Stoichiometry of proton translocation by respiratory complex I and its mechanistic implications

Abstract

Complex I (NADH-ubiquinone oxidoreductase) in the respiratory chain of mitochondria and several bacteria functions as a redoxdriven proton pump that contributes to the generation of the protonmotive force across the inner mitochondrial or bacterial membrane and thus to the aerobic synthesis of ATP. The stoichiometry of proton translocation is thought to be 4 H þ per NADH oxidized (2 e − ). Here we show that a H þ ∕2 e − ratio of 3 appears more likely on the basis of the recently determined H þ ∕ATP ratio of the mitochondrial F 1 F o -ATP synthase of animal mitochondria and of a set of carefully determined ATP∕2 e − ratios for different segments of the mitochondrial respiratory chain. This lower H þ ∕2 e − ratio of 3 is independently supported by thermodynamic analyses of experiments with both mitochondria and submitochondrial particles. A reduced H þ ∕2 e − stoichiometry of 3 has important mechanistic implications for this proton pump. In a rough mechanistic model, we suggest a concerted proton translocation mechanism in the three homologous and tightly packed antiporterlike subunits L, M, and N of the proton-translocating membrane domain of complex I.cell respiration | proton pumping | conformational changes | phosphorylation potential C omplex I is the entry point of the respiratory chain in mitochondria and many bacteria and structurally by far the most complicated of the three respiratory chain complexes with protonmotive activity, viz. I, III, and IV. Complex I is an L-shaped integral membrane protein. Its hydrophilic arm, which protrudes out of the membrane on the negatively charged N side, contains the NADH-oxidizing FMN site, a set of iron sulfur centers, and a binding site for ubiquinone (1-3). The membrane domain includes seven subunits that in mitochondria are all encoded by mtDNA and that are present also in bacteria (see Fig. 1). Three of these subunits*, L, M, and N, are homologous to one another as well as to certain bacterial proton/cation antiporters (2, 3, 6). They share structural features, such as two disrupted transmembrane α-helices (4) and conserved lysines within the membrane domain, which appear to be important in the proton-pumping mechanism (7-9).When considering possible mechanisms of redox-linked proton translocation by complex I, some kind of conformational coupling has appeared obvious due to the long distance between the redoxactive cofactors in the hydrophilic domain and the likely protontranslocating subunits in the membrane domain. The crystal structures (4, 10) revealed a most uncommon long amphipathic α-helix that is part of the distal subunit L. It passes along almost the entire N surface of the membrane domain and is followed by a transmembrane helix on the proximal side of subunit N, thus "wrapping up" the three antiporter-like subunits. This helix has been suggested to communicate redox-dependent conformational changes in the hydrophilic domain to the membrane subunits (4, 10).Here we first reexamine the stoichiometry of redox-coupled proton translocation in complex I and...

Electrostatics, hydration, and proton transfer dynamics in the membrane domain of respiratory complex I

“…The reaction cycle in complex I is initiated by reduction of Q, which results in a large charge imbalance (2,4,12,20) that is expected to induce proton charge redistribution and in turn a cooperative hydration of the antiporter-like subunits. The newly released X-ray structure of complex I reveals that the Q-binding site is located in the hydrophilic NuoC (Nqo4) subunit above the interface with the membrane domains and is coupled to the latter by several charged residues within the NuoH (Nqo8) subunit.…”

“…In contrast, in complex I the electron and proton transfers are separated both kinetically and spatially. To explain this long-range coupling, both "direct" (redox-driven) and "indirect" (conformational-driven) mechanisms have been suggested, but the molecular principles of these mechanisms remain elusive (1,2,4,(17)(18)(19)(20)(21)(22).…”

“…By transferring electrons from reduced NADH to quinone (Q), it functions as a primary entry point for electrons into the mitochondrial and bacterial respiratory chains (1,2). Complex I couples the Q reduction to translocation of three to four protons across the mitochondrial or bacterial membrane (3,4), thus contributing to the electrochemical proton-motive force subsequently used for synthesis of ATP by F o F 1 -ATPase and for active transport of solutes (5).…”

Redox-induced activation of the proton pump in the respiratory complex I

“…1) (8,11). Biochemical and structural studies suggest that the reduction of Q activates the proton pump via a conformationaldriven coupling mechanism, accompanied by electrostatic gating (2, 6-8, [12][13][14].…”

Energy Transducing Roles of Antiporter-like Subunits in Escherichia coli NDH-1 with Main Focus on Subunit NuoN (ND2)

“…Complex I catalyzes the electron transfer from NADH to quinone, which is coupled to the translocation of protons through the inner mitochondrial membrane (2). This enzyme complex, made up of ϳ45 different polypeptides, is the largest enzyme of the respiratory chain, with a molecular mass of ϳ1,000 kDa (1,3).…”