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.     

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.     

A dynamic model of nitric oxide inhibition of mitochondrial cytochrome c oxidase

Nitric oxide can inhibit mitochondrial cytochrome oxidase in both oxygen competitive and uncompetitive modes. A previous model described these interactions assuming equilibrium binding to the reduced and oxidised enzyme respectively (Mason, et al. Proc. Natl. Acad. Sci. U S A 103 (2006) 708-713). Here we demonstrate that the equilibrium assumption is inappropriate as it requires unfeasibly high association constants for NO to the oxidised enzyme. Instead we develop a model which explicitly includes NO binding and its enzyme-bound conversion to nitrite. Removal of the nitrite complex requires electron transfer to the binuclear centre from haem a. This revised model fits the inhibition constants at any value of substrate concentration (ferrocytochrome c or oxygen). It predicts that the inhibited steady state should be a mixture of the reduced haem nitrosyl complex and the oxidized-nitrite complex. Unlike the previous model, binding to the oxidase is always proportional to the degree of inhibition of oxygen consumption. The model is consistent with data and models from a recent paper suggesting that the primary effect of NO binding to the oxidised enzyme is to convert NO to nitrite, rather than to inhibit enzyme activity (Antunes et al. Antioxid. Redox Signal. 9 (2007) 1569-1579).     

The catabolic fate of nitric oxide: the nitric oxide oxidase and peroxynitrite reductase activities of cytochrome oxidase

Stimulation of cardiomyocytes to endogenously evolve nitric oxide is shown by microsensor measurements on single cells to lead to transient nitric oxide concentrations of a few hundred nanomolar. At these submicromolar concentrations, no evidence could be found for the expected reaction between nitric oxide generated and the oxymyoglobin present in the cells: nitric oxide + oxymyoglobin --> nitrate + metmyoglobin. No metmyoglobin formation was detected by electron paramagnetic resonance spectroscopy, and microsensor measurements revealed near quantitative conversion of the nitric oxide to nitrite rather than nitrate ion. Moreover, the rate of nitrite formation is shown to be too rapid to be accounted for by non-enzymatic means. The essentially quantitative and rapid catabolism of nitric oxide to nitrite ion can plausibly be explained on the basis of a cycle of reactions catalyzed by cytochrome c oxidase. It is demonstrated with the purified hemoproteins in vitro that the terminal oxidase can outcompete oxymyoglobin for available nitric oxide. It is proposed that under normal physiological and most pathological (non-inflammatory) conditions, reaction with cytochrome c oxidase is the major route by which NO is removed from mitochondria-rich cells.     

Regulation of xanthine oxidase by nitric oxide and peroxynitrite

Xanthine oxidase (XO) is a central mechanism of oxidative injury as occurs following ischemia. During the early period of reperfusion, both nitric oxide (NO(*)) and superoxide (O-*(2)) generation are increased leading to the formation of peroxynitrite (ONOO(-)); however, questions remain regarding the presence and nature of the interactions of NO(*) or ONOO(-) with XO and the role of this process in regulating oxidant generation. Therefore, we determined the dose-dependent effects of NO(*) and ONOO(-) on the O-*(2) generation and enzyme activity of XO, respectively, by EPR spin trapping of O-*(2) using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide and spectrophotometric assay. ONOO(-) markedly inhibited both O-*(2) generation and XO activity in dose-dependent manner, while NO(*) from NO(*) gas in concentrations up to 200 microM had no effect. Furthermore, we observed that NO(*) donors such as NOR-1 also inhibited O-*(2) generation and XO activity; however, these effects were O-*(2)-dependent and blocked by superoxide dismutase or ONOO(-) scavengers. Finally, we found that ONOO(-) totally abolished the Mo(V) EPR spectrum. These changes were irreversible, suggesting oxidative disruption of the critical molybdenum center of the catalytic site. Thus, ONOO(-) formed in biological systems can feedback and down-regulate XO activity and O-*(2) generation, which in turn may serve to limit further ONOO(-) formation.     

