Regulation of Mitochondrial Respiration by Oxygen and Nitric Oxide

121

Regulation of Mitochondrial Respirationby Oxygen and Nitric Oxide

ALBERTO BOVERIS,

a,b

LIDIA E. COSTA,

c

JUAN J. PODEROSO,

d

MARIA C. CARRERAS,

d

AND ENRIQUE CADENAS

e

a

Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry

c

Institute of Cardiological Research, and

d

University Hospital, School of Medicine,University of Buenos Aires, Buenos Aires, Argentina

e

Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, Los Angeles, California, USA

A

BSTRACT

: Although the regulation of mitochondrial respiration and energyproduction in mammalian tissues has been exhaustively studied and extensive-ly reviewed, a clear understanding of the regulation of cellular respiration hasnot yet been achieved. In particular, the role of tissue pO

2

as a factor regulat-ing cellular respiration remains controversial. The concept of a complex andmultisite regulation of cellular respiration and energy production signaled bycellular and intercellular messengers has evolved in the last few years and isstill being researched. A recent concept that regulation of cellular respirationis regulated by ADP, O

2

and NO preserves the notion that energy demandsdrive respiration but places the kinetic control of both respiration and energysupply in the availability of ADP to F

1

-ATPase and of O

2

and NO to cyto-chrome oxidase. In addition, recent research indicates that NO participates inredox reactions in the mitochondrial matrix that regulate the intramitochon-drial steady state concentration of NO itself and other reactive species such assuperoxide radical (O

2

−

) and peroxynitrite (ONOO

−

). In this way, NO acquiresan essential role as a mitochondrial regulatory metabolite. NO exhibits a richbiochemistry and a high reactivity and plays an important role as intercellularmessenger in diverse physiological processes, such as regulation of blood flow,neurotransmission, platelet aggregation and immune cytotoxic response.

INTRODUCTION

The regulation of mitochondrial respiration and energy production in mammaliantissues has been exhaustively studied and extensively reviewed. However, a clear un-derstanding of the regulation of cellular respiration is not yet complete. In particular,the role of tissue pO

2

as a factor regulating cellular respiration is a matter of contro-versy. It was considered that maximal rates of mitochondrial respiration could bemaintained in a wide range of tissue pO

2

, from the usual 5 to 30

µ

M O

2

to about 0.8

µ

M O

21–3

based on the [O

2

]

0.5

values (the [O

2

] that sustains half maximal respira-tory rate) of 0.02 to 0.3

µ

M O

2

that were measured with isolated mitochondria andcells.

4–5

Considering this high affinity of mitochondrial respiration for O

2

, the rates

b

Author to whom correspondence should be addressed.

122 ANNALS NEW YORK ACADEMY OF SCIENCES

at which mitochondria perform oxidative phosphorylation were supposed to be rath-er independent of tissue pO

2

and limitation of respiration by [O

2

] was thought to oc-cur only under severe hypoxia. However, recent [O

2

]

0.5

values carefully determinedturned out to be higher than the ones previously measured, especially for active (state3) respiration.

6–10

The [O

2

]

0.5

determined by Costa

et al

.

10

using high-resolutionrespirometry in liver and heart mitochondria were 0.30–0.40

µ

M in state 4 and 1.57–1.69

µ

M in state 3,

10

which implies that the intracellular [O

2

] prevailing in some tis-sues,

e.g

., 3–8

µ

M in the heart,

2–3

would be regulatory under normoxia with respi-ration slowed below its maximal rate. Defining a critical rate (Vc) as 80

%

of V

max9

and considering a classical Michaelis-Menten kinetics it follows that the critical [O

2

]will be reached at 6

µ

M O

2

(V

c

=

V

max

[O

2

]/([O

2

]

0.5

+

[O

2

]) and that limitation ofactive mitochondrial respiration may occur at physiological normoxia in the heart;higher values of pO

2

, in the range of 22 to 32

µ

M O

2

and well above the critical [O

2

],have been reported for skeletal muscle and liver, however.

1,2,4,11

Cytochrome oxidase was not usually considered a regulatory enzyme, yet evi-dence has been obtained that non-catalytic subunits alter enzyme kinetics.

12

Directspectral analysis have indicated that a substantial fraction of cytochrome oxidase isreduced in intact tissues,

13

even though this reduction is not observed in isolated mi-tochondria. It has been suggested that this difference is due to regulatory mecha-nisms that are lost

in vitro.

14

It is worth noting that the reduced cytochrome oxidase-NO complex is spectroscopically similar to reduced cytochrome oxidase.

