The cytochrome oxidase g'=12 EPR signal

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#Glynn Research Institute, Bodmin, Cornwall, PL30 4AU, UK. Purified mitochondria1 cytochrome c oxidase can exist in at least two forms. One form ("resting") ...
Biochemical SocietyTransactions ( 1 99 1 ) 19 The cytochrorne oxidase g'=12 EPR signal CHRIS E. COOPER, A. JOHN MOODY#, PETER R. RICH#, JOHN M. WRIGGLESWORTH AND NIKOLAOS IOANNIDIS. Division of Biomolecular Science, King's College London, Campden Hill Road, London W8 7AH, UK #Glynn Research Institute, Bodmin, Cornwall, PL30 4AU, UK. Purified mitochondria1 cytochrome c oxidase can exist in at least two forms. One form ("resting") has slow intramolecular electron transfer rates and cyanide bindng kinetics. Reduction and re-oxidation of the resting enzyme produces a "pulsed" enzyme that has faster intramolecular electron transfer and cyanide binding kinetics [ I ] . This form eventually decays to the "resting" enzyme. The rate of this decay is increased by lowering the pH 121 and decreased by any source of electrons. The addition of formate to "pulsed" oxidase also induces the formation of a resting-type enzyme. Cytochrome c oxidase contains four EPR detectable metal centres - CUA, hnem a , haem (13 and Cue. In the fully oxidized enzyme signals are always observed from CUAand haem a [3]. In pulsed (fast) enzyme the binuclear haem a3 /Cue centre is EPR silent, presumably due to antiferromngnetic coupling between the two nearby metal centres. Reduction of CUBbreaks this coupling and allows detection of the EPR signal from high spin haem a3 . It is also possible to observe Cue signals under conditions where CUBis oxidized and haem a3 reduced, allowing detection of the copper (11) EPR signal [4,5]. In resting (slow) forms of the enzyme an EPR signal is observed from the binuclear haem aJCu, centre. This is a broad low field resonance - the so-called "g'=12" signal [61. It is atributed to an S=2 spin system in the binuclear centre. All enzyme preparations that contain this signal exhibit a slow rate of intramolecuhr electron transfer from haeni a /CUAto the binuclear centre following dithionite addition. Therefore if dithionite is added to these enzymes and they are frozen rapidly (10-15 seconds) optical spectra show that both haem a and CUAare reduced, whilst the vast majority of haem a3 remains oxidized [7]. EPR spectra show that CUAis fully reduced under these conditions and that there is no significant decrease in the g'=12 signal. However, despite the complete appearance of reduced haem a when observed optically, there remains an EPR signal at g=2.9, close to where the oxidized low spin haem a EPR signal is observed (g=3). The same signal is seen upon addition of reductant to the formate-inhibited enzyme [8]. In an attempt to resolve this anomaly we have studied the power and temperature dependence of this broad g=2.9 EPR signal. Figure 1 shows a microwave power saturation study of the different EPR signals present in the formate-inhibited enzyme immediately following dithionite addition. It can be seen that, like the g=12 signal, even at temperatures as low as 5K the g=2.9 signal is barely saturated at high power. For comparison, data from the the g=6 haem a3 signal and the g=3 haem a signal are shown. Both these saturate more readily than the g=12/g=2.9 signal. Furthermore like the g=12 signal (and unlike the g=3 signal) the g=2.9 signal is not detectable at high temperatures (>30K). The g=2.9 component is therefore likely to be part of the anisotropic S=2 g'=12 signal. We conclude that the EPR spectra are in agreement with the optical spectra and that following dithionite reduction of haem a there is complete disappearance of the g=3 EPR signal. This allows the smaller g=2.9 signal underlying the g=3 signal to be observed. Indeed it is possible to detect the g=2.9 signal in fully oxidized enzyme as a distortion of the lineshape of the g=3 signal. This distortion only occurs at the lower temperatures and higher microwave powers where there is a significant g'=12 signal.The g'=12/g=2.9 signal is a marker for the resting enzyme and correlates with an enzyme population where haem a3 reacts slowly with added ligands (e.g. cyanide). Therefore the fact that the halfreduced enzyme still contains this signal argues against a model where the reduction of haem a and CUAalone converts the enzyme from a "closed" to an "open" conformation [9]. Our current hypothesis is that this conversion requires the further reduction of Cue. This reduction displaces an intrinsic (resting) or added (formate-inhibited) bridging ligitnd between haem a3 and CUB, making haem a3 available for rapid ligwd binding.

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Fig. 1 Microwave Dower saturation curves of cvttxhrome oxidase EPR signals at SK Formate-inhibited cytochrome oxidase was prepared by incubation of "fast" enzyme with 5mM potassium formate for 1 hour at pH 6.4. Then 20mM buffered sodium dithionite (pH 6.5) w and the sample in the EPR tube was frozen 1 0 seconds later i n liquid methanol cooled in a liquid nitrogen bath. EPR signals were observed at g=12, g=2.9 and g=6. The power saturation curves were obtained by varying the microwave power at SK and measuring the signal intensity. When log signal/(PowerI/*) is independent of log power there is no power saturation. The g=3 curve was obtained by using a sample of fast oxidase (where no g=12 or g=2.9 signal is present). The data were normalized such that all points are coincidental at log power = 0.5. Similar correlations were observed when "resting" rather than formateinhibited enzyme was used. This work was supported by SERC grant GR/F/17605 to PRR and a King's College Research Fellowship to CEC. 1 . Antonini, E., Brunori, M., Colosimo, A,, Greenwood, C. and Wilson, M. (1981) Proc. Natl. Acad. Sci. USA 78, 71 15-

7118. 2. Baker, G.M., Noguchi, M. and Palmer, G. (1987) J . Biol. Chem. 262, 595-604. 3. Aasa, R., Albracht, S.P.J., Falk, K-E., Lanne, B. and Vanngaard, T. (1976) Biochini. Biophys. Acta. 4 2 2 , 260272. 4. Reinhammer, B., Malkin, R., Jensen, P., Karlsson, B., AndrCasson, L-E., Aase, R., VBnngiard, T. and MalmstrBm, B.G. (1980) J. Biol. Chem. 255, 5000-5003. 5 . Witt, S.N., Blair, D.F., and Chan, S.I. (1986) J. Biol. Chem. 261. 8104-8107. 6. Brudvig, G..W., Stevens, T.H., Morse, R.H. and Chan, S.I. (1981) Biochemistry, 20, 3912-3921. 7 . Wrigglesworth, J.M., Elsden, J., Chapman, A,, Van der Water, N.and Grahn, M.F. (1988) Biochim. Biophys. Acta. 936, 452-464. 8 . Boelens, R., and Wever, R. (1979) Biochim. Biophys. Acta. 547, 296-310. 9 . Jensen, P., Wilson, M.T., Aasa, R . and Malmstriim, B.G. (1984) Biochem. J. 224, 829-837.