Control of cytochrome c oxidase activity by nitric oxide

Over the past decade it was discovered that, over-and-above multiple regulatory functions, nitric oxide (NO) is responsible for the modulation of cell respiration by inhibiting cytochrome c oxidase (CcOX). As assessed at different integration levels (from the purified enzyme in detergent solution to intact cells), CcOX can react with NO following two alternative reaction pathways, both leading to an effective, fully reversible inhibition of respiration. A crucial finding is that the rate of electron flux through the respiratory chain controls the mechanism of inhibition by NO, leading to either a "nitrosyl" or a "nitrite" derivative. The two mechanisms can be discriminated on the basis of the differential photosensitivity of the inhibited state. Of relevance to cell pathophysiology, the pathway involving the nitrite derivative leads to oxidative degradation of NO, thereby protecting the cell from NO toxicity. The aim of this work is to review the information available on these two mechanisms of inhibition of respiration.     

Crp‐dependent cytochrome bd oxidase confers nitrite resistance to Shewanella oneidensis

Shewanella oneidensis is able to respire on a variety of organic and inorganic substrates, including nitrate and nitrite. Conversion of nitrate to nitrite and nitrite to ammonium is catalysed by periplasmic nitrate and nitrite reductases (NAP and NRF) respectively. Global regulator Crp (c yclic AMP r eceptor p rotein) is essential for growth of S. oneidensis on both nitrate and nitrite. In this study, we discovered that crp mutants are not only severely deficient in nitrate or nitrite respiration, but are also hypersensitive to nitrite. This hypersusceptibility phenotype is independent of nitrite respiration. Using random transposon mutagenesis, we obtained 73 Δcrp suppressor strains resistant to nitrite. Transposon insertion sites in 24 suppressor strains were exclusively mapped in the region upstream of the cyd operon encoding a cytochrome bd oxidase, resulting in expression of the operon now driven by a Crp‐independent promoter. Further investigation indicated that the promoter in suppressor strains comes from the transposon. Mutational analysis of the cydB gene (encoding the essential subunit II of the bd oxidase) confirmed that the cytochrome bd oxidase confers nitrite resistance to S. oneidensis.     

Polarographische Messung der Nitritreduktion von Chlorella im monochromatischen Licht

Summary In green plant cells nitrite is reduced by two systems, one dependent on photosynthesis and the other upon respiration. Using a polarographic method for continuous measurement of nitrite uptake, the relationship between light driven and respiration linked nitrite reduction of Chlorella cells was studied.Photosynthetic nitrite reduction is characterized by a pronounced increase in the velocity of nitrite uptake upon illumination. After the light is turned off the velocity immediately returns to the preillumination value. Photosynthetic nitrite reduction of Chlorella is separated from respiration linked nitrite reduction by illumination with red light under anaerobic conditions; it is stimulated by CO₂ and is inhibited by DCMU, findings which confirm earlier observations.In white light a special blue light stimulation of nitrite uptake is overlapped by photosynthetic nitrite reduction. In contrast to photosynthetic nitrite reduction this type of light stimulation is characterized by a lag period of about I min from the onset of illumination; it continues about 10 min when the light is turned off. It is separated from photosynthetic nitrite reduction by irradiation of the algae with low intensities of short wavelength light (<500 nm). Blue light stimulation of nitrite uptake of Chlorella is strongly dependent on the developmental stage of the cells. It is observed with young cells (autospores) of synchronized algae only.There is no evidence for any connection between blue light stimulation of nitrite uptake and photosynthesis. From the sensitivity of this process towards anaerobic conditions and antimycin A it is concluded to be a stimulation of respiration linked nitrite reduction.Under conditions of low exogenous nitrite concentration a temporary inhibition of steady state dark nitrite reduction appears immediately after the light is turned off. From several observations it is concluded that the inhibition already exists during the preceding illumination and decreases the rate of total nitrite uptake in the light. This process is suppressed by inhibition of respiration as well as by the inhibitor of photosynthesis, DCMU.If nitrate is the source of nitrogen an excretion of nitrite is found following illumination. The kinetics of this process agree with those observed for the light induced inhibition of steady state dark nitrite reduction immediately after illumination.     

