Interactions of cytochrome c oxidase with nitric oxide: reactions of the ...

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... J. Torres, C. E. Cooper and M. A. Sharpe. Department of Biological and Chemical Sciences, University of Essex, Wivenhoe Park, Colchester C 0 4 3SQ, Essex,.
Nitric Oxide. Mitochondria and Metabolism

Interactions of cytochrome c oxidase with nitric oxide: reactions of the ‘turnover’ intermediates M. T. Wilson, J. Torres, C. E. Cooper and M. A. Sharpe Department of Biological and Chemical Sciences, University of Essex, Wivenhoe Park, Colchester C 0 4 3SQ, Essex, U.K.

Introduction It is now recognized that in addition to its other well-documented functions NO may act as a powerful, yet reversible, inhibitor of cellular respiration through its interactions with the terminal electron acceptor of the mitochondrion, cytochrome c oxidase [l-31. Whether this role for NO has widespread importance in vivo is still to be established but available data strongly suggest that, in pathological conditions in which NO concentrations are elevated (e.g. sepsis), this inhibition could be significant, even in the presence of O2 at concentrations in excess of 50 pM. Furthermore quantitative analysis of the system in terms of the Ki for NO, KMfor O2 and reasonable estimates of the in vivo concentrations of these gases suggests that NO could modulate the activity of cytochrome c oxidase under normal physiological conditions. The mechanism through which this inhibition is exerted is not fully understood, although it is known that NO competes effectively with O2 for the Fe/Cu binuclear centre of cytochrome c oxidase [2,3] and that the final inhibited form of the enzyme contains the ferrous cytochrome a3.N0 adduct [3]. Previously, we have shown that the reaction of NO with cytochrome c oxidase is complex [4]. This may have been expected because of the rich opportunities for reaction afforded by the ligand-binding/02reduction site of the enzyme. This site comprises two metal centres, the haem iron atom of cytochrome a3 and a copper atom, CuB,which are in close proximity (3..5& [5]. Either metal, and indeed both simultaneously, may in principle bind NO in either of their normal valence states, i.e. ferric or ferrous, cupric or cuprous. It is relatively easy therefore to envisage a number of reactions in which binding of NO to a metal may lead to blocking of access to O2 and/or stabilization of the redox state of this metal, thus impairing electron transfer and giving rise to inhibition. In addition, NO itself may participate in redox reactions at the site depending on the redox state of the metal to which it binds and the availability of electrons donated from the other two metal sites within the enzyme, namely cytochrome a

and Cu,. The picture is further complicated by the fact that once the enzyme enters turnover the binuclear centre harbours a number of partially reduced and possibly reactive O2 intermediates. Two of these intermediates are termed, respectively, ‘P’, a species having an absorption band at 607 nm in the visible difference spectrum (P minus oxidized), and ‘F’, having a peak at 580 nm. The terms ‘P’ and ‘F’ are derived from the assignment of these species as peroxy (P), containing a bound peroxide, i.e. Fe3+Oi-, and ferryl, containing an oxyferryl iron, i.e. [Fe = 01’’ [6]. There remains, however, some controversy over these assignments [7-91. Recently we have attempted to throw light on the complex interactions of NO with cytochrome c oxidase in turnover by exploiting rapidscan stopped-flow methods that allow full spectral information to be collected in the millisecond time range. Here we extend these studies and show that experiments of this type may be useful in identifying intermediates in turnover and help to assign chemical structures to spectral features.

