₂). Intracellular PO₂ also declined, as reflected in the slight drop in the MbO₂ signal (100%, 96%, and 82%). MO₂ increased (100%, 114%, 165%). The addition of 3% CO in the stimulated hearts further decreased the available MbO₂ (46%, 44%, and 29%). In all cases, CO inactivation of Mb does not induce any change in the respiration rate, contractile function, and high-energy phosphate levels. Moreover, the MbCO/MbO₂ partition coefficient shifts dramatically from its in vitro value during hypoxia and increased work. The observation suggests a modulation of an intracellular O₂ gradient. Overall, the experimental observations provide no evidence of a facilitated diffusion role for Mb in perfused myocardium and implicate a physiologically responsive intracellular O₂ gradient. nuclear magnetic resonance; respiration; carbon monoxide; myocardium; oxidative phosphorylation     

Myoglobin: Oxygen Depot or Oxygen Transporter to Mitochondria? A Novel Mechanism of Myoglobin Deoxygenation in Cells (review)

In this review, we shortly summarize the data of our studies (and also corresponding studies of other authors) on the new mechanism of myoglobin (Mb) deoxygenation in a cell, according to which Mb acts as an oxygen transporter, and its affinity for the ligand, like in other transporting proteins, is regulated by the interaction with the target, in our case, mitochondria (Mch). We firstly found that contrary to previously formulated and commonly accepted concepts, oxymyoglobin (MbO₂) deoxygenation occurs only via interaction of the protein with respiring mitochondria (low \({p_{{O_2}}}\) values are necessary but not sufficient for this process to proceed). Detailed studies of the mechanism of Mb–Mch interaction by various physicochemical methods using natural and artificial bilayer phospholipid membranes showed that: (i) the rate of MbO₂ deoxygenation in the presence of respiring Mch fully coincides with the rate of O₂ uptake by mitochondria from a solution irrespectively of their state (native coupled, freshly frozen, or FCCP-uncoupled), i.e. it is determined by the respiratory activity of Mch; (ii) Mb nonspecifically binds to membrane phospholipids of the outer mitochondrial membrane, while any Mb-specific protein or phospholipid sites on it are lacking; (iii) oxygen uptake by Mch from a solution and the uptake of Mb-bound oxygen are two different processes, as their rates are differently affected by proteins (e.g. lysozyme) that compete with MbO₂ for binding to the mitochondrial membrane; (iv) electrostatic forces significantly contribute to the Mb–membrane interactions; the dependence of these interactions on ionic strength is provided by the local electrostatic interactions between anionic groups of phospholipids (the heads) and invariant Lys and Arg residues near the Mb heme pocket; (v) interactions of Mb with phospholipid membranes promote conformational changes in the protein, primarily in its heme pocket, without significant alterations in the protein secondary and tertiary structures; and (vi) Mb–membrane interactions lead to decrease in the affinity of myoglobin for O₂, which could be monitored by the increase in the MbO₂ autooxidation rate under aerobic conditions and under anaerobic ones, by the shift in the MbO₂/Mb(2) equilibrium towards the ligand-free protein. The decrease in the affinity of Mb for the ligand should facilitate O₂ dissociation from MbO₂ at physiological \({p_{{O_2}}}\) values in cells.     

Hemoglobin and myoglobin as reducing agents in biological systems. Redox reactions of globins with copper and iron salts and complexes

