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Mammalian Complex I Pumps 4 Protons per 2 Electrons at High and Physiological Proton Motive Force in Living Cells*

M. Ripple,Namjoon Kim,R. Springett

Published 2013 in Journal of Biological Chemistry

ABSTRACT

Background: The H+/2e− stoichiometry of complex I has been questioned based on recent crystal structures. Results: A new method of quantitating proton motive force and ubiquinone redox potential was used to determine the thermodynamic poise of complex I in living cells. Conclusion: The poise is consistent with pumping 4 not 3H+/2e−. Significance: This new methodology can quantitate electron transport chain function in living cells. Mitochondrial complex I couples electron transfer between matrix NADH and inner-membrane ubiquinone to the pumping of protons against a proton motive force. The accepted proton pumping stoichiometry was 4 protons per 2 electrons transferred (4H+/2e−) but it has been suggested that stoichiometry may be 3H+/2e− based on the identification of only 3 proton pumping units in the crystal structure and a revision of the previous experimental data. Measurement of proton pumping stoichiometry is challenging because, even in isolated mitochondria, it is difficult to measure the proton motive force while simultaneously measuring the redox potentials of the NADH/NAD+ and ubiquinol/ubiquinone pools. Here we employ a new method to quantify the proton motive force in living cells from the redox poise of the bc1 complex measured using multiwavelength cell spectroscopy and show that the correct stoichiometry for complex I is 4H+/2e− in mouse and human cells at high and physiological proton motive force.

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