Reactivity of Nitric Oxide with Cytochrome c Oxidase: Interactions with the Binuclear Centre and Mechanism of Inhibition

Nitric oxide (NO) has recently been recognized as an important biological mediator that inhibits respiration at cytochrome c oxidase (CcO). This inhibition is reversible and shows competition with oxygen, the K _i being lower at low oxygen concentrations. Although the species that binds NO in turnover has been suggested to contain a partially reduced binuclear center, the exact mechanism of the inhibition is not clear. Recently, rapid (ms) redox reactions of NO with the binuclear center have been reported, e.g., the ejection of an electron to cytochrome a and the depletion of the intermediates P and F. These observations have been rationalized within a scheme in which NO reacts with oxidized Cu_B leading to the reduction of this metal center and formation of nitrite in a very fast reaction. Electron migration from Cu_B to other redox sites within the enzyme is proposed to explain the optical transitions observed. The relevance of these reactions to the inhibition of CcO and metabolism of NO are discussed.     

Inhibition of peroxisomal fatty acyl-CoA oxidase by antimycin A.

Peroxisomal fatty acyl-CoA oxidase was inhibited by micromolar concentrations of antimycin A, an inhibitor of mitochondrial respiration. The inhibition was observed with all three substrates tested, i.e. palmitoyl-CoA, trihydroxycoprostanoyl-CoA and hexadecanedioyl-CoA. The peroxisomal D-amino acid oxidase was also inhibited by antimycin, but the peroxisomal L-alpha-hydroxyacid oxidase and uric acid oxidase and the mitochondrial monoamine oxidase were not. The degree of inhibition of acyl-CoA oxidase by antimycin was strongly dependent on the amount of cellular protein present in the assay mixture: at a fixed antimycin concentration, the inhibition was gradually lost with increasing protein concentrations. At a fixed cellular protein concentration in the assay mixtures, the mitochondrial oxidation of glutamate or palmitoylcarnitine was inhibited at antimycin concentrations that were much lower than those required for the inhibition of fatty acyl-CoA oxidase. Our results, nevertheless, demonstrate that antimycin A must be used with caution, when it is added to homogenates or subcellular fractions in order to distinguish between mitochondrial and peroxisomal fatty acid oxidation.     

Terminal oxidases of Crithidia oncopelti

Abstract Washed cell suspensions of Crithidia oncopelti oxidizing a variety of substrates gave complex plots for the inhibition of respiration by potassium cyanide or azide. The data indicated the presence of at least two and possibly three terminal oxidases on the basis of their differential sensitivity to these inhibitors. The oxidase most sensitive to cyanide, azide and CO accounted for approx. 65–70% of whole cell respiration and is probably cytochrome oxidase a/a₃. A second oxidase exhibiting low affinity for CO required high concentrations of KCN or azide for inhibition. This haemoprotein had the spectral characteristics of cytochrome o and accounted for 15–20% of cell respiration. Incomplete inhibition of respiration by high concentrations of KCN or azide suggested the presence of a third oxidase which was CO-unreactive.     

Oxygen regulation of nitrate uptake in denitrifying Pseudomonas aeruginosa

Oxygen had an immediate and reversible inhibitory effect on nitrate respiration by denitrifying cultures of Pseudomonas aeruginosa. Inhibition of nitrate utilization by oxygen appeared to be at the level of nitrate uptake, since nitrate reduction to nitrite in cell extracts was not affected by oxygen. The degree of oxygen inhibition was dependent on the concentration of oxygen, and increasing nitrate concentrations could not overcome the inhibition. The inhibitory effect of oxygen was maximal at approximately 0.2% oxygen saturation. The inhibition appeared to be specific for nitrate uptake. Nitrite uptake was not affected by these low levels of aeration, and nitrite reduction was only partially inhibited in the presence of oxygen. The regulation of nitrate respiration at the level of transport by oxygen may represent a major mechanism by which the entire denitrification pathway is regulated in P. aeruginosa.     