In addition to reversible O₂ binding, respiratory proteins of the globin family, hemoglobin (Hb) and myoglobin (Mb), participate in redox reactions with various metal complexes, including biologically significant ones, such as those of copper and iron. HbO₂ and MbO₂ are present in cells in large amounts and, as redox agents, can contribute to maintaining cell redox state and resisting oxidative stress. Divalent copper complexes with high redox potentials (E ₀, 200-600 mV) and high stability constants, such as [Cu(phen)₂]²⁺, [Cu(dmphen)₂]²⁺, and CuDTA oxidize ferrous heme proteins by the simple outer-sphere electron transfer mechanism through overlapping π-orbitals of the heme and the copper complex. Weaker oxidants, such as Cu²⁺, CuEDTA, CuNTA, CuCit, CuATP, and CuHis (E ₀≤ 100-150 mV) react with HbO₂ and MbO₂ through preliminary binding to the protein with substitution of the metal ligands with protein groups and subsequent intramolecular electron transfer in the complex (the site-specific outer-sphere electron transfer mechanism). Oxidation of HbO₂ and MbO₂ by potassium ferricyanide and Fe(3) complexes with NTA, EDTA, CDTA, ATP, 2,3-DPG, citrate, and pyrophosphate PP_i proceeds mainly through the simple outer-sphere electron transfer mechanism via the exposed heme edge. According to Marcus theory, the rate of this reaction correlates with the difference in redox potentials of the reagents and their self-exchange rates. For charged reagents, the reaction may be preceded by their nonspecific binding to the protein due to electrostatic interactions. The reactions of LbO₂ with carboxylate Fe complexes, unlike its reactions with ferricyanide, occur via the site-specific outer-sphere electron transfer mechanism, even though the same reagents oxidize structurally similar MbO₂ and cytochrome b ₅ via the simple outer-sphere electron transfer mechanism. Of particular biological interest is HbO₂ and MbO₂ transformation into met-forms in the presence of small amounts of metal ions or complexes (catalysis), which, until recently, had been demonstrated only for copper compounds with intermediate redox potentials. The main contribution to the reaction rate comes from copper binding to the “inner” histidines, His97 (0.66 nm from the heme) that forms a hydrogen bond with the heme propionate COO^– group, and the distal His64. The affinity of both histidines for copper is much lower than that of the surface histidines residues, and they are inaccessible for modification with chemical reagents. However, it was found recently that the high-potential Fe(3) complex, potassium ferricyanide (400 mV), at a 5 to 20% of molar protein concentration can be an efficient catalyst of MbO₂ oxidation into metMb. The catalytic process includes binding of ferrocyanide anion in the region of the His119 residue due to the presence there of a large positive local electrostatic potential and existence of a “pocket” formed by Lys16, Ala19, Asp20, and Arg118 that is sufficient to accommodate [Fe(CN)₆]^4–. Fast, proton-assisted reoxidation of the bound ferrocyanide by oxygen (which is required for completion of the catalytic cycle), unlike slow [Fe(CN)₆]^4– oxidation in solution, is provided by the optimal location of neighboring protonated His113 and His116, as it occurs in the enzyme active site.     

Oxygen consumption during leaf nitrate assimilation in a C3 and C4 plant: the role of mitochondrial respiration

Measurements of net fluxes of CO₂ and O₂ from leaves and chlorophyll a fluorescence were used to determine the role of mitochondrial respiration during nitrate (NO₃^–) assimilation in both a C₃ (wheat) and a C₄ (maize) plant. Changes in the assimilatory quotient (net CO₂ consumed over net O₂ evolved) when the nitrogen source was shifted from NO₃^– to NH₄⁺ (ΔAQ) provided a measure of shoot NO₃^– assimilation. According to this measure, elevated CO₂ inhibited NO₃^– assimilation in wheat but not maize. Net O₂ exchange under ambient CO₂ concentrations increased in wheat plants receiving NO₃^– instead of NH₄⁺, but gross O₂ evolution from the photosynthetic apparatus (J_O2) was insensitive to nitrogen source. Therefore, O₂ consumption within wheat photosynthetic tissue (ΔΟ₂), the difference between J_O2 and net O₂ exchange, decreased during NO₃^– assimilation. In maize, NO₃^– assimilation was insensitive to changes in intercellular CO₂ concentration (C_i); nonetheless, ΔΟ₂ at low C_i values was significantly higher in NO₃^–‐fed than in NH₄⁺‐fed plants. Changes in O₂ consumption during NO₃^– assimilation may involve one or more of the following processes: (a) Mehler ascorbate peroxidase (MAP) reactions; (b) photorespiration; or (c) mitochondrial respiration. The data presented here indicates that in wheat, the last process, mitochondrial respiration, is decreased during NO₃^– assimilation. In maize, NO₃^– assimilation appears to stimulate mitochondrial respiration when photosynthetic rates are limiting.     

