The reaction of halides with cytochrome bo from ...

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series, i e fluoride, chloride. bromide and iodide, are ligands for cytochrome ho ... obtained for potassium chloride. sodium bromide and potassium iodide,.

62s Biochemical Society Transactions (1997) 25 The reaction of halides with cytochrome ho from Escherichia coli.

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A JOHN MOODY and PETER R RICH Glynn Research Foundation, Glynn, Bodmin, Cornwall, PL30 4AU. U K Like bovine cytochrome c oxidase. E. colr cytochrome ho is a member of the superfamily of terminal oxidases in which the site of oxygen binding and reduction is a binuclear haedCu centre Fluoride is a well-known ligand of the binuclear centre haem iron in both bovine cytochrome oxidase and 6. coh cytochrome ho [1.2]. and we have previously described the effects of chloride on the nearUVhisible spectrum and the reactivity of the binwlear centre of bovine cytochrome oxidase [3,4.5]. We have also proposed that an equivalent chlorideligated form of cytochrome ho may be present in some purified preparations of the enzyme [6] Here, using the same sorts of criteria, we report that all the halide series, i e fluoride, chloride. bromide and iodide, are ligands for cytochrome ho Cytochromebo was purified [7] in the fast form from the over-producing 6;. coh strain RG I45 Fig. I shows the binding spectra of fluoride and chloride The binding spectrum of chloride is almost identical to that of wide in the visible region In contrast, the binding spectrum of fluoride in the visible region is the inverse of the chloride-binding spectrum The binding spectra of bromide and iodide are essentially identical to that of chloride The position of the "630 nm" charge transfer band is shifted to the blue by fluoride and to the red by chloride, bromide and iodide, but its retention in all cases indicates that the halides form high spin complexes with cymchrome ho Incubation of cytochrome ho with 24 mM potassium sulphate has no significant effect on the spectrum of the enzyme Fluoride binding to cytochrome ho (monitored using AA~*)~-II I ,-J is monophasic The effect of pH on the binding kinetics is consistent with !qq,being modulated by an acidhase group with a pK, of 3, i e. the pK. of hydrofluoric acid and. on this basis, values of k, of 4 3 (i0 8) x 10' and 0 59 (* 0 25) M I s.'. are obtained for hydrofluoric acid and fluoride, respectively In contrast, is pHindependent within experimental error (3-6 x 10" s.') When monitored in the Soret region. e.g using the kinetics of chloride. bromide and iodide binding are clearly biphasic. whereas in the visible region. e.g. using A&j6420mr the kinetics are monophasic. and correspond to the slower of the two kinetic phases seen in the Soret region Both the spectral changes associated with the fast phase in the Soret region. and the response of the kinetics of this phase to halide concentration are uncertain at present The rate of the slow phase, however, is certainly affected by the halide concentration The order of reactivity is chloride > bromide > iodide. For example at pH 5 5. with

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Fig I PitTerence spectra Tor the bindine of fluoride. chloride and Pzide

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PH Fig 2 chloride bindinn to E. co/i cvtochrome ho. A Plots of k 6 5 6 4 2 0 - (0) and k t b (0)versus [KCI] at pH 5 5. and 20 "C B Plots of k6E6J6620,,", (0) and kchs(0) versus pH with 30 mM KCI at 20 "C

1 15 mM halide. values of kA of 3 9 x 10.". I I x 10" and 0 S x 10'' s-' are obtained for potassium chloride. sodium bromide and potassium iodide, respectively Under the same conditions, assuming that the spectral changes induced by chloride. bromide and iodide are identical. the percentage saturation of binding is 100, 90 and 80, respectively For chloride at least, the binding kinetics are not affected by the cation. LiCI, KCI. and NH4CI all give the same result Fig. 2A shows the effect at pH 5 . 5 of chloride concentration on the observed rate constant (k&) and on the k 6 5 6 4 2 0 observed at equilibrium Although ksb. and'hence kcm,shows saturation behaviour. fitting a rectangular hyperbola to the latter data gives an approximate value (2 I ?r 0 3 mM) for the dissociation constant, K d , of chloride. Extrapolation of the k,, versus [KCI] data on this basis gives a value for koK of 2 1 x lo4 s-'. Fig 2B shows the effect at 30 mM chloride of pH on k,,b and A E ~ ~The ~ decrease ) ~ ~in kch . as pH increases from 5 S to 7 0 is consistent with the rate of reaction being principally determined by the concentration of HCI Chloride-ligated cytochrome ho binds cyanide about 10 fold slower than the "unligated enzyme for which k, at pH 6 0 with 0 2-1 mM KCN IS about 37 M i s.' at 20 "C Hence, the rate of cyanide binding can exceed k,,,, for chloride This is reminiscent of the effect on chloride on cyanide binding to bovine cytochrome oxidase 131 In summary, all four halides form high-spin complexes with cytochrome ho, the net bound species in case probably being the protonated form The different spectral effect of fluoride compared with the rest is unsurprisiny given the "anomalous" behaviour of first row elements

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This work was fknded by the BBSRC (grant ref GWJ28 148)

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& coh' [email protected] b q A baseline scan using buffer (50 mM potassium phosphate, pH 7 0. for fluoride and azide. or 50 mM K-MES, pH 5 . 5 . for chloride, both containing 0 5 mM potassium EDTA) was taken. Purified fast enzyme was then diluted to 3-6 pM taken to be 233 mM".cm") in the buffer and a scan taken. Finally the ligand was added and a hrther scan taken after sufficient time for complete binding. 2 min with 20 mM NaF, immediately with 2 mM NaN,, and 25 min with 1 I5 mM KCI,all at 20 "C

Muijsers. A 0 , Van Buuren. K J H & van Gelder. B F ( 1974) Biochim Biophys Acta 333.430-438 Watmough. N J , Cheesman. M R , Gennis, R B . Greenwood, C & Thomson. A J ( 1993) FEBS Lett 3 19. I 5 I I 54 Moody, A J , Cooper, C E & Rich. P R (1991) Biochim Biophys Acta 1059. 189-207 Moody, A J , kchardson, M , Spencer, J P E , Brandt U & Rich P R (1994) Biochem J 302, 821-826 Moody, A J (1996) Biochim Biophys Acta . in press Moody. A I , Rumbley. J N . Gennis. R B . Ingledew. W J & Rich, P R (1993) Biochim Biophys Acta 1141. 321-329 Moody, A J & Rich, P R (1994) Eur J Biochem 226. 73 1-737 ~

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