15–16

The concept of a complex and multisite regulation of cellular respiration and en-ergy production signaled by cellular and intercellular messengers has evolved in thelast few years and is still under development. The classical and elegant concept ofthe regulation of cellular oxygen uptake by ADP put forward by Lardy and Well-man,

17

Chance and Williams,

18

and Estabrook

19

considers that energy needs driverespiration and that availability of ADP to mitochondrial F

1

-ATPase exerts the kinet-ic control of respiration and energy production over a wide range of O

2

concentra-tion that certainly includes the physiological conditions (F

IG

. 1). The new conceptof regulation of cellular respiration by ADP, O

2

and NO keeps the idea that energy

FIGURE 1. Classical view and new concept of the regulation of cellular O2 uptake.

123BOVERIS

et al

.: REGULATION OF MITOCHONDRIAL RESPIRATION

demands drive respiration but places the kinetic control of both respiration and en-ergy supply in the availability of ADP to F

1

-ATPase and of O

2

and NO to cytochromeoxidase (F

IG

. 1). In addition, recent reports by Poderoso

et al

.

20–22

and by Boveris

et al

.

23

indicate that NO participates in redox reactions in the mitochondrial matrixthat regulate the intramitochondrial steady state concentration of NO itself and otherreactive species such as superoxide radical (O

2

−

) and peroxynitrite (ONOO

−

).In this way, NO acquires an essential role as a mitochondrial regulatory metabo-

lite. Nitric oxide, a diatomic gas with an unpaired electron in its external orbitals,exhibits a rich biochemistry and a high reactivity and plays important roles as inter-cellular messenger in diverse physiological processes, such as regulation of bloodflow, neurotransmission, platelet aggregation and immune cytotoxic response.

24

En-dothelium produced NO stimulates the guanylate cyclase activity of the underlyingvascular smooth muscle cells and the increased level of cGMP produces muscle re-laxation and vasodilation.

25–26

This action slows down blood flow and increases theequilibration time of HbO

2

with tissue O

2

resulting in better tissue oxygenation. Thecoupling of the endothelial production of NO and blood flow to changes in tissuepO

2

may be thought as dependent on cellular metabolites that are produced when mi-tochondrial metabolism becomes O

2

limited and that reach endothelial cells. Since Granger and Lehninger

27

early recognized macrophage cytotoxicity as ex-erted partly on mitochondrial respiration, the recognition of NO production in acti-vated macrophages and neutrophils

28–29

prompted the assay of NO effects onmitochondrial function. Two British research groups, Cleeter

et al

.

30

and Brown andCooper,

31

simultaneously reported the inhibitory effects of NO on cytochrome oxi-dase activity using skeletal muscle mitochondria

30

and brain synaptosomes.

31

Theinhibition is reversible and competitive with O

231

suggesting that tissue NO mayraise the [O

2

]

0.5

and therefore become the first known physiological regulator to actdirectly on the mitochondrial respiratory chain.

Cytosolic Ca

2

+

has been frequently suggested as a regulator of mitochondrialfunction, likely as part of a role as intracellular second messenger that signals acti-vation of diverse and specialized cell responses, many of which require increased en-ergy production.

14

However, cytosolic Ca

2

+

, as a cation able to dischargemitochondrial membrane potential, seems to act as antagonist for the coordinated ac-tivation of mitochondrial respiration and energy supply.

32

OXYGEN DEPENDENCE OF MITOCHONDRIAL RESPIRATION

The dependence of the respiratory rate of isolated mitochondria and cells on [O

2

]is hyperbolic; therefore it is conveniently described by [O

2

]

0.5

, the oxygen concen-tration that provides half-maximal respiration rate. The [O

2

]

0.5

values are also some-times referred to as K

0.5

and Km

O

2

; however, since, owing to their operational naturethey are different with mitochondria in state 4 or in state 3 and depend on [NO], it isconvenient to use only the [O

2

]

0.5

notation and concept.The use of high resolution respirometers is advisable to precisely measure the

rates of O2 uptake that occur in the 0.01 to 5 µM O2 range which is essential to de-termine [O2]0.5 values. The [O2]0.5 of active (state 3) and resting (state 4) respirationwere recently reexamined in tightly coupled mitochondria isolated from rat liver and


FIGURE 2. Oxygen concentration ([O2]) and its first derivative (rate of O2 uptake;d[O2]/dt) during the respiratory activity of rat liver mitochondria (0.35 mg prot/ml) sus-pended in 0.25 M sucrose, 0.5 mM EGTA, 5 mM MgCl2, 1.5% BSA, 10 mM HEPES, and6 mM phosphate buffer (pH 7.35). ADP pulse: 0.3 mM. A: initial rate of O2 uptake, ∆[O2]/∆t, used to produce one data point in FIGURE 3. B: time course of the differential rates of O2uptake, d[O2]/dt), that generate the data points of FIGURE 4. Modified from Costa et al.10

TABLE 1. Oxygen dependence of active (state 3) mitochondrial respiration determinedby four approaches

