c oxidasc. A JOHN MOODY, FRITHJOF von GERMAR'. WERNER MANTELE' and PETER R. RICH. Glynn Research Foundation, Bodmin, Cornwall, PL30 4AU.
BiochemicalSocietyTransactions ( 1 995) 23
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Rapid-scan FITR spectnncopic studies on the photolysb and recombination of the cyanide adduct of fully reduced bovine cytochrome c oxidasc
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A JOHN MOODY, FRITHJOF von GERMAR'. WERNER MANTELE' and PETER R. RICH
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Glynn Research Foundation, Bodmin, Cornwall, PL30 4AU. U K. *Institut fiir Physikalische und Theoretische Chemie. Universitat Erlangen. 91058 Erlangen, Germany. The site of oxygen reduction in bovine cytochrome c oxidase is a binuclear centre consisting of haem q and CUB.Much of our knowledge of the relationship between the haem and the Cu in bovine cytochrome oxidase, as well as the other members of the superfamily of h a d C u terminal oxidases, has come from the ligand-binding dynamics of the binuclear centre For this purpose, carbon monoxide, which binds to the haem when the binuclear centre is fully reduced, has been particularly usefid because the Fe-CO bond can be photolysed with a quantum yield of one (seee.g. [ 11). The Fe-CK bond of the cyanide adduct of the fully reduced oxidase can also be photolysed, and. although the quantum yield is much lower [2], this system is potentially more interesting because of the requirema of a charge compensating proton movement 131. We report here the preliminary results of studies using Fourier Transform I&-Red (FTIR) spectrscopy on the photolysis and recombination of the ferrous cyanide adduct. Cytochrome oxidase (about 200 pM, nominal pH 8 5) was concentrated on ice to about 1 mM using a gentle flow of argon Concentrated enzyme (50 pI) was placed in a small glass tube (internal diameter about 2 mm) and 5 pl of freshly prepared 1 M sodium dithionite were added to the top The surface was flushed with argon and the tube sealed with Parafilm M e r mixing, the enzyme was allowed to reduce for 15 min on ice Potassium cyanide (10 pI of 1 M, pH 8-8.5) was then added in the same way as for the dithionite. For the FTIR sample from which the results shown here were obtained, 3 pl of this stock were placed on a CaF2 plate and spread under a gentle stream of argon. M e r about 30 s, when the enzyme had concentrated further, the second plate was placed on top and the unit sealed. The FTIR measurements were carried out at 1 "C using a customised Bruker spectroneter Photolysis of the cyanide adduct was achieved using a UV and
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Time (s) Fig 2 Liuand stretch modes after ohotolvsis of the cvamde adduct of fully mochrome oxidase A Averaged FTIR difference spectra for 75 flashes at the times indicated after each flash The offset section shows the peak at 1963 cm I that can be seen in the spectra averaged between 5 44 and 14 80 s B The time course for the disappearance of the troughs at 2059 and 2045 cm
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IR-filtered Xenon flash. Optical spectra taken before and after the FTIR measurements confirm the presence of the cyanide adduct of the fblly reduced enzyme (the shoulder at 590 nm. compare Fig IA with Fig IB) At a nominal concentration of 150 mM we would expect complete formation of the adduct (Kd is between 0 1 and 1 KIM [3,4]). However, the saturation appears to be only 70-80% This discrepancy is probably caused by partial oxidation of haem a3. M e r photolysis of the cyanide adduct two negative absorption bands are seen at 2059 and 2045 cm.' in the kinetic FTIR spectra (Fig 2A) These both decay at a rate consistent with the kinetics of cyanide recombination observed by optical spectroscopy [3]. One of them matches the band at 2058 cm.', Seen by Yoshikawa & Caughey [ 5 ] in static IR spectra, which was was lost in the presence of CO. and hence was ascribed to haem-bound C N The other matches the band seen at 2042 cm'l in static spectra, which was ascribed to CIT bound to an intrinsic metal [5]. There appears to be a positive absorption band following photolysis in the range 2075-2100 cm.', which could be caused by the appearance of HCN (2093 cm") in the bulk phase. However. the kinetic spectra in this region may be complicated by changes in a band at 2092cm" ascribed by Yoshikawa & Caughey [5] to Cu$-CN A small positive absorption seen at 1963 cm'l is in the same position as that reported for the CO adduct of fully reduced cytochrome oxidase [6], and may be caused by the transient binding of adventitious CO to haemu, aAer the photolysis of the cyanide adduct.
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A.J M. is grateful to the BBSRC for a grant under the 1994 International Scientific Interchange Scheme (Ref B6 131123) 1.
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of r e d u xi d i Fig 1 Q&al -duct A The FTIR sample bath-I = 13.5 pm, therefore [enzyme] = 1 7 mM B Control spectrum (solid line) at pH 7 5, w t h 40 mM KCN and 4 pM enzyme @ath length = 1 cm) The dashed spectra are calculated using a specuum from the same sample but wrthout cyanide (not shown)
Lemon. D.D., Calhoun, M.W. Gennis, R.B & Woodruff, W.H (1993)
Biochemistry 32, 11953-11956 2. Hill, B.C. & Marmor, S (1991) Biochem I. 279. 355-360 3. Mitchell, R., Moody, A J & Rich, P R (1995) Biochemistry, in press 4. Jones, M.G., Bickar, D.. Wilson, M.T., Brunori, M., Colosimo, A. & Sarti, P. (1984) Biochem. J. 220.57-66 5. Yoshikawa. S. & Caughey, W S. (1990) J. Biol. Chem. 265. 7945-7958. 6. Caughey, W . S . Dong, A.. Sampath, V.. Yoshikawa, S . & Zhao,X.-J (1993)1.Bioenerg. Biomemb. 25. 81-91