Assessing the sources and magnitude of diurnal nitrate variability in the San Joaquin River (California) with an in situ optical nitrate sensor and dual nitrate isotopes

1. We investigated diurnal nitrate (NO₃⁻) concentration variability in the San Joaquin River using an in situ optical NO₃⁻ sensor and discrete sampling during a 5‐day summer period characterized by high algal productivity. Dual NO₃⁻ isotopes (δ¹⁵N_NO3 and δ¹⁸O_NO3) and dissolved oxygen isotopes (δ¹⁸O_DO) were measured over 2 days to assess NO₃⁻ sources and biogeochemical controls over diurnal time‐scales. 2. Concerted temporal patterns of dissolved oxygen (DO) concentrations and δ¹⁸O_DO were consistent with photosynthesis, respiration and atmospheric O₂ exchange, providing evidence of diurnal biological processes independent of river discharge. 3. Surface water NO₃⁻ concentrations varied by up to 22% over a single diurnal cycle and up to 31% over the 5‐day study, but did not reveal concerted diurnal patterns at a frequency comparable to DO concentrations. The decoupling of δ¹⁵N_NO3 and δ¹⁸O_NO3 isotopes suggests that algal assimilation and denitrification are not major processes controlling diurnal NO₃⁻ variability in the San Joaquin River during the study. The lack of a clear explanation for NO₃⁻ variability likely reflects a combination of riverine biological processes and time‐varying physical transport of NO₃⁻ from upstream agricultural drains to the mainstem San Joaquin River. 4. The application of an in situ optical NO₃⁻ sensor along with discrete samples provides a view into the fine temporal structure of hydrochemical data and may allow for greater accuracy in pollution assessment.     

A nitrogen dioxide delivery system for biological media

Nitrogen dioxide is formed endogenously via the oxidation of NO by O₂ or O₂⁻ and from NO₂⁻ via peroxidases, among other pathways. This radical has many potential biological targets and its concentration, like that of NO and other reactive nitrogen species, is thought to be elevated at sites of inflammation. To investigate the specific cytotoxic or mutagenic effects of NO₂, it is desirable to be able to maintain its concentration at constant, predictable, and physiological levels in cell cultures, in the absence of NO. To do this, a delivery system was constructed in which NO₂-containing gas mixtures contact a liquid within a small (110 ml) stirred reactor. In such gas mixtures NO₂ is present in equilibrium with its dimer, N₂O₄. The uptake of NO₂ and N₂O₄ was characterized by measuring the accumulation rates of NO₂⁻ and NO₃⁻, the stable products of N₂O₄ hydrolysis, in buffered aqueous solutions. In some experiments NO₂-reactive 2,2′-azino-bis(3-ethyl-benzothiazoline-6-sulfonate) (ABTS) was included and formation of the stable ABTS radical was measured. A reaction–diffusion model was developed that predicts the accumulation rates of all three products to within 15% for gas-phase concentrations of NO₂ spanning 3 orders of magnitude. The model also provides estimates for the NO₂ concentration in the liquid. This system should be useful for exposing cells to NO₂ concentrations similar to those in vivo.     