Materials and methods A ‘fast’ form of cytochrome c oxidase [lo] was used in the experiments, although the results were not different using a ‘slow’ preparation. Experiments were performed in a SX-18MV stopped-flow apparatus (Applied Photophysics, Leatherhead, Surrey, U.K.), and spectra were collected using a photodiode array detector with a resolution of 3.3 ms. The data were analysed using Global analysis (Applied Photophysics) or by obtaining internal difference spectra. Both methods gave consistent results. NO was obtained by mixing 1 M H2S04with NaNOz in a Kipps apparatus. After passage through a solution of KI, NaOH, water and a cold trap covered with solid COz, the gas was dissolved in degassed buffer to saturation (approx. 2 mM). The buffer used throughout was 100 mM Hepes/O.S% Tween 80, pH 7.4. The enzyme in turnover was obtained by first fully reducing the enzyme. Cytochrome c oxidase, ruthenium hexamine and sodium ascorbate

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were mixed in the above buffer. When the enzyme was fully reduced, the solution was mixed with 0.5 ml of aerated buffer to reoxidize the enzyme and initiate a steady-state (final concentrations: 8.2 pM cytochrome c oxidase, 1.6 pM ruthenium hexamine and 5 mM ascorbate). After the initiation of this steady-state (typically, the turnover number was 0.5-1 s-I), this solution was rapidly mixed in the stopped-flow apparatus with the solution containing 2 mM NO. Compound P was formed by incubating fast oxidized resting enzyme with CO overnight, after which time the sample was exposed to a flash in the presence of 02.The amount of P present in this sample was typically 80%. Immediately, the sample containing P was mixed in the stoppedflow apparatus with either buffer (control) or the solution containing NO. The concentrations of P and F were calculated using A E ~= l~l 000 ~ -M-'-cm-' ~ ~ ~ and A E ~= 5500 ~ ~ M-':cm-' - ~ ~ respectively ~ [6].

Results T o study the enzyme in 'turnover', the reduced anaerobic enzyme in the presence of excess reductant was mixed with aerobic buffer. This procedure ensures that the enzyme enters turnover from the pulsed or fast state. The rate of

turnover and hence the length of the steady-state (until O2 exhaustion) can be varied by suitable choice of the concentration of the ruthenium hexamine mediator. The species populated in 'turnover' were characterized by recording the spectrum of the steady-state referenced against the pulsed enzyme. By subtracting the spectral contribution of the fraction of the cytochrome a which is reduced in steady-state, the spectrum of the binuclear centre was constructed and is shown in Figure 1. The major feature is a band at 607 nm characteristic of P and constituting some 30% of the enzyme. The remainder of the enzyme appears to be in the oxidized '0' state. This agrees with our earlier EPR results [ 111. On mixing the enzyme in steady-state with NO, a series of rapid spectral transitions was observed. These may be listed as follows. (1) Reduction of a fraction (-10%) of the cytochrome a (k-100 s-I), which we attribute to reaction of NO at the binuclear centre resulting in the ejection of an electron, see Figure 2 (top) [4]. The species responsible for this reaction we have identified as 0, the oxidized centre (Fe3+.Cui+) in the pulsed enzyme. This was confirmed by independent experiments in which freshly prepared pulsed enzyme was mixed with NO and which yielded approx. 50% reduced

Difference spectra obtained after the subtraction of the pulsed oxidized enzyme from cytochrome c oxidase (4 pM) in slow turnover (-0.5 sC') before (----) and after -( ) the subtraction of a spectrum a reduced minus oxidized (-

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cytochrome a and 20% reduced CuA with the same rate constant as observed with the steadystate preparation. (2) There follows a slower reaction (k -8 s-') in which the 607 nm and 580 nm bands are simultaneously bleached (Figure 2, bottom). Reaction of a preparation of compound P with NO leads to the same time course. A remarkable feature of the bottom panel of Figure 2, when compared with Figure 1, is that we see the bleaching of a substantial band at 580 nm on mixing the enzyme in turnover with NO, whereas no such band was evident before mixing. We conclude therefore that a rapid reaction must occur in the dead time of the instrument (-1 ms) which forms F and hence the 580 nm band

Difference spectra obtained after mixing enzyme in turnover (see Figure I ) with 2 mM NO 9, 21 and 40 ms after mixing (with respect to 3 ms) (top) and 200, 300, 550 and 740 ms after mixing (with respect to I60 ms) (bottom)