Aerobic mitochondria Anaerobic mitochondria

ADP pulses O2 pulses

Initial rates Differential rates

Air-saturated medium

H2O2

Liver mitochondria

Vmax (nmol O2/sec ⋅mg prot)

1.80 ± 0.02 1.78 ± 0.09 1.84 ± 0.07 1.82 ± 0.08

[O2]0.5 (µM) 2.45 ± 0.32 1.69 ± 0.09 1.04 ± 0.02 1.09 ± 0.04

Heart mitochondria

Vmax (nmol O2/sec ⋅mg prot)

1.47 ± 0.10 1.55 ± 0.09 1.51 ± 0.09 1.53 ± 0.10

[O2]0.5 (µM) 3.02 ± 0.30 1.45 ± 0.10 1.07 ± 0.04 1.12 ± 0.10

125BOVERIS et al.: REGULATION OF MITOCHONDRIAL RESPIRATION

heart by Costa et al.10 The [O2]0.5 values corresponding to state 4 respiration (0.30–0.40 µM) were not different from previously reported values obtained using ad-vanced instrumentation6–9 and are given, as well as methodological details, else-where.10,23 The new [O2]0.5 values corresponding to state 3 respiration weredetermined using four different experimental approaches and opened the way to thenew concept of respiratory regulation by pO2.

10,23

In the first approach, termed “initial rates,” ADP pulses were added to mitochon-dria in the resting state 4, at a different [O2] in different runs, collecting one datapoint in each run, i.e. the maximal rate, ∆O2/∆t, usually constant during several secafter ADP addition (FIG. 2). Hyperbolic fitting of the individual data points gave the[O2]0.5 (FIG. 3 and TABLE 1, initial rates), which tend to be overestimated becauseonly a few points could be obtained in the range of limiting [O2].

In the second approach, termed “differential rates,” ADP pulses were also addedto mitochondria in resting state 4, with excess ADP to exhaust O2 in the reaction me-dium and taking advantage of the 0.2–1 sec period in which dO2/dt can be measuredin high resolution respirometers (FIG. 2). The dO2/dt values were fitted to a singlehyperbola as a function of [O2] and gave the highest possible accuracy for the deter-mination of [O2]0.5 (FIG. 4 and TABLE 1, differential rates). The third approach,termed “O2 pulses,” consisted of the injection of air-saturated reaction medium tomitochondria incubated in anaerobiosis for 3–5 min in the presence of non-limiting[ADP] (FIG. 5). In order to reach easily anaerobiosis without accumulation of meta-bolic by-products, mitochondria were suspended in a reaction medium in which O2

FIGURE 3. Oxygen dependence of the state 3 respiration of rat liver mitochondriashowing the hyperbolic fitting of the initial rates (∆[O2]/∆t). For details see text andFIGURE 2. The triangle indicates the mean value of the O2 uptake rate measured in air-satu-rated buffer.


FIGURE 4. Oxygen dependence of the state 3 respiration of rat liver mitochondria.The dotted line is the hyperbolic fitting of the data points (d[O2]/dt) collected at 1 sec in-tervals after an ADP pulse. For details see text and FIGURE 2. Different symbols indicatedifferent runs. The calculated [O2]0.5 is 1.7 µM. Modified from Costa et al.10

FIGURE 5. Oxygen concentration ([O2]) and its first derivative (d[O2]/dt) during therespiration of rat liver mitochondria (0.30 mg prot/ml) that were incubated in anaerobiosisfor 3–5 min and added with an O2 pulse. For details see text. Experimental conditions as inFIGURE 2.


was diminished to 10–20 µM O2 by flushing N2. The non-linear regression fitting ofdO2/dt as a function of [O2] from two consecutive O2 pulses and from an ADP pulseshow that the [O2]0.5 was significantly lower in the case of the O2 pulses than withthe ADP pulse (FIG. 6, TABLE 1). Because of the relatively low [O2] in air-saturatedreaction medium and the fast rate of state 3 respiration, a fourth approach deliveringmore O2 to the reaction medium and termed “H2O2 pulse” was introduced. In thiscondition 10 µM O2, enough to reach the Vmax, is easily added to anaerobic catalase-supplemented mitochondria by the injection of a few µl of a H2O2 solution. The[O2]0.5 values obtained by this approach were similar to the ones obtained by the O2pulses (TABLE 1). The lower [O2]0.5 values obtained after anaerobiosis suggest theexistence of an O2-dependent system that is able to modulate mitochondrial respira-tion at low [O2] and that the regulator is competitive with O2, since [O2]0.5 is in-creased in its presence while Vmax is not changed. The production of NO bymitochondrial nitric oxide synthase (mtNOS)33–34 during active state 3 respirationseems to afford the referred regulation.35