Oxidation of oxymyoglobin by nitric oxide through dissociation from cobalt nitrosyls

Kinetic evaluation of the oxidation of oxymyoglobin (MbO₂) to metmyoglobin (Mb⁺) by bis(dimethylglyoximato)cobalt nitrosyl [Co(NO)(DMGH)₂] has established that the mechanism of this transformation involves initial dissociation of nitric oxide from Co(NO)(DMGH)₂, followed by direct oxidation of MbO₂ by nitric oxide. Nitrate formation accompanies the production of Mb⁺ and is proposed to arise from isomerization of the initially formed peroxynitrite ion. By comparative kinetic determinations with nitrosyl transfer from the cobalt nitrosyl reagent to deoxyhemoglobin, the rate constant for oxidation of MbO₂ by nitric oxide is calculated to be 31 X 10⁶ M^?1sec^?1 at 10.0°C in phosphate-buffered media at pH 7.0. Bis(dimethylglyoximato)cobalt(II), the cobalt complex formed by nitric oxide dissociation from Co(NO)(DMGH)₂, is an effective trap for dioxygen liberated from MbO₂. The resulting μ-peroxo- or μ-superoxo-dicobaloxime(III) oxidizes deoxymyoglobin to metmyoglobin at a rate that is competitive with oxidation induced by Co(NO)(DMGH)₂.     

An integrated network analysis reveals that nitric oxide reductase prevents metabolic cycling of nitric oxide by Pseudomonas aeruginosa

Nitric oxide (NO) is a chemical weapon within the arsenal of immune cells, but is also generated endogenously by different bacteria. Pseudomonas aeruginosa are pathogens that contain an NO-generating nitrite (NO₂⁻) reductase (NirS), and NO has been shown to influence their virulence. Interestingly, P. aeruginosa also contain NO dioxygenase (Fhp) and nitrate (NO₃⁻) reductases, which together with NirS provide the potential for NO to be metabolically cycled (NO→NO₃⁻→NO₂⁻→NO). Deeper understanding of NO metabolism in P. aeruginosa will increase knowledge of its pathogenesis, and computational models have proven to be useful tools for the quantitative dissection of NO biochemical networks. Here we developed such a model for P. aeruginosa and confirmed its predictive accuracy with measurements of NO, O₂, NO₂⁻, and NO₃⁻ in mutant cultures devoid of Fhp or NorCB (NO reductase) activity. Using the model, we assessed whether NO was metabolically cycled in aerobic P. aeruginosa cultures. Calculated fluxes indicated a bottleneck at NO₃⁻, which was relieved upon O₂ depletion. As cell growth depleted dissolved O₂ levels, NO₃⁻ was converted to NO₂⁻ at near-stoichiometric levels, whereas NO₂⁻ consumption did not coincide with NO or NO₃⁻ accumulation. Assimilatory NO₂⁻ reductase (NirBD) or NorCB activity could have prevented NO cycling, and experiments with ΔnirB, ΔnirS, and ΔnorC showed that NorCB was responsible for loss of flux from the cycle. Collectively, this work provides a computational tool to analyze NO metabolism in P. aeruginosa, and establishes that P. aeruginosa use NorCB to prevent metabolic cycling of NO.     

