Respiratory Complex I inBos taurusandParacoccus denitrificansPumps Four Protons across the Membrane for Every NADH Oxidized.
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 (fromBos taurus) and from the bacteriumParacoccus 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 ofP. denitrificanscomplex I as a model system for the mammalian enzyme.P. denitrificansis the first system described in which mutagenesis in any complex I core subunit may be combined with quantitative proton-pumping measurements for mechanistic studies.
From the Medical Research Council Mitochondrial Biology Unit, Cambridge, CB2 0XY, United Kingdom.
Andrew J Y
AJ
Jones
From the Medical Research Council Mitochondrial Biology Unit, Cambridge, CB2 0XY, United Kingdom.
James N
JN
Blaza
From the Medical Research Council Mitochondrial Biology Unit, Cambridge, CB2 0XY, United Kingdom.
Febin
F
Varghese
From the Medical Research Council Mitochondrial Biology Unit, Cambridge, CB2 0XY, United Kingdom jh@mrc-mbu.cam.ac.uk.
Judy
J
Hirst
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002417:Cattle / Q000378:metabolism*; D004579:Electron Transport; D042967:Electron Transport Complex I / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D010085:Oxidative Phosphorylation; D010231:Paracoccus denitrificans / Q000201:enzymology* / Q000378:metabolism; D018892:Proton-Motive Force; D011522:Protons
D011522:Protons; D009243:NAD; D000255:Adenosine Triphosphate; D042967:Electron Transport Complex I
complex I; electron transfer complex; mitochondria; mitochondrial respiratory chain complex; oxidative phosphorylation; proton motive force