Nitric oxide and cytochrome oxidase: reaction mechanisms from the enzyme to the cell

doi:10.1016/S0891-5849(02)01326-6

Free Radical Biology and Medicine

Volume 34, Issue 5, 1 March 2003, Pages 509-520

https://doi.org/10.1016/S0891-5849(02)01326-6 Get rights and content

Abstract

The aim of this work is to review the information available on the molecular mechanisms by which the NO radical reversibly downregulates the function of cytochrome c oxidase (CcOX). The mechanisms of the reactions with NO elucidated over the past few years are described and discussed in the context of the inhibitory effects on the enzyme activity. Two alternative reaction pathways are presented whereby NO reacts with the catalytic intermediates of CcOX populated during turnover. The central idea is that at “cellular” concentrations of NO (≤ μM), the redox state of the respiratory chain results in the formation of either the nitrosyl- or the nitrite-derivative of CcOX, with potentially different metabolic implications for the cell. In particular, the role played by CcOX in protecting the cell from excess NO, potentially toxic for mitochondria, is also reviewed highlighting the mechanistic differences between eukaryotes and some prokaryotes.

Introduction

In 1990 Carr and Ferguson showed that nitric oxide (NO) catalytically generated by nitrite reductase was able to inhibit the respiration of bovine heart submitochondrial particles [1]. Clear-cut evidence showing that NO controls cell respiration was published in 1994 2, 3, 4. Subsequent work showed that NO can react with several respiratory chain complexes 5, 6, 7, 8, 9. Among these, the interaction with Cytochrome c Oxidase (CcOX) is of particular interest, being rapid (micro-milliseconds time scale) and reversible 10, 11 as it is for many functional modulators, whereas the reaction with the other complexes is slower (time scale of several minutes to hours) or occurs at higher, nonphysiological, NO concentrations (for some complexes in the mM range) 12, 13, 14.

A number of reviews on the biomedical relevance of the NO chemistry has been published in the last few years 15, 16, 17, 18, 19, including an extensive analysis of the interactions between NO and metalloproteins 20, 21. The aim of this work is to review the information available on the molecular mechanisms by which NO reacts with CcOX either isolated in detergent solution, or in respiring cells. Since a role for CcOX in NO degradation has been envisaged 22, 23, this issue will be also reviewed focusing on both eukaryotic and prokaryotic oxidases.

A great body of evidence has been accumulated showing that NO inhibits cell respiration 3, 24, 25, 26 by reacting with CcOX at all integration levels, from the purified enzyme in detergent solution 10, 27 to mitochondria 2, 3, 4, cells 28, 29, 30, 31 and tissues 32, 33, up to in vivo 34, 35. The reaction mechanism(s) involved in the inhibition have been elucidated, although the extent to which each of these is effective at the different integration levels is still a matter of study.

We shall focus on the following main topics:

1)
The mechanisms underlying the control exerted by NO on mitochondrial CcOX.
2)
The role of mitochondrial and prokaryotic oxidases in the degradation of NO.

Section snippets

The structure

Since 1995, the 3D crystallographic structure of several heme-copper oxidases has been solved. This set of structures includes one eukaryotic cytochrome c oxidase (the aa₃ from beef heart, see Fig. 1 and [36]), three prokaryotic cytochrome c oxidases (the aa₃ oxidases from Paracoccus denitrificans [37] and from Rhodobacter sphaeroides [38] and the ba₃ from the Thermus thermophilus [39]), and a quinol oxidase (the bo₃ from Escherichia coli [40]). The view emerging from the structural data is

The mechanisms of interaction of NO with CcOX

In 1955, Wanio first reported that NO reacts with CcOX [56]. In the early 1960s Gibson and Greenwood described the reaction of NO with the reduced enzyme [57], and later Chan and co-workers observed that NO also reacts with the oxidized enzyme [58]. The physiological relevance of these reactions, however, was unknown and like CO, NO was simply used as an efficient but dull electron trapping ligand in heme-proteins reactions 59, 60. More than 10 years later, it was proposed that NO controls the

Attempting to depict an overall picture

All together, the evidence summarized above suggests that the electron transfer rate through the mitochondrial respiratory chain is an important parameter driving the NO-to-CcOX interaction towards accumulation of either the nitrosyl- or the nitrite-derivative of the enzyme. These adducts are both inhibited but follow different metabolic fates. To better clarify this point, we shall distinguish two extreme electron flux regimes (high and low) through the respiratory chain (Fig. 7).

No scavenging in the cell

Cells producing NO as a messenger, activator, or modulator are faced with its potential mitochondrial toxicity. It may be recalled that a consequence of the pathway leading to accumulation of the nitrosyl-adduct is that, once dissociated from the active site, NO would be released in the cell cytoplasm as such. NO free in the cell environment for a long enough time can ultimately induce apoptosis and cell death 18, 93. This is expected based on the finding that NO not only downregulates the

The contribution of CcOX to NO degradation

While the capacity of CcOX to oxidize NO to nitrite has never been controversial, doubts have been raised about the property of CcOX, particularly the mammalian, to metabolize NO to N₂O via a reductive pathway. The existence of a marginal NO- and NO₂⁻-reductase activity of mammalian CcOX was first proposed by Brudvig et al. [58] and later revisited by others 22, 104, 105, stimulated by the newly discovered implications of NO in the mitochondrial physiology. The capability of CcOX to catalyze