Oxygen limitation of N2 fixation in stem-girdled and nitrate-treated soybean

The effects of increasing rhizosphere pO₂on nitrogenase activity and nodule resistance to O₂diffusion were investigated in soybean plants [Glycine max (L.) Merr. cv. Harosoy 63] in which nitrogenase (EC 1.7.99.2) activities were inhibited by (a) removal of the phloem tissue at the base of the stem (stem girdling), (b) exposure of roots to 10 mM NO₃over 5 days (NO₃-treated), or (c) partial inactivation of nitrogenase activity by an exposure of nodulated roots to 100 kPa O₂(O₂-inhibitcd). In control plants and in plants which had been treated with 100 kPa O₂, increasing rhizosphere O₂concentrations in 10 kPa increments from 20 to 70 kPa did not alter the steady-state nitrogenase activity. In contrast, in plants in which nitrogenase activities were depressed by stem girdling or by exposure to NO₃, increasing rhizosphere pO₂resulted in a recovery of 57 or 67%, respectively, of the initial, depressed rates of nitrogenase activity. This suggests that the nitrogenase activity of stem-girdled and NO₃-treated soybeans was O₂-limited. For each treatment, theoretical resistance values for O₂diffusion into nodules were estimated from measured rates of CO₂exchange, assuming a respiratory quotient of 1.1 and 0 kPa of O₂in the infected cells. At an external partial pressure of 20 kPa O₂, the stem-girdled and NO₃^--treated plants displayed resistance values which were 4 to 8.6 times higher than those in the nodules of the control plants. In control and O₂-inhibited plants, increases in pO₂from 20 to 70 kPa in 10 kPa increments resulted in a 2.5- to 3.9-fold increase in diffusion resistance to O₂, and had little effect on either respiration or nitrogenase activity. In contrast, in stem-girdled and NO₃^--treated plants, increases in external pO₂had little effect on diffusion resistance to O₂, but resulted in a 2.3- to 3.2-fold increase in nodule respiration and nitrogenase activity. These results are consistent with stem-girdling and NO₃^--inhibition treatments limiting phloem supply to nodules causing an increase in diffusion resistance to O₂at 20 kPa and an apparent insensitivity of diffusion resistance to increases in external pO₂.     

Diagnostic parameters for selecting against novel spruce (Picea abies) decline: II. Response of photosynthesis and transpiration to acute NOx exposures

The dose- and time-response effects of sequential 3 h+3 h NO→NO₂ day time exposures [0–9 μl l^?1 (ppm) NO, 0–7.5 μl l^?1 NO₂] followed by 3 h+3 h NO→NO₂ night-time exposures (0–9.5 μl l^?1 NO, 0–9 μl l^?1 NO₂) on photosynthesis, transpiration and dark respiration were examined for nine Carpatho-Ukrainian (‘Rachovo’) half-sib families and for two populations, one from the FRG (‘Westerhof’) and one from the GDR (‘Schmiedefeld’) of Norway spruce [Picea abies (L.) Karst.], all in their 4th growing season. In a second exposure series the exposure sequence was reversed. None of the treatments induced needle scorching. The higher NO_x (NO or NO₂) concentrations reduced photosynthesis and transpiration within 1 h. The physiology of the different spruce types was affected significantly differently, the most sensitive spruce having its photosynthesis suppressed 6.6 times and its transpiration 5.5 times more than the most tolerant. ‘Westerhof’ was more sensitive to NO₂ than the average ‘Rachovo’ half-sibs. The gradients of different photosynthesis and transpiration sensitivities among the half-sibs (and ‘Westerhof’) demonstrated a significant, positive, mutual correlation, but significant negative correlations with the gradient of novel decline symptoms among their parents growing in Danish forests. The relative photosynthesis and transpiration sensitivies may thus serve as diagnostic parameters for laboratory selection of the most resistant trees to novel spruce decline. The average NO₂ flux density was three times larger than the average NO flux density. Only for NO₂ and in light was stomatal NO_x uptake larger than the total NO_x uptake. Both night transpiration and dark respiration were stimulated by high concentrations of night NO_x, preceded by day NO_x exposures.     

Enzymatic reduction of iron oxide by fungi

The occurrence of the iron-reducing phenomenon among some common fungi was studied. Results indicated that (i) the reduction of ferric iron to the ferrous state by fungi seems to be restricted to nitrate reductase-inducible strains such as Actinomucor repens, Alternaria tenuis, Fusarium oxysporum, and F. solani and (ii) the amount of dissolved ferrous iron may be reduced progressively by increasing the amount of nitrate added to the medium. Compared with a complex medium (Sabouraud medium), less iron became reduced if NO₃^- was the only nitrogen source (Czapek Dox medium). These data strongly support the view that ferric iron is acting as an hydrogen acceptor in respiration, competing with nitrate for electrons that are mediated by the enzyme nitrate reductase. The significance of this property from an ecological viewpoint is discussed.     

