Abstract
Respiratory complex I couples electron transfer between NADH and ubiquinone to proton translocation across an energy-transducing membrane to support the proton-motive force that drives ATP synthesis. The proton-pumping stoichiometry of complex I (i.e. the number of protons pumped for each two electrons transferred) underpins all mechanistic proposals. However, it remains controversial and has not been determined for any of the bacterial enzymes that are exploited as model systems for the mammalian enzyme. Here, we describe a simple method for determining the proton-pumping stoichiometry of complex I in inverted membrane vesicles under steady-state ADP-phosphorylating conditions. Our method exploits the rate of ATP synthesis, driven by oxidation of NADH or succinate with different sections of the respiratory chain engaged in catalysis as a proxy for the rate of proton translocation and determines the stoichiometry of complex I by reference to the known stoichiometries of complexes III and IV. Using vesicles prepared from mammalian mitochondria (from Bos taurus) and from the bacterium Paracoccus denitrificans, we show that four protons are pumped for every two electrons transferred in both cases. By confirming the four-proton stoichiometry for mammalian complex I and, for the first time, demonstrating the same value for a bacterial complex, we establish the utility of P. denitrificans complex I as a model system for the mammalian enzyme. P. denitrificans is the first system described in which mutagenesis in any complex I core subunit may be combined with quantitative proton-pumping measurements for mechanistic studies.
Highlights
Ery and are conserved in all species of complex I, and a cohort of 31 supernumerary subunits that is particular to the mammalian complex [1,2,3]
A fundamental property of the mechanism of complex I catalysis is the stoichiometry of proton translocation: how many protons does complex I transport across the membrane for each oxidation of NADH? The proton stoichiometry for complex I is considered historically to be four, and so four proton channels have been proposed in structural models [4, 5, 8]
These “different species” include the model systems exploited in mechanistic investigations of complex I catalysis, which are assumed to be relevant to the mammalian complex
Summary
P/O, phosphate (ATP formation) to oxygen (1/2 O2 consumption) ratio; FCCP, 4-(trifluoromethoxy)phenylhydrazone; Q1, ubiquinone-1; RCR, respiratory control ratio; SBP, sub-bacterial particle; SMP, submitochondrial particle. The possibility that different species of complex I adopt different stoichiometries cannot be excluded: the complex I proton-pumping machinery is modular, marked variations between the core subunits exist between species, and some species use alternative quinones with much lower reduction potentials that imply an altered quantitative scale for bioenergetics. These “different species” include the model systems exploited in mechanistic investigations of complex I catalysis, which are assumed to be relevant to the mammalian complex. Our results reaffirm the relevance of using simpler model systems for mechanistic studies and enable accurate stoichiometry measurements in a genetically tractable model system for the future testing of mechanistic hypotheses
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