Electron competition in yeast mitochondria is governed by dehydrogenases catalytic efficiencies.

In Saccharomyces cerevisiae, cytosolic NADH generated during biomass production must be efficiently reoxidized to sustain metabolic flux. Under aerobic conditions, this process is primarily mediated by the oxidative phosphorylation and specifically via external NADH dehydrogenases (Nde1p/Nde2p) and the glycerol-3-phosphate (G3P) shuttle. Although these pathways perform equivalent redox functions, their relative contributions vary with physiological conditions, indicating the existence of regulatory mechanisms governing electron partitioning. Here, we combined experimental measurements and kinetic modeling to investigate the mechanisms underlying electron competition between NADH and G3P oxidation in isolated yeast mitochondria. Using wild-type and Δnde1 strains, we determined the kinetic parameters of the relevant dehydrogenases and analyzed their dependence on the quinone redox state. A mathematical model incorporating these parameters reproduced the experimentally observed preferential utilization of NADH in wild-type mitochondria as well as the simultaneous oxidation of NADH and G3P in the Δnde1 mutant. Our results demonstrate that, under conditions where the downstream respiratory chain is rate-limiting, electron partitioning is governed by the ratio of catalytic efficiencies (Vmax/K0.5) of NADH and G3P dehydrogenases with respect to the quinone redox ratio (Q/QH₂). These findings identify a simple kinetic principle underlying substrate competition in mitochondrial electron transport and provide a quantitative framework for understanding metabolic flux distribution in eukaryotic cells.