Biphasic regulation of leukocyte superoxide generation by nitric oxide and peroxynitrite

Activation of the NADPH oxidase-derived oxidant burst of polymorphonuclear leukocytes (PMNs) is of critical importance in inflammatory disease. PMN-derived superoxide (O(2)) can be scavenged by nitric oxide (NO( small middle dot)) with the formation of peroxynitrite (ONOO(-)); however, questions remain regarding the effects and mechanisms by which NO( small middle dot) and ONOO(-) modulate the PMN oxidative burst. Therefore, we directly measured the dose-dependent effects of NO( small middle dot) and ONOO(-) on O(2) generation from human PMNs stimulated with phorbol 12-myristate 13-acetate using EPR spin trapping. Pretreatment with low physiological (microm) concentrations of NO( small middle dot) from NO( small middle dot) gas had no effect on PMN O(2) generation, whereas high levels (> or =50 microm) exerted inhibition. With ONOO(-) pretreatment, however, a biphasic modulation of O(2) generation was seen with stimulation by microm levels, but inhibition at higher levels. With the NO( small middle dot) donor NOR-1, which provides more sustained release of NO( small middle dot) persisting at the time of O(2) generation, a similar biphasic modulation of O(2) generation was seen, and this was inhibited by ONOO(-) scavengers. The enhancement of O(2) generation by low concentrations of ONOO(-) or NOR-1 was associated with activation of the ERK MAPKs and was blocked by their inhibition. Thus, low physiological levels of NO( small middle dot) present following PMN activation are converted to ONOO(-), which enhances O(2) generation through activation of the ERK MAPK pathway, whereas higher levels of NO( small middle dot) or ONOO(-) feed back and inhibit O(2) generation. This biphasic concentration-dependent regulation of the PMN oxidant burst by NO( small middle dot)-derived ONOO(-) may be of critical importance in regulating the process of inflammation.     

Membrane potential-controlled inhibition of cytochrome c oxidase by zinc

Like many voltage-sensitive ion pumps, cytochrome c oxidase is inhibited by zinc. Binding of zinc to the outside surface of Rhodobacter sphaeroides cytochrome c oxidase inhibits the enzyme with a K(I) of < or = 5 microm when the enzyme is reconstituted into phospholipid vesicles in the presence of a membrane potential. In the absence of a membrane potential and a pH gradient, millimolar concentrations of zinc are required to inhibit. This differential inhibition causes a dramatic increase in the respiratory control ratio from 6 to 40 for wild-type oxidase. The external zinc inhibition is removed by EDTA and is not competitive with cytochrome c binding but is competitive with protons. Only Cd(2+) of the many metals tested (Mg(2+), Mn(2+), Ca(2+), Ba(2+), Li(2+), Cs(2+), Hg(2+), Ni(2+), Co(2+), Cu(2+) Tb(3+), Tm(3+)) showed inhibitory effects similar to Zn(2+). Proton pumping is slower and less efficient with zinc. The results suggest that zinc inhibits proton movement through a proton exit path, which can allow proton back-leak at high membrane potentials. The physiological and mechanistic significance of proton movement in the exit pathway and its blockage by zinc is discussed in terms of regulation of the efficiency of energy transduction.     

Tyrosine nitration by peroxynitrite formed from nitric oxide and superoxide generated by xanthine oxidase

Peroxynitrite (ONOO(-)) is a potent nitrating and oxidizing agent that is formed by a rapid reaction of nitric oxide (NO) with superoxide anion (O(2)). It appears to be involved in the pathophysiology of many inflammatory and neurodegenerative diseases. It has recently been reported (Pfeiffer, S., and Mayer, B. (1998) J. Biol. Chem. 273, 27280-27285) that ONOO(-) generated at neutral pH from NO and O(2) (NO/O(2)) was substantially less efficient than preformed ONOO(-) at nitrating tyrosine. Here we re-evaluated tyrosine nitration by NO/O(2) with a shorter incubation period and a more sensitive electrochemical detection system. Appreciable amounts of nitrotyrosine were produced by ONOO(-) formed in situ (2.9 micrometer for 5 min; 10 nm/s) by NO/O(2) flux obtained from propylamine NONOate (CH(3)N[N(O)NO](-) (CH(2))(3)NH(2)(+)CH(3)) and xanthine oxidase using pterin as a substrate in phosphate buffer (pH 7.0) containing 0.1 mm l-tyrosine. The yield of nitrotyrosine by this NO/O(2) flux was approximately 70% of that produced by the same flux of preformed ONOO(-) (2.9 micrometer/5 min). When hypoxanthine was used as a substrate, tyrosine nitration by NO/O(2) was largely eliminated because of the inhibitory effect of uric acid produced during the oxidation of hypoxanthine. Tyrosine nitration caused by NO/O(2) was inhibited by the ONOO(-) scavenger ebselen and was enhanced 2-fold by NaHCO(3), as would be expected, because CO(2) promotes tyrosine nitration. The profile of nitrotyrosine and dityrosine formation produced by NO/O(2) flux (2.9 micrometer/5 min) was consistent with that produced by preformed ONOO(-). Tyrosine nitration predominated compared with dityrosine formation caused by a low nanomolar flux of ONOO(-) at physiological concentrations of free tyrosine (<0.5 mm). In conclusion, our results show that NO generated with O(2) nitrates tyrosine with a reactivity and efficacy similar to those of chemically synthesized ONOO(-), indicating that ONOO(-) can be a significant source of tyrosine nitration in physiological and pathological events in vivo.     