Oxygen regulation of nitrate uptake in denitrifying Pseudomonas aeruginosa.

Oxygen had an immediate and reversible inhibitory effect on nitrate respiration by denitrifying cultures of Pseudomonas aeruginosa. Inhibition of nitrate utilization by oxygen appeared to be at the level of nitrate uptake, since nitrate reduction to nitrite in cell extracts was not affected by oxygen. The degree of oxygen inhibition was dependent on the concentration of oxygen, and increasing nitrate concentrations could not overcome the inhibition. The inhibitory effect of oxygen was maximal at approximately 0.2% oxygen saturation. The inhibition appeared to be specific for nitrate uptake. Nitrite uptake was not affected by these low levels of aeration, and nitrite reduction was only partially inhibited in the presence of oxygen. The regulation of nitrate respiration at the level of transport by oxygen may represent a major mechanism by which the entire denitrification pathway is regulated in P. aeruginosa.     

Protease inhibitors block the macrophage-mediated inhibition of tumor cell mitochondrial respiration

The antitumor activity of activated macrophages toward tumor cells, in vitro, appears to involve the production of toxic nitrogen intermediates. These intermediates, particularly nitric oxide, have been shown to cause the inhibition of cell division and to decrease cellular respiration by inhibiting electron transport. We studied the effects of proteolytic inhibitors on macrophage-mediated inhibition of L1210 tumor cell respiration and DNA synthesis, and found that chloromethyl ketone derivatives, which covalently modify serine proteases, can block macrophage cytotoxicity. Furthermore, these inhibitors decrease nitrite production by activated macrophages suggesting that the mechanism of action involves the inhibition of nitric oxide production.     

Investigations into the Mechanism of Action of a Novel Nitric Oxide Generator on Cellular Respiration

Abstract: Nitric oxide may regulate cellular respiration by competition with oxygen at mitochondrial cytochrome oxidase. Using an astrocyte-derived cell line, we have compared the mechanism of action of the nitric oxide-generating compound Roussin's black salt with that of sodium nitroprusside on cellular oxygen consumption. Intense light exposure induced the release of large quantities of nitric oxide from both of the donor compounds. However, in room light only Roussin's black salt generated low levels of the radical. Simultaneous measurement of oxygen consumption and of nitric oxide production demonstrated that sodium nitroprusside only had inhibitory actions when exposed to intense light (nitric oxide release), whereas Roussin's black salt had inhibitory actions in room light. Extracellular haemoglobin did not prevent the inhibition of respiration rate induced by Roussin's black salt even though stimulation of nitric oxide release on light exposure was markedly reduced. Preincubation of cells with Roussin's black salt and subsequent measurement of levels of light-liberated nitric oxide demonstrated that the compound was rapidly internalised. The uptake of sodium nitroprusside was minimal. These data suggest that, in contrast to sodium nitroprusside, the cellular internalisation of Roussin's black salt allows site-directed nitric oxide release and very effective inhibition of cellular respiration.     

