The presteady state reaction of chemically modified cytochromes c with cytochrome oxidase (original) (raw)

Journal of Biological Chemistry

Determination of the presteady state kinetics of the reaction of purified beef cytochrome oxidase and of the high affinity complex between native horse cytochrome c and the beef oxidase with native 4-carboxy-2,6-dinitrophenyllysine horse cytochromes c singly modified at residue 7, 13, 25, 27, 60, 72, 86, or 87 and 2,4,6-trinitrophenyllysine horse cytochrome c modified at residue 13, demonstrated that 1) the high affinity interaction domain on the surface of cytochrome c mapped in this fashion is indistinguishable from that determined by the steady state polarographic kinetic analysis (Ferguson-Miller, S., Brautigan, D. L., and Margoliash, E. (1978) J. Biol. Chem. 253, 149-159). 2) The low affinity enzymic interaction domain is indistinguishable from the high affinity domain. 3) The inhibition of the low affinity reaction, observed with the modified cytochromes c as measured by the presteady state assay is greater than that determined by the steady state polarographic kinetics. This results from the fact that the two sites are interacting (Osheroff, N., Speck, S. H., Margoliash, E., Verrman, E. C. I., Wilms, J., Konig, B. W., and Muijsers, A. 0. (1983) J. Biol. Chem. 258,5731-5738) and that for the presteady state kinetics the high affinity site is occupied by a tightly bound native cytochrome c, while for the polarographic assay it is occupied by a more weakly bound modified cytochrome c. 4) The bimjolecular rate constant (k,) for the reaction of 4-carboxy-2,6-dinitrophenyllysine 13 horse cytochrome c with beef cytochrome oxidase varies with ionic strength. From a plot of In k1 as a function of P" the rate constants at zero and infinite ionic strength were estimated. These values were used to calculate that the effective charge on purified beef cytochrome oxidase is in the range of -8 to -10.5e. 5) The effect of 32 30 29 28 29 24 23 21 Determined polarographically using submitochondrial particle cytochrome oxidase (15,16); 25 m~ %/acetate, pH 7.8, 250 m~ sucrose, 25 "C. e Determined using purified cytochrome oxidase; 25 m~ Tris/acetate, pH 7.8,0.25% Tween 20,25 "C (16). Determined in the presence of excess cytochrome c . cytochrome oxidase complex; cf. Fig. 3; 5 m~ potassium 1 cal = 4.184 J. Monophasic kinetics was observed (15). phosphate, pH 7.0,0.5% Tween 20, 10 "C. by guest, on January 5, 2012 www.jbc.org Downloaded from