Mitochondrial mechanistic P/O ratios are still in question. The major studies since 1937 are summarized and various systematic errors are discussed. Values of about 2.5 with NADH-linked substrates and 1.5 with succinate are consistent with most reports after apparent contradictions are explained. Variability of coupling may occur under some conditions but is generally not significant. The fractional values result from the coupling ratios of proton transport. An additional revision of P/O ratios may be required because of a report of the structure of ATP synthase (D. Stock, A.G.W. Leslie, J.E. Walker, Science 286 (1999) 1700-1705) which suggests that the H+/ATP ratio is 10/3, rather than 3, consistent with P/O ratios of 2.3 with NADH and 1.4 with succinate, values that are also possible.

In this paper we examine the non-linearity of the relationship between the proton electrochemical gradient across the mitochondrial inner membrane (delta p) and oxygen consumption of non-phosphorylating mitochondria in situ in hepatocytes. Models proposing to explain the non-linear relationship were tested experimentally. It was shown that the mitochondrial proton conductance and the number of protons pumped to the cytosolic side of the mitochondrial inner membrane by the electron transport complexes per oxygen atom consumed (H+/O ratio) are independent of electron transport rate in mitochondria in isolated hepatocytes. The non-linearity of the plot of delta p against the non-phosphorylating oxygen consumption is due to either a potential-dependent slippage of the proton pumps of the mitochondrial inner membrane and/or a potential-dependent leakage of protons back across the mitochondrial inner membrane.

Significance The electron transport chain of mitochondria is initiated by the respiratory complex I that converts chemical energy into a proton motive force to power synthesis of adenosine triphosphate. On a chemical level, complex I catalyzes elementary electron and proton transfer processes that couple across large molecular distances of >300 Å. However, under low oxygen concentrations, the respiratory chain operates in reverse mode and produces harmful reactive oxygen species. To avoid cell damage, the mitochondrial complex I transitions into a deactive state that inhibits turnover by molecular principles that remain elusive. By combining large-scale molecular simulations with cryo-electron microscopy data, we show here that complex I deactivation blocks the communication between proton pumping and redox modules by conformational and hydration changes. Cellular respiration is powered by membrane-bound redox enzymes that convert chemical energy into an electrochemical proton gradient and drive the energy metabolism. By combining large-scale classical and quantum mechanical simulations with cryo-electron microscopy data, we resolve here molecular details of conformational changes linked to proton pumping in the mammalian complex I. Our data suggest that complex I deactivation blocks water-mediated proton transfer between a membrane-bound quinone site and proton-pumping modules, decoupling the energy-transduction machinery. We identify a putative gating region at the interface between membrane domain subunits ND1 and ND3/ND4L/ND6 that modulates the proton transfer by conformational changes in transmembrane helices and bulky residues. The region is perturbed by mutations linked to human mitochondrial disorders and is suggested to also undergo conformational changes during catalysis of simpler complex I variants that lack the “active”-to-“deactive” transition. Our findings suggest that conformational changes in transmembrane helices modulate the proton transfer dynamics by wetting/dewetting transitions and provide important functional insight into the mammalian respiratory complex I.

Quinones are found in the lipid membranes of prokaryotes like E. coli and cyanobacteria, and are also abundant in eukaryotic mitochondria and chloroplasts. They are intricately involved in the reaction mechanism of redox phosphorylations. In the Mitchellian chemiosmotic school of thought, membrane-lodged quinones are perceived as highly mobile conveyors of two-electron equivalents from the first leg of Electron Transport Chain (ETC) to the 'second pit-stop' of Cytochrome bc₁ or b₆f complex (CBC), where they undergo a regenerative 'Q-cycle'. In Manoj's murburn mechanism, the membrane-lodged quinones are perceived as relatively slow-moving one- or two- electron donors/acceptors, enabling charge separation and the CBC resets a one-electron paradigm via 'turbo logic'. Herein, we compare various purviews of the two mechanistic schools with respect to: constraints in mobility, protons' availability, binding of quinones with proteins, structural features of the protein complexes, energetics of reaction, overall reaction logic, etc. From various perspectives, the murburn mechanism appeals as a viable alternative explanation well-rooted in thermodynamics/kinetics and one which lends adequate structure-function correlations for the roles of quinones, lipid membrane and associated proteins.

Mitochondria tightly couple the transport of electrons and proton pumping with oxidative phosphorylation to synthesize ATP. However, such energetic processes can “leak” electrons to oxygen forming reactive oxygen species (ROS). Increased ROS associated with oxidative stress has been implicated in numerous pathological conditions. To better understand the complex system regulating ROS, we present a mechanistic mitochondrial model that includes the electron transport chain, tricarboxylic acid cycle kinetics, mitochondrial calcium, sodium and proton handling, membrane ion transport processes that is coupled to oxidative phosphorylation, generation of ROS by complexes I and III, and matrix and extramitochondrial antioxidant defenses. Consistent with earlier study we found that in heart mitochondria respiring in presence of complex-II linked substrate succinate there is an increased ROS production with increasing membrane potential. Studies from experiments suggest that this relationship between ROS production and ΔѰm due to substrate modification is attributed to the process of Reverse Electron Transport. However, the model suggests that ROS generation with increasing membrane potential in presence of succinate is not because of reverse electron flow, but occurs because of the fact that as the mitochondrial membrane potential increases, it becomes harder for the protons to be pumped out to the p-side, thereby decreasing proton pumpingflux, and the most favorable pathway for the e

Mitochondrial disorders are among the most frequent inborn errors of metabolism, their primary cause being the dysfunction of the oxidative phosphorylation system (OXPHOS). OXPHOS is composed of the electron transport chain (ETC), formed by four multimeric enzymes and two mobile electron carriers, plus an ATP synthase [also called complex V (cV)]. The ETC performs the redox reactions involved in cellular respiration while generating the proton motive force used by cV to synthesize ATP. OXPHOS biogenesis involves multiple steps, starting from the expression of genes encoded in physically separated genomes, namely the mitochondrial and nuclear DNA, to the coordinated assembly of components and cofactors building each individual complex and eventually the supercomplexes. The genetic cause underlying around half of the diagnosed mitochondrial disease cases is currently known. Many of these cases result from pathogenic variants in genes encoding structural subunits or additional factors directly involved in the assembly of the ETC complexes. Here, we review the historical and most recent findings concerning the clinical phenotypes and the molecular pathological mechanisms underlying this particular group of disorders.

through the electron transport chain releases the energy to pump protons out of the mitochondrial matrix and into the intermembrane space. This pumping generates Adenosine triphosphate (ATP) is a nucleoside triphosphate that provides free energy of approximately 58 kJ/mol (0.6 eV) to drive and support many processes in living cells, such as muscle contraction, nerve impulse propagation, and chemical synthesis. Found in all known forms of life, it is often referred to as the "molecular unit of currency" for intracellular energy transfer. When consumed in a I…