Impacts of Nitrate and Nitrite on Physiology of Shewanella oneidensis

Shewanella oneidensis exhibits a remarkable versatility in anaerobic respiration, which largely relies on its diverse respiratory pathways. Some of these are expressed in response to the existence of their corresponding electron acceptors (EAs) under aerobic conditions. However, little is known about respiration and the impact of non-oxygen EAs on the physiology of the microorganism when oxygen is present. Here we undertook a study to elucidate the basis for nitrate and nitrite inhibition of growth under aerobic conditions. We discovered that nitrate in the form of NaNO₃ exerts its inhibitory effects as a precursor to nitrite at low concentrations and as an osmotic-stress provider (Na⁺) at high concentrations. In contrast, nitrite is extremely toxic, with 25 mM abolishing growth completely. We subsequently found that oxygen represses utilization of all EAs but nitrate. To order to utilize EAs with less positive redox potential, such as nitrite and fumarate, S. oneidensis must enter the stationary phase, when oxygen respiration becomes unfavorable. In addition, we demonstrated that during aerobic respiration the cytochrome bd oxidase confers S. oneidensis resistance to nitrite, which likely functions via nitric oxide (NO).     

Effects of Pesticides on Nitrite Oxidation by Nitrobacter agilis

The influence of pesticides on the growth of Nitrobacter agilis in aerated cultures and on the respiration of N. agilis cell suspensions and cell-free extracts was studied. Two pesticides, aldrin and simazine, were not inhibitory to growth of Nitrobacter, but five compounds [isopropyl N-(3-chlorophenyl) carbamate (CIPC), chlordane, 1,1-dichloro-2,2-bis (p-chlorophenyl) ethane (DDD), heptachlor, and lindane] prevented growth when added to the medium at a concentration of 10 mug/ml. Whereas CIPC and eptam prevented nitrite oxidation by cell suspensions, the addition of DDD and lindane resulted in only partial inhibition of the oxidation. Heptachlor and chlordane also caused only partial inhibition of oxidation, but were more toxic with cell-free extract nitrite oxidase. None of the pesticides inhibited the nitrate reductase activity of cell-free extracts, but most caused some repression of cytochrome c oxidase activity. Heptachlor was the most deleterious compound.     

Effect of organophosphorus insecticides on the oxidative processes in rat brain synaptosomes

Abstract: This paper describes the effect of four organophosphorus insecticides: Dipterex, DDVP, Ronnel and its oxygen analogue on the respiration of rat brain synaptosomes. Dipterex and DDVP in the concentrations used, 5, 50, or 500 μM, did not change the rate of oxygen uptake and oxidative phosphorylation in rat brain synaptosomes. Ronnel in the highest concentration (500 μM) inhibited respiration in state 3 conditions and abolished respiratory control by ADP. This inhibition was correlated with a change of cytochrome c oxidase activity. The oxygen analogue of Ronnel (OAR) in micromolar concentrations (50 μM) increased the rate of respiration of synaptosomes utilizing glutamate plus malate as substrate. Higher concentrations of OAR produced inhibition of respiration, cytochrome c oxidase and NADH: cytochrome c reductase activities. These observations are typical for uncouplers of oxidative phosphorylation. Noteworthy is the fact that the uncoupling activity of OAR was observed at concentrations which did not inhibit acetylcholinesterase activity. These findings seem to suggest that disturbances in oxidative processes could play an important role in the toxicity of organophosphorus insecticides. The relation between chemical structure and the ability of insecticides to affect oxidative phosphorylation is discussed.     

Nitrite reduction: a ubiquitous function from a pre‐aerobic past

In eukaryotes, small amounts of nitrite confer cytoprotection against ischemia/reperfusion‐related tissue damage in vivo, possibly via reduction to nitric oxide (NO) and inhibition of mitochondrial function. Several hemeproteins are involved in this protective mechanism, starting with deoxyhemoglobin, which is capable of reducing nitrite. In facultative aerobic bacteria, such as Pseudomonas aeruginosa, nitrite is reduced to NO by specialized heme‐containing enzymes called cd₁ nitrite reductases. The details of their catalytic mechanism are summarized below, together with a hypothesis on the biological role of the unusual d₁‐heme, which, in the reduced state, shows unique properties (very high affinity for nitrite and exceptionally fast dissociation of NO). Our results support the idea that the nitrite‐based reactions of contemporary eukaryotes are a vestige of earlier bacterial biochemical pathways. The evidence that nitrite reductase activities of enzymes with different cellular roles and biochemical features still exist today highlights the importance of nitrite in cellular homeostasis.     