THE EFFECTS OF NO ON THE MITOCHONDRIALRESPIRATORY CHAIN

The rich biochemical properties of NO make possible its multiple effects on themitochondrial respiratory chain: (a) NO inhibits cytochrome oxidase activity com-petitively with oxygen; (b) NO inhibits electron transfer between cytochromes b and

FIGURE 6. Oxygen dependence of the state 3 respiration of rat liver mitochondria thatwere pulsed with ADP in aerobiosis (�) or with O2 after anaerobiosis (�, �). The datapoints (d[O2]/dt) were collected at 1 sec intervals. For details see text and FIGURES 2 and 5.The calculated [O2]0.5 are 1.60 µM O2 (ADP pulse) and 1.05 µM O2 (O2 pulse).


c and increases the mitochondrial production of O2−; and (c) NO inhibits electron

transfer and NADH-dehydrogenase function in Complex I.

Cytochrome Oxidase (Complex IV)

Concerning the inhibition of cytochrome oxidase activity, NO binds to the en-zyme in its reduced and oxidized forms; in the reduced form NO binds to the binu-clear reaction center formed by cytochrome a3 heme and CuB. The cytochromeoxidase activity of the isolated enzyme15,31 and of rat heart submitochondrial parti-cles,21 as well as the active respiration of mitochondria isolated from rat muscle ,30

liver,36 heart21 and brown adipose tissue37 and of rat brain synaptosomes31 are ef-fectively inhibited by 0.05–2 µM NO (see FIG. 7). Binding and inhibition are revers-ible and removable by washing21,30 or by addition of excess myoglobin orhemoglobin.21,36 The degree of inhibition of cytochrome oxidase activity by NO de-pends on the O2 concentration in the reaction medium 31,36,37; NO and O2 competefor the binding site at the reaction center of cytochrome oxidase. Then, the inhibitionof mitochondrial respiration by NO can be expressed as a function of the ratio [O2]/[NO]; half-maximal inhibition of state 3 respiration is reached at a ratio of 150 O2/NO (FIG. 7, inset) which clearly indicates the very high affinity of NO for cyto-chrome oxidase. Ratios of 400–500 O2/NO and 500–1000 O2/NO have been report-ed to inhibit 50% the respiration of rat brain synaptosomes31 and of rat brownadipose tissue,37 respectively.

FIGURE 7. Inhibition of cytochrome oxidase activity and active respiration by NO.The inhibition of uptake, normalized for the different experiments, is plotted against theratio of O2 and NO concentrations. Rat liver mitochondria in the presence of: �, ADP (state3), and �, 20 µM dinitrophenol (state 3u); data from Takahara et al.36 Rat heart mitochon-dria, ∆, in the presence of ADP; data from Poderoso et al.20

O2–


The Ubiquinol-Cytochrome b-Cytochrome c Space (Complex III)

Rat heart submitochondrial particles added with NO show a marked inhibition oftheir succinate-cytochrome c activity with half maximal effect at about 0.7 mM NO(FIG. 8 and ref. 22) with a NO-induced reduction of cytochrome b.21,22 This secondeffect of NO on the mitochondrial respiratory chain results in increased O2

− produc-tion in submitochondrial particles and H2O2 generation in whole mitochondria, be-ing about 0.5 mM NO the concentration required for half maximal effect (FIG. 8 andref. 21). The interaction of NO with the NO-reactive component of the ubiquinone-cytochrome b area of the mitochondrial respiratory chain, likely an iron-sulfur cen-ter, is also reversible but is not affected by the O2/NO ratio. The time course of theinhibition of cytochrome oxidase produced by a 1 mM NO pulse as a function of[O2] shows a marked O2 dependence (a steeper slope) when ascorbate-TMPD, re-ductants of cytochrome oxidase, were added, and a lower O2 dependence (a lessmarked slope) when succinate and antimycin, reductants of the ubiquinone-cytochrome b isopotential pool, were supplied (FIG. 8, inset). In the first case, themarked O2 dependence indicates the competition of NO and O2 for cytochrome ox-idase. In the latter case, the lower O2 dependence of the inhibition of the ubiquinol-cytochrome c electron transfer reflects the O2 dependence of O2

− production byubisemiquinone autoxidation.38,39

FIGURE 8. Inhibition of succinate–cytochrome c reductase activity and increase in thegeneration of O2

− produced by NO in rat heart submitochondrial particles. Modified fromPoderoso et al.20 Inset: Effect of [O2] on the NO-dependent inhibition of cytochrome oxi-dase activity.


