“…Our results confirm that the proton-pumping stoichiometry of mammalian complex I is 4 H + /2 e − , consistent with the consensus of earlier measurements (16, 17, 19), not 3 H + /2 e − as proposed recently by Wikström and Hummer (45). Furthermore, we demonstrate, for the first time, that complex I from a bacterial species, P. denitrificans , also has a stoichiometry of 4 H + /2 e − .…”
Section: Discussionsupporting
confidence: 93%
“…Recently, however, Wikström and Hummer (45) reappraised both Wikström's and Hinkle's earlier analyses in response to the 8 H + /3 ATP stoichiometry of mammalian ATP synthase inferred from the 8 c -subunits observed in the crystal structure (18) and concluded that the proton stoichiometry of complex I is three, not four. Subsequently, Ripple and co-workers (19) utilized the b -hemes of complex III in intact cells to determine the redox span (Δ E ) and proton-motive force (Δ p ) across complex I and then extrapolated to the position of zero net catalysis at which 2Δ E = n Δ p . The data gave a proton stoichiometry ( n value) close to four.…”
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.
“…Our results confirm that the proton-pumping stoichiometry of mammalian complex I is 4 H + /2 e − , consistent with the consensus of earlier measurements (16, 17, 19), not 3 H + /2 e − as proposed recently by Wikström and Hummer (45). Furthermore, we demonstrate, for the first time, that complex I from a bacterial species, P. denitrificans , also has a stoichiometry of 4 H + /2 e − .…”
Section: Discussionsupporting
confidence: 93%
“…Recently, however, Wikström and Hummer (45) reappraised both Wikström's and Hinkle's earlier analyses in response to the 8 H + /3 ATP stoichiometry of mammalian ATP synthase inferred from the 8 c -subunits observed in the crystal structure (18) and concluded that the proton stoichiometry of complex I is three, not four. Subsequently, Ripple and co-workers (19) utilized the b -hemes of complex III in intact cells to determine the redox span (Δ E ) and proton-motive force (Δ p ) across complex I and then extrapolated to the position of zero net catalysis at which 2Δ E = n Δ p . The data gave a proton stoichiometry ( n value) close to four.…”
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.
“…The rates of ATP synthesis depend linearly on the rates of NADH oxidation (Figure 4), and comparison of their gradients showed that 2.51 ± 0.09 times as much ATP per NADH is synthesized by the CI/CIII/CIV pathway compared to the CI/AOX pathway. Applying the established 6H + /2 e – stoichiometry for CIII/CIV catalysis (Nicholls and Ferguson, 2013) then yields a CI stoichiometry of 3.96 ± 0.24 H + /2 e – , in excellent agreement with most previous studies (Galkin et al., 2006, Jones et al., 2017, Ripple et al., 2013, Wikström, 1984). Supplementing membrane vesicles by AOX may thus prove useful in future studies of CI H + /2 e – stoichiometries.Figure 4Addition of AOX to SMPs to Determine the H + /2 e – Stoichiometry of Complex IThe rate of ATP synthesis driven by NADH oxidation through CI/CIII/CIV or CI/AOX is shown as a function of the rate of NADH oxidation.…”
SummaryMitochondrial respiratory supercomplexes, comprising complexes I, III, and IV, are the minimal functional units of the electron transport chain. Assembling the individual complexes into supercomplexes may stabilize them, provide greater spatiotemporal control of respiration, or, controversially, confer kinetic advantages through the sequestration of local quinone and cytochrome c pools (substrate channeling). Here, we have incorporated an alternative quinol oxidase (AOX) into mammalian heart mitochondrial membranes to introduce a competing pathway for quinol oxidation and test for channeling. AOX substantially increases the rate of NADH oxidation by O2 without affecting the membrane integrity, the supercomplexes, or NADH-linked oxidative phosphorylation. Therefore, the quinol generated in supercomplexes by complex I is reoxidized more rapidly outside the supercomplex by AOX than inside the supercomplex by complex III. Our results demonstrate that quinone and quinol diffuse freely in and out of supercomplexes: substrate channeling does not occur and is not required to support respiration.
“…However, in highly metabolizing tissues at physiological oxygen concentrations [17,19,37], a significant fraction of the enzyme (5-15%) was present in the D-form. It can be concluded that in situ, part of the energy released during steady-state NADH oxidation is used to maintain the catalytically competent A-form [4]. Energy-dependent maintenance of the fraction of the enzyme in the D-form would permit a fast response to changes in conditions such as oxygen availability and ATP demand resembling the so-called excess capacity of cytochrome c oxidase [38,39].…”
In recent years, there have been significant advances in our understanding of the functions of mitochondrial complex I other than the generation of energy. These include its role in generation of reactive oxygen species, involvement in the hypoxic tissue response and its possible regulation by nitric oxide (NO) metabolites. In this review, we will focus on the hypoxic conformational change of this mitochondrial enzyme, the so-called active/deactive transition. This conformational change is physiological and relevant to the understanding of certain pathological conditions including, in the cardiovascular system, ischaemia/reperfusion (I/R) damage. We will discuss how complex I can be affected by NO metabolites and will outline some potential mitochondria-targeted therapies in I/R damage.
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