0.020

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(see also [12]). This hypothesis was confirmed by mixing samples of the enzyme rich in P with NO. It is not possible to prepare 100% P and our samples contained, at most, 80%, the remainder being uncharacterized. On mixing such a preparation with NO we observed that, within the dead time, a spectral change occurred at 580 nm having the features expected for the formation of F (Figure 3) while leaving the 607 nm band, P, unchanged. In a series of experiments of this trpe we found that, when using preparations containing a lower fraction of P or preparations of P that had been allowed to decay partially with time, the fraction of F formed was increased (up to 60% F being formed). We conclude therefore that the 580 nm species, F, is not formed from the 607 nm, P, species but from some uncharacterized species that is spectrally similar to, but chemically distinct from, 0, as 0 itself does not generate F on reaction with NO.

Discussion These complex reactions are difficult to disentangle because of the uncertainty in assigning spectra to species, the fact that distinct chemical species may display similar optical spectra and our lack of knowledge of the products of the reaction. Despite these problems we are able to rationalize the reactions we have described by assuming they share an important common feature, namely that the reaction of NO with the

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Absolute spectra corresponding to a 4.7 pM sample and containing compound P ( 77%) before (-) and difference after mixing with I mM NO (----), spectrum (secondary axis)

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binuclear centre makes an electron available which may then be transferred either to another metal centre or to a bound intermediate. This is best illustrated by the reduction of cytochrome a after the reaction of 0 with NO. A possible mechanism may be as shown in Scheme 1. The oxidized centre binds NO to C u p forming a nitrosonium complex which (by analogy with the Fe2+NO+complexof haemoglobin) on hydration yields H N 0 2 and a proton and leaves the binuclear centre containing a single electron. The proton may be expelled from the hydrophobic interior of the site and, in order to maintain electrical neutrality, an important feature of this site according to Rich [13], an electron must accompany it. The rate of the process (100 s-') may in fact be limited by the rate of proton exit. Under other circumstances the available

NO

Fe3' 0 2 % CUB'' +~-

electron may reduce a bound O2 intermediate. In this way species F can be formed by reduction of a more oxidized species, e.g. peroxide. See Scheme 2 for an example. Here the first step is the same as in Scheme 1 but instead of the electron being transferred to cytochrome a it is transferred to the bound peroxide. This process, because of the vicinity of the peroxide, is rapid, in our case within the dead time of our experiments. A difficulty with this mechanism is that we must postulate a peroxide intermediate, other than P (because the 607 nm-absorbing species does not itself react with NO to give F, see above). An alternative mechanism is to suppose that a species exists in turnover formed from P but spectrally distinct from P (Scheme 3). The species containing Cu;' has been postulated previously. Here we must propose that in the Soret-

Fe3' 02"CUB+NO'

2W

H20 ,ub F e 012' CUB'++ HN02

Spectrally similar to "0'

F (580)

2H' Fe" 0;-C u c ~ P (607)

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[Fe O]2+Cuc b L-, Spectrally similar to "0'

H201 [Fe 012' CuCNO'

H '

[Fe 012'CuB2' F (580)

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and a-band regions of the spectrum, this species is more similar to 0 than to F. This may be considered a weakness of the mechanism. A very similar argument may be invoked to account for the reaction of F with N O which results in the bleaching of the 580 nm band (k-8 s-I). Here, the electron on the CuBatom is transferred to the oxyferryl iron reducing this to the ferric form and leaving the binuclear centre in the 0 state. In the above we have concentrated on the complex reactions between NO and intermediates formed at the binuclear centre and have attempted to rationalize these in terms of a common redox reaction between NO and CuB. T h i s reaction makes available an electron that may be transferred to a variety of sites depending on the state of the binuclear centre. These are, however, only a subset of the total ensemble of reactions. For example, we have paid little attention to other more stable metal-NO complexes, such as F e 2 + N 0 , which we know also play an important role in the inhibition of the enzyme [3,14]. A full appreciation of all the reactions is yet to be achieved, but it is tempting to suppose that within this wealth of reactions are several that play a role in moderating cellular respiration and/or are related to N O metabolism. J.T. acknowledges a fellowship from the Spanish Ministerio de Educacion y Ciencia, and C.E.C. a University Award Fellowship from the Wellcome Trust. We thank Dr. Soulimane and Dr. Buse for the gift of samples of their preparations of cytochrome c oxidase.