NADH-Dehydrogenase (Complex I)

Prolonged exposure of cells to NO results in a persistent inhibition of complex Iactivity 26,40 simultaneously with a decrease in the cellular content of reduced glu-tathione. The inhibition is reversible by exposing the cells to high intensity light andappears to result from S-nitrosylation of thiol groups in the enzyme.40 It has beenclaimed that S-nitrosylation of complex I may play a role in neurodegenerativediseases.40

MITOCHONDRIAL PRODUCTION OF NO AND INHIBITION OFCYTOCHROME OXIDASE ACTIVITY

Nitric oxide is produced during the oxidation of L-arginine (Arg) to citrulline cat-alyzed by nitric oxide synthase (NOS). The recent finding of a mitochondrial en-zyme (mtNOS) in the inner mitochondrial membrane by Giulivi et al.33,34 and byGhadoufar and Richter41 supports the idea of a physiological role for NO in mito-chondrial respiration. The production of NO has been measured in whole mitochon-dria and in mitochondrial membranes isolated from a few rat and mouse organs(FIG. 9 and TABLE 2). Giulivi35 assayed the effects of the endogenous NO productionon the rates of mitochondrial respiration and cytochrome oxidase activity by supple-menting mitochondria with either the substrate Arg or the inhibitor of NOS N(G)-monomethyl-L-arginine (NMMA). The [O2]0.5 of the respiration of tightly coupledmitochondria oxidizing succinate in the presence of ADP ranged from 2 to 3 µM inagreement with those obtained by the ADP pulses (TABLE 1, initial rates). In thepresence of Arg the rates of O2 uptake decreased significantly at all measured O2

FIGURE 9. Mitochondrialproduction of NO. Rat thymusmitochondrial membranes (0.25mg protein/ml) were supplement-ed with 0.1 mM NADPH, 1 mMarginine, 1 mM Cl2Ca, 1 µM su-peroxide dismutase, 0.5 µM cata-lase and 10 µM oxyhemoglobin in50 mM phosphate buffer (pH7.4). a: complete reaction mix-ture; b: as in a plus NMMA.


concentrations. Two concentrations of Arg were used: 5 µM (about the Km ofmt-NOS for Arg33) and 0.1 mM. The observed values of [O2]0.5 were 18 and 40 µMat low and high concentrations of Arg, respectively. Conversely, addition of NMMAto intact mitochondria increased the O2 uptake by 40–50% of the control values atlow O2 levels and decreased the [O2]0.5 to 1.2 µM. Concerning cytochrome oxidase,the enzyme activity as a function of [O2] was determined in mitochondria supple-mented with either Arg or NMMA. The effects of NOS substrate and inhibitor weresimilar to those observed in mitochondrial state 3 respiration. The [O2]0.5 were 0.9,5 and 45 µM with NMMA, endogenous and exogenous Arg, respectively. Giuliviplotted the data concerning the effect of Arg and NMMA on state 3 respiration andcytochrome oxidase with the Eadie-Hofstee treatment and found them to fit to a sin-gle line indicating that cytochrome oxidase inhibition by endogenous NO entirelyaccounts for respiratory regulation. The unchanged Vmax and the different [O2]0.5 in-dicate a competitive inhibition kinetics in which NO inhibited the respiratory chaincompeting with O2 for cytochrome oxidase.35 Furthermore, the rate of ATP synthesisin intact mitochondria, evaluated by measuring the amount of ATP in aliquots takenat different time points during state 3 respiration, was similarly decreased in thepresence of Arg, as well as the respiratory rates in state 4.35

Assuming the classical view of “all or nothing,” where mitochondria are in state 3or in state 4, and that tissue O2 uptake is accounted by the sum of the O2 uptakes ofmitochondria respiring in state 4 and in state 3, the fraction of mitochondria instate 3 and in state 4 under physiological conditions were estimated as 28% and72%, respectively, for rat heart.42 Alternatively, considering for heart mitochondriaan [O2] of 6 µM and a [NO] of 30 nM, the corresponding ratio 200 O2//NO indicatesan inhibition of 33% for both state 3 and state 4 respiration (FIG. 7, inset) and theactual respiratory rate can be estimated as 0.67 × maximal respiratory rate measuredat saturating [O2]. In such case, with kinetic NO control, the fraction of mitochondriain state 3 (X) and in state 4 (1−X) are estimated as: (X) × (NO-inhibited state 3 O2uptake) + (1−X) × (NO-inhibited state 4 O2 uptake) = O2 uptake of the perfused or-gan / content of mitochondria in the tissue. The data used for the calculation are(0.67 × 135 nmol O2/min.mg prot) and (0.67 × 28 nmol O2/min.mg prot) for NO-inhibited state 3 and state 4 respiration,42 3.05 µ mol O2/min.g heart21 and 53 mgmitochondrial prot/g heart.43 The fraction of mitochondria in state 3 and state 4 are54% and 46%, respectively.

TABLE 2. Mitochondrial production of nitric oxide

aGiulivi et al.33

bGhafourifar and Richter.41

cJ. Bustamante, personal communication.dS. Lores Arnaiz, personal communication.