Reactions of nitric oxide with copper containing oxidases; cytochrome c oxidase and laccase

The reactions of nitric oxide with copper containing oxidases such as cytochrome c oxidase and laccase are described and discussed in the present review.     

Measurement of free radical oxygen generation by cytochrome c reduction requirement for cytochrome c oxidase blockade

Data is presented showing that one commercial preparation of cytochrome c, used to trap and measure free radical superoxide anion, can be contaminated with cytochrome c oxidase activity. This activity can vary from lot to lot, can introduce variability into the measurement of superoxide anion and can result in falsely low estimations of free radical formation. This cytochrome c oxidase activity can be inhibited by low (0.2 mM) concentrations of KCN. Blockade of the cytochrome c oxidase activity allows reproducible measurement of superoxide anion formation at low levels by red cells.     

Heme-mediated oxygen activation in biology: cytochrome c oxidase and nitric oxide synthase.

Major advances have been made in our understanding of cytochrome c oxidase owing to continued crystallographic work on important intermediates. This, together with a wealth of data derived from selective mutations and sophisticated spectroscopic probes, has provided significant new insights into oxidase dioxygen chemistry and proton pumping activities. Recent advances have also been made for nitric oxide synthase, owing to the crystal structure determination of the heme domain for two of three nitric oxide synthase isoforms.     

Biochemical properties of cytochrome c nitrated by peroxynitrite

Nitration of tyrosine residues is taken as evidence for intracellular formation of peroxynitrite. Cytochrome c (cyt c) can be nitrated by peroxynitrite and nitrated cyt c has been observed in cells and tissues under stress conditions. Here we studied the biochemical properties of nitrated cyt c in order to understand its potential roles in nitrative stress. Nitration of cyt c resulted in disruption of the heme-methionine bond and rapid binding to cyanide. Equilibrium unfolding by guanidine hydrochloride showed that cyt c was slightly destabilized upon nitration but the unfolding transition of nitrated cyt c was highly cooperative indicating that the overall folding was largely preserved. Nitrated cyt c could not be reduced by superoxide and did not support electron transfer between ascorbate and cyt c oxidase. Nitration of cyt c resulted in a tremendous increase in peroxidase activity so that nitrated cyt c rapidly oxidized dihydrodichlorofluorescein even in the presence of a high concentration of glutathione. Enhanced peroxidase activity of nitrated cyt c was responsible for H2O2-induced oxidation of phospholipid membranes and H2O2/NO2--mediated nitration of other proteins. These results suggest that nitration of cyt c by peroxynitrite may exacerbate oxidative damage to mitochondrial proteins and membranes.     

Reversible redox titrations of cytochrome c and cytochrome c oxidase using detergent solubilized electrochemically generated mediator-titrants

The repetitive, $\text{reversible}$ equilibrium redox cycling of cytochrome $\text{c}$, cytochrome $\text{c}$ oxidase, or mixtures thereof has been made possible by the use of the oxidant, ferricinium ion. This ion is electrochemically generated by the use of non-ionic detergent solubilized ferrocene which is apparently incorporated as micelles and readily electron transfers with an electrode. The $\text{ferricinium-ferrocene}$ couple equilibrates rapidly with these heme proteins. Electrochemically generated benzylviologen radical cations are used as the reductant. The E^O′ values for cytochrome $\text{c}$ oxidase at pH 7.0 are 209 ± 15 mv (2e^?) and 340 ± 15 mv (2e^?).     