1 Cleeter, M. W. J., Cooper, J. M., Darley-Usmar, V. M., Moncada, S. and Schapira, A. H.V. (1994) FEBS Lett. 354,50-54 2 Brown, G. C. and Cooper, C. E. (1994)FEBS Lett. 356,295-298 3 Torres, J., Darley-Usmar, V. M. and Wilson, M. T. (1995)Biochem. J. 312, 169-173 4 Torres, J., Cooper, C. E. and Wilson, M. T. (1996) Biochem. SOC.Trans. 450S,24 5 Tsukihara, T., Aoyama, H., Yamashita, E., Tomizaki, T., Yamaguchi, H., Shinzawa-Itoh, K., Nakashima, R., Yaono, R. and Yoshikawa, S. (1995) Science 269, 1069-1074 6 Wikstrom, M. and Morgan, J. E. (1992)J. Biol. Chem. 267, 10266-10273 7 Weng, L. and Baker, G. M. (1991)Biochemistry 30,5727-5733 8 Fabian, M. and Palmer, G. (1995)Biochemistry 34, 13802- 13810 9 Proshlyakov, D. A., Ogura, T., Shinzawa-Itoh, K., Yoshikawa, S. and Kitagawa, T. (1996) Biochemistry 35,8580-8586 10 Soulimane, T. and Buse, G. (1995)Eur. J. Biochem. 227,588-595 1 1 Wilson, M. T., Jensen, P., Aasa, R., Malmstrom, B. G. and Vanngard, T. (1982)Biochem. J. 203, 483-492 12 Torres, J.and Wilson, M. T. (1997)Biochem. SOC. Trans. 25,402s 13 Rich, P. R. (1995)Aust. J. Plant Physiol. 22, 479-486 14 Giuffre, A., Sarti, P., D'Itri, E., Buse, G., Soulimane, T. and Brunori, M.(1996)J. Biol. Chem. 271,33404-33408 Received 13 March 1997

Induction of the mitochondria1 permeability transition by peroxynitrite M. A. Packer, J. L. Scarlett, S. W. Martin and M. P. Murphy* Department of Biochemistry, University of Otago, Box 56, Dunedin, N e w Zealand

Introduction T h e superoxide radical (0;') is produced as a harmful by-product of metabolism [ 1,2]; however, 0;' itself is not particularly reactive [3]. Abbreviations used: MnSOD, manganese superoxide 1-methyl-4-phenylpyridinium; dismutase; MPP, MPTP, l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine; N O , nitric oxide; NO;, nitrogen dioxide; nNOS, neuronal nitric oxide synthase; Oc', superoxide radical; OH', hydroxyl radical, ONOO-, peroxynitrite, this refers to both the anion and its conjugate acid. *To whom correspondence should be addressed.

Shortly after the discovery that nitric oxide (NO') was a widespread biological messenger [4],Beckman suggested that much of the oxidative damage caused by 0,' followed its reaction with N O to form the damaging oxidant peroxynitrite (ONOO-) [5]. Both 0;' and N O are free radicals therefore they react extremely rapidly (k = 6 . 7 lo9 ~ M-'-s-' [6]) to form ONOO-. T h i s reaction rate is close to the diffusion limit, therefore NO' competes effectively with superoxide dismutases (SODS) for 0;' [7]. As N O is relatively long-lived and membrane-permeant, it diffuses through lipid bilayers to react with 0;'

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