Mitochondria NO production(nmol/min.mg prot)

Reference

Rat liver mitochondria 1.4 a

Rat liver mitochondrial membranes 0.9–4.2 a, b, c

Rat thymus mitochondrial membranes 0.12–0.35 c

Mouse brain mitochondrial membranes 1.6 d


THE INTRAMITOCHONDRIAL STEADY STATE CONCENTRATION OF NITRIC OXIDE

The understanding of the NO regulation of mitochondrial respiration requiresknowledge of its mitochondrial metabolism and intramitochondrial steady state lev-el. For the following analysis only mitochondrial NO production and utilization areconsidered disregarding NO production by endothelial NOS and the fast reactions ofNO with muscle cytosolic myoglobin and blood hemoglobin that certainly have aneffect on mitochondria1 and cellular NO steady state concentrations.

Concerning NO and O2− metabolism it is convenient to consider the intramito-

chondrial space as a specialized intracellular compartment due to the selective per-meability of the inner mitochondrial membrane (FIG. 10). The key features are theimpermeability of the inner membrane to O2

− and H+, the relative impermeability ofthe same membrane to ONOO−, and the presence of Mn-SOD at a content which isabout five times lower than CuZn-SOD in the cytosol. The reactions of O2

− with NO(k = 2 × 1010 M−1 s−1) and with Mn-SOD (k = 2.4 × 109 M−1 s−1) are apparently theonly ones that occur in the mitochondrial matrix at rates that effectively contributeto O2

− utilization. Considering intramitocondrial MnSOD as 3 µM44 and intramito-condrial [NO] as 30 nM,45 it can be calculated that the intramitocondrial productionof ONOO− will account for 8% of O2

− utilization with the remaining 92% yieldingH2O2 as final product. Under conditions of endothelium NOS activation by bradiki-nin, intramitochondrial NO reaches 100 nM21 and consequently ONOO− formationmay account for as much as 27% of O2

− utilization. Moreover, in conditions of mt-NOS induction, intramitochondrial NO may reach 0.3 µM NO and the production ofONOO− will account for more than 50% of O2

− utilization. Besides this oxidativepathway, intramitochondrial NO is metabolized through reductive one-electrontransfer reactions from cytochrome oxidase,46 and ubiquinol.22 The two reductive

FIGURE 10. Mitochondrial metabolism of NO and its effect regulating cytochromeoxidase activity.


reactions yield nitroxyl anion (NO−) as intermediate and N2O as final stable product.According to our preliminary data the oxidative reaction yielding ONOO− accountsfor 88% of mitochondrial NO utilization and the two reductive reactions for theremaining 12%.

The fine regulation of the steady state concentration of NO in the mitochondria1matrix, accomplished by its reactions with O2

−, cytochrome oxidase, and ubiquinol,modulates cytochrome oxidase activity. The knowledge of the intracellular or intra-mitochondrial signaling that activates mtNOS is essential to fully understand theoverall regulatory process.

REFERENCES

1. GAYESKI, T.E.J. & C.R. HONIG. 1988. Intracellular pO2 in long axis of individualfibers in working dog gracilis muscle. Am. J. Physiol. 254: H1179–H1186.

2. WITTENBERG, B.A. & J.B. WITTENBERG. 1989. Transport of oxygen in muscle. Annu.Rev. Physiol. 51: 857–878.

3. GAYESKI, T.E.J. & C.R. HONIG. 1991. Intracellular pO2 in individual cardiac myocytesin dogs cats rabbits, ferrets, and rats. Am. J. Physiol. 260: H522–H531.

4. SUGANO, T., N. OSHINO & B. CHANCE. 1974. Mitochondrial functions under hypoxicconditions. The steady states of cytochrome c reduction and of energy metabolism.Biochim Biophys. Acta 347: 340–358.

5. DE GROOT, H., T. NOLL & H. SIES. 1985. Oxygen dependence and subcellular parti-tioning of hepatic menadione-mediated oxygen uptake. Studies with isolated hepato-cytes, mitochondria, and microsomes from rat liver in an oxystat system. Arch.Biochem. Biophys. 243: 556–562.

6. WILSON, D.F., W.L. RUMSEY, T.J. GREEN & J.M. VANDERKOOI. 1988. The oxygendependence of mitochondrial oxidative phosphorylation measured by a new opticalmethod for measuring oxygen concentration. J. Biol. Chem. 263: 2712–2718.

7. RUMSEY, W.L., C. SCHLOSSER, E.M. NUUTINEN, M. ROBIOLIO & D.F. WILSON. 1990.Cellular energetics and the oxygen dependence of respiration in cardiac myocytesisolated from adult rat. J. Biol. Chem. 265: 15392–15399.

