KINETIC AND STRUCTURAL MODEL FOR THE BINDING OF FORMATE TO THE RAPID FORM OF CYTOCHROME-C-OXIDASE

The binding of formate to the rapid form of cytochrome c oxidase from bovine heart has been examined at pH 8.8 and high ionic strength. The optical changes included (1) a transient decrease at 414 nm, followed by a biphasic increase, and (2) an isosbestic wavelength. The apparent blue shift in the Soret envelope, following the transient, was consistent with a 430 --> 414 transition in cytochrome a(3), described previously for acid jump conditions in the absence of formate [Papadopoulos, P. G., Waiter, S. A., Li, J., and Baker, G. M. (1991) Biochemistry 30, 840-850]. A two-step binding mechanism was implied by the biphasic increase, but the k(obs) values for each phase, when plotted against formate concentration, were unable to statistically discriminate two rival kinetic models. Both models postulated a rapid 430 + L reversible arrow 414.L step (where L = HCOOH + HCOO-), but they differed in whether the slower step depended on L. The equilibrium dissociation constant, K-D(app), the overall binding reaction was 0.3 mM. An analysis of the rival mechanisms indicated this value to be consistent with a step that was independent of L. A slow 414.L reversible arrow 414'.L conversion was therefore postulated, and the K-eq for this step was found to be approximate to 9. The analysis to this point assumed that cytochrome a(3) was entirely in the 430 state at the time of formate addition. Modeling of the transient could not be achieved, however, unless cytochrome a(3) was present as a rapid equilibrium mixture of 414 and 430 states. The 414, 414.L, and 414'.L states were assumed to be electronically identical to account for the isosbestic wavelength. Numerical simulations of a four-state binding model provided a good fit at both 1 and 25 mM formate and further suggested that the 414'.L state may be the slow conformer that is detected by cyanide. A structural model consistent with these kinetic constraints is proposed.