In Vitro Nonenzymatic Glycation Enhances the Role of Myoglobin as a Source of Oxidative Stress

Metmyoglobin (Mb) was glycated by glucose in a nonenzymatic in vitro reaction. Amount of iron release from the heme pocket of myoglobin was found to be directly related with the extent of glycation. After in vitro glycation, the unchanged Mb and glycated myoglobin (GMb) were separated by ion exchange (BioRex 70) chromatography, which eliminated free iron from the protein fractions. Separated fractions of Mb and GMb were converted to their oxy forms -MbO² and GMbO², respectively. H²O²-induced iron release was significantly higher from GMbO² than that from MbO². This free iron, acting as a Fenton reagent, might produce free radicals and degrade different cell constituents. To verify this possibility, degradation of different cell constituents catalyzed by these fractions in the presence of H²O² was studied. GMbO² degraded arachidonic acid, deoxyribose and plasmid DNA more efficiently than MbO². Arachidonic acid peroxidation and deoxyribose degradation were significantly inhibited by desferrioxamine (DFO), mannitol and catalase. However, besides free iron-mediated free radical reactions, role of iron of higher oxidation states, formed during interaction of H²O² with myoglobin might also be involved in oxidative degradation processes. Formation of carbonyl content, an index of oxidative stress, was higher by GMbO². Compared to MbO², GMbO² was rapidly auto-oxidized and co-oxidized with nitroblue tetrazolium, indicating increased rate of Mb and superoxide radical formation in GMbO². GMb exhibited more peroxidase activity than Mb, which was positively correlated with ferrylmyoglobin formation in the presence of H²O². These findings correlate glycation-induced modification of myoglobin and a mechanism of increased formation of free radicals. Although myoglobin glycation is not significant within muscle cells, free myoglobin in circulation, if becomes glycated, may pose a serious threat by eliciting oxidative stress, particularly in diabetic patients.     

Role of nitrification and denitrification for NO metabolism in soil

Release and uptake of NO was measured in a slightly alkaline (pH 7.8) and an acidic (pH 4.7) cambisol. In the alkaline soil under aerobic conditions, NO release was stimulated by ammonium and inhibited by nitrapyrin. Nitrate accumulated simultaneously and was also inhibited by nitrapyrin.¹⁵NO was released after fertilization with¹⁵NH₄NO₃ but not with NH₄ ¹⁵NO₃. The results indicate that in aerobic alkaline cambisol NO was mainly produced during nitrification of ammonium. The results were different under anaerobic conditions and also in the acidic cambisol. There, NO release was stimulated by nitrate and not by ammonium, and was inhibited by chlorate and not by nitrapyrin indicating that NO production was exclusively due to reduction of nitrate. The results were confirmed by¹⁵NO being released mainly from NH₄ ¹⁵NO₃ rather than from¹⁵NH₄NO₃. The observed patterns of NO release were explained by the NO production processes being stimulated by either ammonium or nitrate in the two different soils, whereas the NO consumption processes being only stimulated by nitrate. NO release was larger than N₂O release, but both were small compared to changes in concentrations of soil ammonium or nitrate.(*request for offprints)     

Interaction of benthic microalgae and macrofauna in the control of benthic metabolism, nutrient fluxes and denitrification in a shallow sub-tropical coastal embayment (western Moreton Bay, Australia)