Regulation of mitochondrial respiration by nitric oxide inhibition of cytochrome c oxidase

Nitric oxide (NO) and its derivatives inhibit mitochondrial respiration by a variety of means. Nanomolar concentrations of NO immediately, specifically and reversibly inhibit cytochrome oxidase in competition with oxygen, in isolated cytochrome oxidase, mitochondria, nerve terminals, cultured cells and tissues. Higher concentrations of NO and its derivatives (peroxynitrite, nitrogen dioxide or nitrosothiols) can cause irreversible inhibition of the respiratory chain, uncoupling, permeability transition, and/or cell death. Isolated mitochondria, cultured cells, isolated tissues and animals in vivo display respiratory inhibition by endogenously produced NO from constitutive isoforms of NO synthase (NOS), which may be largely mediated by NO inhibition of cytochrome oxidase. Cultured cells expressing the inducible isoform of NOS (iNOS) can acutely and reversibly inhibit their own cellular respiration and that of co-incubated cells due to NO inhibition of cytochrome oxidase, but after longer-term incubation result in irreversible inhibition of cellular respiration due to NO or its derivatives. Thus the NO inhibition of cytochrome oxidase may be involved in the physiological and/or pathological regulation of respiration rate, and its affinity for oxygen.     

Carbon monoxide sensitivity of cytochrome oxidase in wheat leaves in relation to dwarfing genes (Rht) in wheat cultivars

Summary Leaves of young seedlings of a number of tall cultivars of wheat, lacking the dwarfing Rht genes, readily responded to a brief 2 min exposure to CO, as assessed by in vivo aerobic assay of nitrate reductase. This test depends on the inhibition of cytochrome c oxidase by CO, which in turn renders cytosolic NADH available for the reduction of nitrate to nitrite in vivo. Semi-dwarf cultivars of wheat (Rht present) did not respond to CO in this way. Since CO forms a complex only with reduced cytochrome a₃, the results indicate differences in the redox state of cytochrome a₃, during in situ respiration of leaves from tall and semi-dwarf plants which are likely to be under genetic control.     

Direct Inhibition of Plant Mitochondrial Respiration by Elevated CO2

Doubling the concentration of atmospheric CO2 often inhibits plant respiration, but the mechanistic basis of this effect is unknown. We investigated the direct effects of increasing the concentration of CO2 by 360 [mu]L L-1 above ambient on O2 uptake in isolated mitochondria from soybean (Glycine max L. cv Ransom) cotyledons. Increasing the CO2 concentration inhibited the oxidation of succinate, external NADH, and succinate and external NADH combined. The inhibition was greater when mitochondria were preincubated for 10 min in the presence of the elevated CO2 concentration prior to the measurement of O2 uptake. Elevated CO2 concentration inhibited the salicylhydroxamic acid-resistant cytochrome pathway, but had no direct effect on the cyanide-resistant alternative pathway. We also investigated the direct effects of elevated CO2 concentration on the activities of cytochrome c oxidase and succinate dehydrogenase (SDH) and found that the activity of both enzymes was inhibited. The kinetics of inhibition of cytochrome c oxidase were time-dependent. The level of SDH inhibition depended on the concentration of succinate in the reaction mixture. Direct inhibition of respiration by elevated CO2 in plants and intact tissues may be due at least in part to the inhibition of cytochrome c oxidase and SDH.     