8. MÉNDEZ, G. & E. GNAIGER. 1994. How does oxygen pressure control oxygen flux inisolated mitochondria? A methodological approach by high-resolution respirometryand digital data analysis. In Modern Trends in Biothermokinetics. E. Gnaiger, F.N.Gellerich & M. Wyss, Eds. 3: 191–194. Innsbruck University Press. Innsbruck.

9. GNAIGER, E., R. STEINLECHNER-MARAN, G. MÉNDEZ, T. EBERL & R. MARGREITER.1995. Control of mitochondrial and cellular respiration by oxygen. J. Bioenerg.Biomembr. 27: 583–596.

10. COSTA, L. E., G. MÉNDEZ & A. BOVERIS. 1997. Oxygen dependence of mitochondrialfunction measured by high-resolution respirometry in long-term hypoxic rats. Am. J.Physiol. 273: C852–C858.

11. RICHMOND, K.N., S. BURNITE & R.M. LYNCH. 1997. Oxygen sensitivity of mitochon-drial metabolic state in isolated skeletal and cardiac myocytes. Am. J. Physiol. 273:C1613–C1622.

12. KADENBACH, B., J. BARTH, R. AKGÜN, R. FREUND, D. LINDER & S. POSSEKEL. 1995.Regulation of mitochondrial energy generation in health and disease. Biochim. Bio-phys. Acta 1271: 103–109.

13. SNOW, T.R., L.H. KLEINMAN, J.C. LAMANNA, A.S. WICHSLER & F.F. JOBSIS. 1981.Response of cyt aa3 in the situ canine heart to transient ischemic episodes. BasicRes. Cardiol. 76: 289–304.

14. JONES D.P., X. SHAN & Y. PARK. 1992. Coordinated multisite regulation of cellularenergy metabolism. Annu. Rev. Nutr. 12: 327–343.

15. TORRES, J. & M.T. WILSON. 1997. Interaction of cytochrome c oxidase with nitricoxide. Meth. Enzymol. 269: 3–11.


16. ROUSSEAU D.L., S. SINGH, Y.C. CHING & M. SASSAROLI. 1988. Nitrosyl cytochrome coxidase. Formation and properties of mixed valence enzyme. J. Biol. Chem. 263:5681–5685.

17. LARDY, H.A. & H. WELLMAN. 1952. Oxidative phosphorylation: role of inorganicphosphate and acceptor systems in control of metabolic rates. J. Biol. Chem. 195:215–224.

18. CHANCE, B. & G.R. WILLIAMS. 1956. The respiratory chain and oxidative phosphory-lation. Adv. Enzymol. 17: 65–134.

19. ESTABROOK, R.W. 1967. Mitochondrial respiratory control and the polarographic mea-surement of ADP:O ratios. Meth. Enzymol. 10: 41–47.

20. PODEROSO, J.J., M.C. CARRERAS, C. LISDERO, N. RIOBÓ, F. SCHOPFER & A. BOVERIS.1996. Nitric oxide inhibits electron transfer and increases superoxide radical produc-tion in rat heart mitochondria and submitochondrial particles. Arch. Biochem. Bio-phys. 328: 85–92.

21. PODEROSO, J.J., J.G. PERALTA, C.L. LISDERO, M.C. CARRERAS, M. RADISIC, F.SCHOPFER, E. CADENAS & A. BOVERIS. 1998. Nitric oxide regulates oxygen uptakeand hydrogen peroxide release by the isolated beating rat heart. Am. J. Physiol. 274:C112–C119.

22. PODEROSO, J. J., M.C. CARRERAS, F. SCHÖPFER, C. L. LISDERO, N. RIOBÓ, C. GIULIVI,A. D. BOVERIS, A. BOVERIS & E. CADENAS. 1999. The reaction of nitric oxide withubiquinol: kinetic properties and biological significance. Free Radical Biol. Med.26: 925–935.

23. BOVERIS, A., L.E. COSTA, E. CADENAS & J.J. PODEROSO. 1999. Regulation of mito-chondrial respiration by adenosine diphosphate, oxygen, and nitric oxide. Meth.Enzymol. 301: 188–198.

24. BREDT, D.S. & S.H. SNYDER. 1994. Nitric oxide: a physiologic messenger molecule.Annu. Rev. Biochem. 63: 175–195.

25. IGNARRO, L.J., G.M. BUGA, K.S. WOOD, R.E. BYRNS & G. CHAUDHURI. 1987. Endot-helium-derived relaxing factor produced and released from artery and vein is nitricoxide. Proc. Natl. Acad. Sci. USA 84: 9265–9269.

26. MONCADA, S., R.M. PALMER & E.A. HIGGS. 1988. The discovery of nitric oxide as theendogenous nitrovasodilator. Hypertension 12: 365–372.

27. GRANGER, D.L. & A.L. LEHNINGER. 1982. Sites of inhibition of mitochondrial electrontransport in macrophage-injured neoplastic cells. J. Cell. Biol. 95: 527–535.