Benthic biogeochemistry and macrofauna were investigated six times over 1 year in a shallow sub-tropical embayment. Benthic fluxes of oxygen (annual mean ?918 μmol O₂ m^?2 h^?1), ammonium (NH₄ ⁺), nitrate (NO₃ ^?), dissolved organic nitrogen, dinitrogen gas (N₂), and dissolved inorganic phosphorus were positively related to OM supply (N mineralisation) and inversely related to benthic light (N assimilation). Ammonium (NH₄ ⁺), NO₃ ^? and N₂ fluxes (annual means +14.6, +15.9 and 44.6 μmol N m^?2 h^?1) accounted for 14, 16 and 53 % of the annual benthic N remineralisation respectively. Denitrification was dominated by coupled nitrification–denitrification throughout the study. Potential assimilation of nitrogen by benthic microalgae (BMA) accounted for between 1 and 30 % of remineralised N, and was greatest during winter when bottom light was higher. Macrofauna biomass tended to be highest at intermediate benthic respiration rates (?1,000 μmol O₂ m^?2 h^?1), and appeared to become limited as respiration increased above this point. While bioturbation did not significantly affect net fluxes, macrofauna biomass was correlated with increased light rates of NH₄ ⁺ flux which may have masked reductions in NH₄ ⁺ flux associated with BMA assimilation during the light. Peaks in net N₂ fluxes at intermediate respiration rates are suggested to be associated with the stimulation of potential denitrification sites due to bioturbation by burrowing macrofauna. NO₃ ^? fluxes suggest that nitrification was not significantly limited within respiration range measured during this study, however comparisons with other parts of Moreton Bay suggest that limitation of coupled nitrification–denitrification may occur in sub-tropical systems at respiration rates exceeding ?1,500 μmol O₂ m^?2 h^?1.     

Myoglobin functions in the heart

The physiological role of myoglobin (Mb) within the heart depends on its oxygenation state. The myocardium exhibits a broad oxygen partial pressure (pO₂) spectrum with a transmural gradient from the epicardial to the subendocardial layer, ranging from arterial values to an average of 19.3 mm Hg down to 0 mm Hg. The function of Mb as an O₂ storage depot is well appreciated, especially during systolic compression. In addition, Mb controls myocardial nitric oxide (NO) homeostasis and thus modulates mitochondrial respiration under physiological and pathological conditions. We recently discovered the role of Mb as a myocardial O₂ sensor; in its oxygenated state Mb scavenges NO, protecting the heart from the deleterious effects of excessive NO. Under hypoxia, however, deoxygenated Mb changes its role from an NO scavenger to an NO producer. The NO produced protects the cell from short phases of hypoxia and from myocardial ischemia/reperfusion injury. In this review we summarize the traditional and novel aspects of Mb and its (patho)physiological role in the heart.     

Exchange between interstitial and surface water: Implications for stream metabolism and nutrient cycling

Metabolism of a Sonoran Desert stream was investigated by both enclosure and whole system oxygen techniques. We used recirculating chambers to estimate surface sediment metabolism and measured deep sediment respiration in isolated sediment cores. Metabolism of the stream ecosystem was determined for a 30-m reach as dark and light oxygen change with and without black plastic sheeting that darkened the stream and prevented diffusion. Average ecosystem respiration for two dates in August (440 mg O₂ m^-2 h^-1) exceeded respiration of either the surface sediment community (155 Mg O₂ m^-2 h^-1) or the hyporheic community (170 mg O₂ m^-2 h^-1) alone. Deep sediments show substantial oxygen and nitrate uptake when isolated. In the stream, this low nitrate interstitial water is exchanged with surface water. Metabolism of the isolated surface community suggests a highly productive and autotrophic system, yet gross production is balanced or exceeded by community respiration when ecosystem boundaries include the hyporheic zone. Thus, despite high rates of gross primary production (600–1200 mg O₂ m^-2 h^-1), desert streams may be heterotrophic (P_G < R) during summer.     