Kinetic studies on autohydrogenotrophic growth of <Emphasis Type="Italic">Ralstonia eutropha</Emphasis> with nitrate as terminal electron acceptor

Autohydrogenotrophic batch growth of Ralstonia eutropha H16 was studied in a stirred-tank reactor with nitrate and nitrite as terminal electron acceptors and the sole limiting substrates. Assuming product inhibition by nitrite, saturation kinetics with the two limiting substrates and a simple switching function, which allows growth on nitrite only at low nitrate concentrations, resulted in a kinetic growth model with nine model parameters. The data of two batch experiments were used to identify the kinetic model. The kinetic model was validated with two additional batch experiments. The model predictions are in very good agreement with the experimental data. The maximum nitrite concentration was estimated to be 30.7 mM (total inhibition of growth). After complete reduction of nitrate, the growth rate decreases almost to zero before it increases again because of the following nitrite respiration. The maximum autohydrogenotrophic growth rate of Ralstonia eutropha with nitrate as a final electron acceptor (0.509 d⁻¹) was found to be reduced by 90–95% compared to the so far reported autohydrogenotrophic growth rates with oxygen.     

Nitric oxide and mitochondrial complex IV

Micromolar nitric oxide (NO) rapidly (ms) inhibits cytochrome c oxidase in turnover with physiological substrates. Two reaction mechanisms have been identified leading, respectively, to formation of a nitrosyl- [a3(2+) -NO] or a nitrite- [a3(3+) -NO2-] derivative of the enzyme. In the presence of O2, the nitrosyl adduct recovers activity slowly, following NO displacement at k' approximately equal to 0.01 s(-1) (37 degrees C); the recovery of the nitrite adduct is much faster. Relevant to pathophysiology, the enzyme does not degrade NO by following the first mechanism, whereas by following the second one it promotes NO oxidation and disposal as nitrite/nitrate. The reaction between NO and cytochrome c oxidase has been investigated at different integration levels of the enzyme, including the in situ state, such as in mouse liver mitochondria or cultured human SY5Y neuroblastoma cells. The respiratory chain is inhibited by NO, either supplied exogenously or produced endogenously via the NO synthase activation. Inhibition of respiration is reversible, although it remains to be clarified whether reversibility is always full and how it depends on concentration of and time of exposure to NO. Oxygraphic measurements show that cultured cells or isolated state 4 mitochondria exposed to micromolar (or less) NO recover from NO inhibition rapidly, as if the nitrite reaction was predominant. Mitochondria in state 3 display a slightly more persistent inhibition than in state 4, possibly due to a higher accumulation of the nitrosyl adduct. Among a number of parameters that appear to control the switch over between the two mechanisms, the concentration of reductants (reduced cytochrome c) at the cytochrome c oxidase site has been proved to be the most relevant one.     

Reconstitution of cytochrome oxidase vesicles and conferral of sensitivity to energy transfer inhibitors

Summary 1. Phospholipids and cytochrome oxidase solubilized with chotate were reconstituted either by dialysis or by dilution of the detergent. The reconstituted cytochrome oxidase vesicles oxidized ascorbate-cytochromec at a rate which was low, insensitive to energy transfer inhibitors and markedly stimulated by uncouplers of oxidative phosphorylation. The rate of reconstitution was dependent on pH, on the concentration of cholate and on the presence of high concentrations of monovalent ions or low concentrations of divalent ions. The integrity of the cytochrome oxidase vesicles was retained after freeze-drying, provided sucrose was present during the process. 2. Reconstitution with pure phospholipids revealed that cardiolipin was required for the marked stimulation of respiration by uncouplers. 3. Cytochrome oxidase vesicles were reconstituted in the presence of hydrophobic mitochondrial proteins which contained oligomycin-sensitive ATPase. The resulting vesicles oxidized ascorbate-cytochromec at a rapid rate which was not enhanced by uncouplers. Addition of an energy transfer inhibitor such as rutamycin resulted in a partial inhibition of respiration which was released by uncouplers. 4. Cytochrome oxidase vesicles reconstituted in the presence of phenol red were rather impermeable to protons and became very permeable on addition of uncouplers. When the reconstitution was performed in the presence of the hydrophobic proteins from mitochondria, proton translocation became partially sensitive to rutamycin. 5. These observations are consistent with some of the formulations of the chemiosmotic hypothesis.