28. YUI, Y., R. HATTORI, K. KOSUGA, H. EIZAWA, K. KIKI & C. KAWAI. 1991. Purificationof nitric oxide synthase from rat macrophages. J. Biol. Chem. 266: 12544–12547.

29. NATHAN, C. 1992. Nitric oxide as a secretory product of mammalian cells. FASEB J.6: 3051–3064.

30. CLEETER, M.W.J., J.M. COOPER, V.M. DARLEY-USMAR, S. MONCADA & A.H.V.SCHAPIRA. 1994. Reversible inhibition of cytochrome c oxidase, the terminal enzymeof the mitochondrial respiratory chain, by nitric oxide. Implications for neurodegen-erative diseases. FEBS Lett. 345: 50–54.

31. BROWN, G.C. & C.E. COOPER. 1994. Nanomolar concentrations of nitric oxide revers-ibly inhibit synaptosomal respiration by competing with oxygen at cytochrome oxi-dase. FEBS Lett. 356: 295–298.

32. DUCHEN, M.R. 1999. Contributions of mitochondria to animal physiology: fromhomeostatic sensor to calcium signalling and cell death. J. Physiol. (London) 516: 1–17.

33. GIULIVI, C., J.J. PODEROSO & A. BOVERIS. 1998. Production of nitric oxide by mito-chondria. J. Biol. Chem. 273: 11038–11043.

34. TATOYAN, A. & C. GIULIVI. 1998. Purification and characterization of a nitric-oxidesynthase from rat liver mitochondria. J. Biol. Chem. 273: 11044–11048.

35. GIULIVI, C. 1998. Functional implications of nitric oxide produced by mitochondria inmitochondrial metabolism. Biochem. J. 332: 673–679.

36. TAKEHARA, Y., T. KANNO, T. YOSHIOKA, M. INOUE & K. UTSUMI. 1995. Oxygen-dependent regulation of mitochondrial energy metabolism by nitric oxide. Arch.Biochem. Biophys. 323: 27–32.


37. KOIVISTO, A., A. MATTHIAS, G. BRONNIKOV & J. NEDERGARD. 1997. Kinetics of theinhibition of mitochondrial respiration by NO. FEBS Lett. 417: 75–80.

38. BOVERIS, A., E. CADENAS & A.O.M. STOPPANI. 1976. Role of ubiquinone in the mito-chondrial generation of hydrogen peroxide. Biochem. J. 156: 435–444.

39. CADENAS, E., A. BOVERIS, C.I. RAGAN & A.O.M. STOPPANI. 1977. Production ofsuperoxide radicals and hydrogen peroxide by NADH-ubiquinone reductase andubiquinol-cytochrome c reductase from beef-heart mitochondria. Arch. Biochem.Biophys. 180: 248–257.

40. CLEMENTI, E., G.C. BROWN, M. FEELISCH & S. MONCADA. 1998. Persistent inhibitionof cell respiration by nitric oxide: crucial role of S-nitrosylation of mitochondrialcomplex I and protective action of glutathione. Proc. Natl. Acad. Sci. USA 95:7631–7636.

41. GHAFOURIFAR, P. & C. RICHTER. 1997. Nitric oxide synthase activity in mitochondria.FEBS Lett. 418: 291–296.

42. BOVERIS, A., L.E. COSTA & E. CADENAS. 1999. The mitochondrial production of oxy-gen radicals and cellular aging. In Understanding the Process of Aging. E. Cadenas& L. Packer, Eds. :1–16. Marcel Dekker. New York.

43. COSTA, L.E., A. BOVERIS, O.R. KOCH & A.C. TAQUINI. 1988. Liver and heart mito-chondria in rats submitted to chronic hypobaric hypoxia. Am. J. Physiol. 255: 123–129.

44. BOVERIS, A. & E. CADENAS. 1997. Cellular sources and steady-state levels of reactiveoxygen species. In Oxygen, Gene Expression and Cellular Function. L. B. Clerch &D. J. Massaro, Eds. :1–25. Marcel Dekker. New York.

45. BOVERIS, A. & J.J. PODEROSO. 1999. Regulation of oxygen metabolism by nitricoxide. In Nitric Oxide. L. Ignarro, Ed. Academic Press, New York. In press.

46. ZHAO, X.J., V. SAMPATH & W. S. CAUGHEY. 1995. Cytochrome c oxidase catalysis ofthe reduction of nitric oxide to nitrous oxide. Biochem. Biophys. Res. Commun.212: 1054–1060.

Date post:	10-Dec-2023
Category:	Documents
Upload:	independent
View:	0 times
Download:	0 times

Regulation of Mitochondrial Respiration by Oxygen and Nitric Oxide

Documents