The rate‐limiting step and nonhyperbolic kinetics in the oxidation of ferrocytochrome c catalyzed by cytochrome c oxidase

Abstract

The level of reduction of cytochrome a and cua during the oxidation of ferrocytochrome c has been determined in stopped‐flow experiments. Both components are partially reduced but become progressively more oxidized as the reaction proceeds. When all cytochrome c has been oxidized, cua is also completely oxidized, whereas cytochrome a is still partially reduced. These results can be simulated on the basis of a model which requires that the intramolecular electron transfer from cytochrome a and cua to cytochrome a 3‐Cub is a two‐electron process and, in addition, that the binding of oxidized cytochrome c to the electron‐transfer site decreases the rate constants for intramolecular electron transfer from cytochrome a. The first requirement is related to the function of the oxidase as a proton pump. Product dissociation is not by itself rate‐limiting, making it less likely that the source of the nonhyperbolic substrate kinetics is an effect on this step from electrostatic interaction with ferricytochrome c bound to a second site. It is pointed out that nonhyperbolic kinetics is, in fact, an intrinsic property of ion pumps.

Electron-transport-driven proton pumps display nonhyperbolic kinetics: Simulation of the steady-state kinetics of cytochrome c oxidase

“…Furthermore, the models ignore the requirement that a pump protein must exist in two different conformational states with different substrate affinities, as also observed experimentally. The models based on a nonproductive binding site (13,16) must assume that product dissociation is strictly rate limiting, but this is excluded by the observation that cytochrome a is always partially reduced in the steady state (14). (25, *).…”

“…Instead the existence of two active sites has generally been assumed (9,12), and an electrostatic model involving a single catalytic site and a second, nonproductive, binding site has also been proposed (13). In a recent paper (14) we have provided experimental evidence against the latter model and furthermore suggested that the nonhyper- bolic kinetics of cytochrome oxidase is a direct consequence of its function as a proton pump. It is not, however, sufficient to demonstrate this obligatory relation between proton pumping and substrate kinetics.…”

Redox‐linked conformational changes in cytochrome c oxidase

“…The chrome a3-CuB site does not occur in the singly reduced oxidashed line represents a forbidden transition. For further explanadase [15,16]. tions, see the text.…”

Kinetic analysis of proton translocation catalyzed by F0F1 ATPase

“…If only one catalytic site is considered [l 11, a control site and/or a reaction scheme similar to that proposed for cytochrome oxidase [12] would be required in order to explain the concave nature of the Eadie Hofstee plot.…”