Nitric oxide from neuronal nitric oxide synthase sensitises neurons to hypoxia-induced death via competitive inhibition of cytochrome oxidase

Hypoxia/ischaemia is known to trigger neuronal death, but the role of neuronal nitric oxide synthase (nNOS) in this process is controversial. Nitric oxide (NO) inhibits cytochrome oxidase in competition with oxygen. We tested whether NO derived from nNOS synergises with hypoxia to induce neuronal death by inhibiting mitochondrial cytochrome oxidase. Sixteen hours of hypoxia (2% oxygen) plus deoxyglucose (an inhibitor of glycolysis) caused extensive, excitotoxic death of neurons in rat cerebellar granule cell cultures. Three different nNOS inhibitors (including the selective inhibitor N-4S-4-amino-5-2-aminoethyl-aminopentyl-N'-nitroguanidine) decreased this neuronal death by half, indicating a contribution of nNOS to hypoxic death. The selective nNOS inhibitor did not, however, block neuronal death induced either by added glutamate or by added azide (an uncompetitive inhibitor of cytochrome oxidase), indicating that nNOS does not act downstream of glutamate or cytochrome oxidase. Hypoxia plus deoxyglucose-induced glutamate release and neuronal depolarisation, and the nNOS inhibitor decreased this. Hypoxia inhibited cytochrome oxidase activity in the cultures, but a selective nNOS inhibitor prevented this inhibition, indicating NO from nNOS was inhibiting cytochrome oxidase in competition with oxygen. These data indicate that hypoxia synergises with NO from nNOS to induce neuronal death via cytochrome oxidase inhibition causing neuronal depolarisation. This mechanism might contribute to ischaemia/stroke-induced neuronal death in vivo.     

Quantization of membrane potential generation by cytochrome c oxidase in small vesicles

Proteoliposomes incorporating cytochrome c oxidase have been prepared by the cholate dialysis method and by sonication. Sonication produces multilamellar vesicles heterogeneous in size in contrast to a more uniform preparation of unilamellar vesicles produced by the dialysis procedure. Respiratory control in both preparations ranges between 4 and 8. From an electron microscopic analysis of proteoliposome size, the average electrical capacitance/vesicle for the dialyzed and sonicated preparations is calculated as 15 X 10(-18) F and 130 X 10(-18) F, respectively. These capacitance values would lead to a quantization of membrane potential generation by the enzyme at 77 mV/turnover for the dialyzed preparation and 9 mV/turnover for sonicated vesicles. It is argued that these differences can explain the dependence of H+ translocation on the number of turnovers of cytochrome c oxidase in dialyzed preparations in contrast to the lack of dependence on number of turnovers in sonicated preparations.     

Dynamics of nitric oxide in the active site of reduced cytochrome c oxidase aa3.

Nitric oxide (NO) is involved in the regulation of respiration by acting as a competitive ligand for molecular oxygen at the binuclear active site of cytochrome c oxidase. The dynamics of NO in and near this site are not well understood. We performed flash photolysis studies of NO from heme a3 in cytochrome c oxidase from Paracoccus denitrificans, using femtosecond transient absorption spectroscopy. The formation of the product state--the unliganded heme a3 ground state--occurs in a similar stepwise manner (period approximately 700 fs) as previously observed for carbon monoxide photolysis from this enzyme and interpreted in terms of ballistic ligand motions in the active site on the subpicosecond time scale [Liebl, U., Lipowski, G., Négrerie, M., Lambry, J.-C., Martin, J.-L., and Vos, M. H. (1999) Nature 401, 181-184]. A fraction (approximately 35% at very low NO concentrations) of the dissociated NO recombines with heme a3 in 200-300 ps. The presence of this recombination phase indicates that a transient bond to the second ligand-binding site, a copper atom (CuB), has a short lifetime or may not be formed. Increasing the NO concentration increases the recombination yield on the hundreds of picoseconds time scale. This effect, unprecedented for heme proteins, implies that, apart from the one NO molecule bound to heme a3, a second NO molecule can be accommodated in the active site, even at relatively low (submicromolar) concentrations. Models for NO accommodation in the active site, based on molecular dynamics energy minimizations are presented. Pathways for NO motion and their relevance for the regulation of respiration are discussed.