Catalase catalyzes nitrotyrosine formation from sodium azide and hydrogen peroxide

Sodium azide (NaN₃) is known as an inhibitor of catalase, and a nitric oxide (NO) donor in the presence of catalase and H₂O₂. We showed here that catalase-catalyzed oxidation of NaN₃ can generate reactive nitrogen species which contribute to tyrosine nitration in the presence of H₂O₂. The formation of free-tyrosine nitration and protein-bound tyrosine nitration by the NaN₃/catalase/H₂O₂ system showed a maximum level at pH 6.0. Free-tyrosine nitration induced by peroxynitrite was inhibited by ethanol and dimethyl-sulfoxide (DMSO), and augmented by superoxide dismutase (SOD). However, free-tyrosine nitration induced by the NaN₃/catalase/H₂O₂ system was not affected by ethanol, DMSO and SOD. NO^-₂ and NO donating agents did not affect free-tyrosine nitration by the NaN₃/catalase/H₂O₂ system. The reaction of NaN₃ with hydroxyl radical generating system showed free-tyrosine nitration, but no formation of nitrite and nitrate. The generation of nitrite (NO^-₂) and nitrate (NO^-₃) by the NaN₃/catalase/H₂O₂ system was maximal at pH 5.0. These results suggested that the oxidation of NaN₃ by the catalase/H₂O₂ system generates unknown peroxynitrite-like reactive nitrogen intermediates, which contribute to tyrosine nitration.     

Nitrate photo-assimilation in tomato leaves under short-term exposure to elevated carbon dioxide and low oxygen

The role of photorespiration in the foliar assimilation of nitrate (NO₃^–) and carbon dioxide (CO₂) was investigated by measuring net CO₂ assimilation, net oxygen (O₂) evolution, and chlorophyll fluorescence in tomato leaves (Lycopersicon esculentum). The plants were grown under ambient CO₂ with ammonium nitrate (NH₄NO₃) as the nitrogen source, and then exposed to a CO₂ concentration of either 360 or 700 µmol mol^?1, an O₂ concentration of 21 or 2%, and either NO₃^– or NH₄⁺ as the sole nitrogen source. The elevated CO₂ concentration stimulated net CO₂ assimilation under 21% O₂ for both nitrogen treatments, but not under 2% O₂. Under ambient CO₂ and O₂ conditions (i.e. 360 µmol mol^?1 CO₂, 21% O₂), plants that received NO₃^– had 11–13% higher rates of net O₂ evolution and electron transport rate (estimated from chlorophyll fluorescence) than plants that received NH₄⁺. Differences in net O₂ evolution and electron transport rate due to the nitrogen source were not observed at the elevated CO₂ concentration for the 21% O₂ treatment or at either CO₂ level for the 2% O₂ treatment. The assimilatory quotient (AQ) from gas exchange, the ratio of net CO₂ assimilation to net O₂ evolution, indicated more NO₃^– assimilation under ambient CO₂ and O₂ conditions than under the other treatments. When the AQ was derived from gross O₂ evolution rates estimated from chlorophyll fluorescence, no differences could be detected between the nitrogen treatments. The results suggest that short‐term exposure to elevated atmospheric CO₂ decreases NO₃^– assimilation in tomato, and that photorespiration may help to support NO₃^– assimilation.     

Stimulation of bull seminal Ca2+, Mg2+-dependent endonuclease by various DNA-binding proteins

We have measured ΔA transient absorption spectra in the Soret region and kinetics of photodissociation of oxymyoglobin (MbO₂) solutions following excitation by pulses of duration 350 fsec and 10 μJ energy at 307 nm. We observed an instantaneous bleaching of the absorbance at 414 nm and the appearance of a broad, red-shifted absorption band in the 438–470 nm region with a time constant of 250 fsec indicative of the formation of a short-lived deliganded Mb^★ species which relaxes to the stable Mb with a constant of 3.5 psec. Following this early relaxation, changes in absorption kinetics indicate also a geminate recombination process of constant τ = 100 psec. These data demonstrate that the well established low quantum yield (φ = 0.03) of photodissociation in MbO₂ is related both to the relaxation of an excited Mb^★ state and to a fast geminate recombination process.