Determination of the upper and lower limits of the mechanistic stoichiometry of incompletely coupled fluxes. Stoichiometry of incompletely coupled reactions.

A rationale is formulated for the design of experiments to determine the upper and lower limits of the mechanistic stoichiometry of any two incompletely coupled fluxes J1 and J2. Incomplete coupling results when there is a branch at some point in the sequence of reactions or processes coupling the two fluxes. The upper limit of the mechanistic stoichiometry is given by the minimum value of dJ2/dJ1 obtained when the fluxes are systematically varied by changes in steps after the branch point. The lower limit is given by the maximum value of dJ2/dJ1 obtained when the fluxes are varied by changes in steps prior to the branch point. The rationale for determining these limits is developed from both a simple kinetic model and from a linear nonequilibrium thermodynamic treatment of coupled fluxes, using the mechanistic approach [Westerhoff, H. V. & van Dam, K. (1979) Curr. Top. Bioenerg. 9, 1-62]. The phenomenological stoichiometry, the flux ratio at level flow and the affinity ratio at static head of incompletely coupled fluxes are defined in terms of mechanistic conductances and their relationship to the mechanistic stoichiometry is discussed. From the rationale developed, experimental approaches to determine the mechanistic stoichiometry of mitochondrial oxidative phosphorylation are outlined. The principles employed do not require knowledge of the pathway or the rate of transmembrane leaks or slippage and may also be applied to analysis of the stoichiometry of other incompletely coupled systems, including vectorial H+/O and K+/O translocation coupled to mitochondrial electron transport.