The reduction of NO to N₂O by prokaryotic heme-copper oxidases

In terms of NO reductase activity, heme-copper oxidases purified from Thermus thermophilus have been found to behave very differently from mammalian CcOX [61]. This microorganism expresses the ba₃ cytochrome oxidase when grown under conditions of low O₂ tension [109]. The Cu_B⁺ in this oxidase displays an unusually high affinity for CO, compared with mesophilic oxidases (K_aff = 10³–10⁴ M⁻¹ vs. K_aff = 10¹–10² M⁻¹) 110, 111. If extended to O₂ and NO, the higher affinity of Cu_B⁺ for these ligands

Abbreviations

CcOX—cytochrome c oxidase
7-N—7-nitro-indazole
NMDA—N-methyl-D-aspartate
NOR—bacterial NO reductase
NOS—NO synthase

Acknowledgements

Work supported by Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) of Italy (PRIN “Bioenergetica: aspetti genetici, biochimici e fisiopatologici” and Prg. Biotecnologie 5% - Neuroscienze). The research grant “Marker periferici e danno mitocondriale da nitrossido nelle demenze”, by the University of Rome “La Sapienza”, is also gratefully acknowledged. We wish to thank Victor Darley-Usmar (University of Alabama, USA) and Andrea Urbani (University of Chieti, Italy) for stimulating

Paolo Sarti and Maurizio Brunori received their M.D. degrees from the University of Rome in 1972 and 1961, respectively; they are full Professors of Chemistry and Biochemistry in the Faculty of Medicine Ist and IInd of the University of Rome “La Sapienza”. Alessandro Giuffr, graduated in Biology at the University of Rome “La Sapienza” in 1993, was awarded the Ph.D. in Biochemistry in 1997; he is Researcher at the Institute of Molecular Biology and Pathology of the Consiglio Nazionale delle

References (117)

G.J Carr et al.
Nitric oxide formed by nitrite reductase of Paracoccus denitrificans is sufficiently stable to inhibit cytochrome oxidase activity and is reduced by its reductase under aerobic conditions
Biochim. Biophys. Acta
(1990)
M.W.J Cleeter et al.
Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide
FEBS Lett
(1994)
G.C Brown et al.
Nanomolar concentration of nitric oxide reversibly inhibit synaptosomal cytochrome oxidase respiration, by competing with oxygen at cytochrome oxidase
FEBS Lett
(1994)
M Schweizer et al.
NO potently and reversibly de-energizes mitochondria at low oxygen tension
Biochem. Biophys. Res. Commun
(1994)
J.B Hibbs et al.
NOa cytotoxic-activated macrophage effector molecule
Biochem. Biophys. Res. Commun
(1988)
J.J Poderoso et al.
The regulation of mitochondrial oxygen uptake by redox reactions involving nitric oxide and ubiquinol
J. Biol. Chem
(1999)
R Welter et al.
The effects of NO on electron transport complexes
Arch. Biochem. Biophys
(1996)
L.L Pearce et al.
Identification of respiratory complexes I and III as mitochondrial sites of damage following exposure to ionizing radiation and nitric oxide
Nitric Oxide
(2001)
C.E Cooper
Nitric oxide and iron proteins
Biochim. Biophys. Acta
(1999)
J Torres et al.
The reactions of copper proteins with nitric oxide
Biochim. Biophys. Acta
(1999)

R.P Patel et al.

Biological aspects of reactive nitrogen species

Biochim. Biophys. Acta

(1999)

N Hogg

Biological chemistry and clinical potential of S-nitrosothiols

Free Radic. Biol. Med

(2000)

F Malatesta et al.

(1998)

A.P Halestrap et al.

Oxidative stress, thiol reagents, and membrane potential modulate the mitochondrial permeability transition by affecting nucleotide binding to the adenine nucleotide translocase

J. Biol. Chem

(1997)

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Original contributionNitric oxide and cytochrome oxidase: reaction mechanisms from the enzyme to the cell

Abstract

Introduction

Section snippets

The structure

The mechanisms of interaction of NO with CcOX

Attempting to depict an overall picture

No scavenging in the cell

The contribution of CcOX to NO degradation

The reduction of NO to N2O by prokaryotic heme-copper oxidases

Abbreviations

Acknowledgements

Biochim. Biophys. Acta

FEBS Lett

FEBS Lett

Biochem. Biophys. Res. Commun

Biochem. Biophys. Res. Commun

J. Biol. Chem

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FEBS Lett

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J. Biol. Chem

J. Inorg. Biochem

J. Mol. Biol

Biophys. Chem

Biochim. Biophys. Acta

J. Biol. Chem

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Free Radic. Biol. Med

J. Biol. Chem

Biochim. Biophys. Acta

J. Biol. Chem

Biochem. Biophys. Res. Commun

FEBS Lett

J. Biol. Chem

Biochem. Biophys. Res. Commun

Biochem. Biophys. Res. Commun

FEBS Lett

Arch. Biochem. Biophys

FEBS Lett

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

Biochem. Biophys. Res. Commun

Biochem. Biophys. Res. Commun

Biochem. Biophys. Res. Commun

Biochem. Biophys. Res. Commun

Biochem. Biophys. Res. Commun

FEBS Lett

J. Biol. Chem

J. Biol. Chem

Biochem. Biophys. Res. Commun

Biochem. Biophys. Res. Commun

Biochim. Biophys. Acta

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

Original contribution
Nitric oxide and cytochrome oxidase: reaction mechanisms from the enzyme to the cell

The reduction of NO to N₂O by prokaryotic heme-copper oxidases