Department of Biological Sciences, University of Essex,. Wivenhoe Park, Colchester, Essex, CO4 3!3Q, U. K. INTRODUCTION. NO is both a cellular messenger ...
Biochemical Society Transactions (1998) 26 24
Nitric oxide as an effector and a substrate for cytochromec oxidme Peter Nicholls, Martyn Sharpe, Jaume Torres, Michael T. Wilson and Christopher E. Cooper Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex, CO4 3!3Q, U. K. INTRODUCTION NO is both a cellular messenger and an inhibitor of cytochrome aa3. Previous studies (1-4)have shown that it can react either with CU$+ or with cyt. a32+. In its final inhibited state the binuclear centre is in the reduced cyt. u~~+NOCUB+ form but the immediate product of NO interaction with the enzyme is the oxidized cyt. a33+NOCugz+form in which the femc iron remains available for ligation by other species such as cyanide. How does the latter species give rise to the former? NO is also metabolised by the oxidase in its steady state. This metabolism has been variously attributed to redox reactions involving either reduction of NO (Eq. 1) or oxidation (Eq.2).Which is the more important pathway? 2NO + 2e- +W+ =-N f l + H 2 0 ..(l) NO + OH- * N@- + e- .. (2) METHODS (a) The enzyme was beef heart cytochrome c oxidase prepared by one of several versions of the method of Yonetani as modified by Kuboyama, Yong and King. The oxidase was 'pulsed' by being treated with Na2S204 followed by removal of excess dithionite on an aerobic G 25 column in the presence of catalase. (b) Nitric oxide was obtained either from compressed NO gas or using a Kipps apparatus with acid-treated sodium nitrite as reagent. In each case the NO gas was passed through a set of alkaline 'saubbers' to remove N 2 Q which gives rise to HN@ in solution before being dissolved at a final concentration of 1-2mM NO in Nzpurged buffer. RESULTS AND DISCUSSION Fig. 1shows the steady state of cytochrome aa3 and the effect of NO addition during that steady state. An immediate reduction of cyt.a is followed more slowly by the appearance of fully reduced a 3 2 + N o c u ~ +
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Cyt. a reduction indicates inhibition of the a -> a3 electron transfer step. Yet the agF$+NO species is not seen and values of k, and Ki indicate a reaction rate - if a$&+ were the NO target - greater than the directly measured rate of = 108 M-lsecrl. What other oxidase species may be involved in reaction with NO? After NO interaction with pulsed enzyme (compound '0')and even in the absence of other reductants, an early event is cyt.a reduction together with some perturbation of the femc a3 spectrum (4). When NO reacts with compound F (the supposed ferry1 enzyme) the product is a perturbed fully oxidized enzyme. What is the species responsible for the spectral change?
"P Fig. 2. Proposed NO & NO2- reactions with cyt. c oxidase. ~
It is identical with N@--complexed femc enzyme ('CY), obtained by adding excess sodium nitrite to pulsed enzyme. NO oxidation in the heme pocket gives rise to the same species obtained by binding exogenous nitrite. Fig. 2 summarises these reactions. For all three oxidized enzyme forms the initial NO reaction is with Cug2+(1-3). One-electron oxidation of NO to NOzgenerates ferric-nitrite enzyme from either the '0' or 'F' species. The NO reaction of compound 'P is more complex (5). In each case the redox step requires the hydration of the NO+ ion by a nearby water or OH- ion. 'The availability of this species may account for differences in reactivity shown by different states of the oxidase, both in our studies and previously. One-electron oxidation of NO may be a adaptive way to remove NO from the system just as NO binding to the reduced binuclear centre may be one way in which NO modulates cell function. Cytochrome oxidase is both possible target and control enzyme for this unique hormone. REFERENCES 1. Brudvig, G., Stevens, T. H. and Chan, S. I. (1980) Biochemistry 19,52755285. 2. Hill, B.C.,Brittain, T., Eglinton, D. G., Gadsby,P. M. A., Greenwood, C., Nicholls, P., Peterson, J., Thomson, A. J. and Woon, T. C. (1983).Biochem. J. 215,574. 3. Boelens, R., Wever, R., Van Gelder, 8. F., and Rademaker, H. (1983). Bioch. Biophys. Acta 724,176-183. 4. Cooper, C. E., Torres, J., Sharpe, M. A. and Wilson, M.T. (1997) FEBS letters 414,281-284. 5.Torres, J.,Cooper, C. E., and Wilson, M.T. (1997) J. Biol. Chem. 273,8756-8766.
