Mitochondrial DNA mutations drive aerobic glycolysis to enhance checkpoint blockade response in melanoma.

The mitochondrial genome (mtDNA) encodes essential machinery for oxidative phosphorylation and metabolic homeostasis. Tumor mtDNA is among the most somatically mutated regions of the cancer genome, but whether these mutations impact tumor biology is debated. We engineered truncating mutations of the mtDNA-encoded complex I gene, Mt-Nd5, into several murine models of melanoma. These mutations promoted a Warburg-like metabolic shift that reshaped tumor microenvironments in both mice and humans, consistently eliciting an anti-tumor immune response characterized by loss of resident neutrophils. Tumors bearing mtDNA mutations were sensitized to checkpoint blockade in a neutrophil-dependent manner, with induction of redox imbalance being sufficient to induce this effect in mtDNA wild-type tumors. Patient lesions bearing >50% mtDNA mutation heteroplasmy demonstrated a response rate to checkpoint blockade that was improved by ~2.5-fold over mtDNA wild-type cancer. These data nominate mtDNA mutations as functional regulators of cancer metabolism and tumor biology, with potential for therapeutic exploitation and treatment stratification.

Human mitochondrial ADP/ATP carrier SLC25A4 operates with a ping-pong kinetic mechanism.

The mitochondrial ADP/ATP carrier (SLC25A4), also called the adenine nucleotide translocase, imports ADP into the mitochondrial matrix and exports ATP, which are key steps in oxidative phosphorylation. Historically, the carrier was thought to form a homodimer and to operate by a sequential kinetic mechanism, which involves the formation of a ternary complex with the two exchanged substrates bound simultaneously. However, recent structural and functional data have demonstrated that the mitochondrial ADP/ATP carrier works as a monomer and has a single substrate binding site, which cannot be reconciled with a sequential kinetic mechanism. Here, we study the kinetic properties of the human mitochondrial ADP/ATP carrier by using proteoliposomes and transport robotics. We show that the Km/Vmax ratio is constant for all of the measured internal concentrations. Thus, in contrast to earlier claims, we conclude that the carrier operates with a ping-pong kinetic mechanism in which substrate exchange across the membrane occurs consecutively rather than simultaneously. These data unite the kinetic and structural models, showing that the carrier operates with an alternating access mechanism.

Tumour mitochondrial DNA mutations drive aerobic glycolysis to enhance checkpoint blockade.

The mitochondrial genome encodes essential machinery for respiration and metabolic homeostasis but is paradoxically among the most common targets of somatic mutation in the cancer genome, with truncating mutations in respiratory complex I genes being most over-represented1. While mitochondrial DNA (mtDNA) mutations have been associated with both improved and worsened prognoses in several tumour lineages1-3, whether these mutations are drivers or exert any functional effect on tumour biology remains controversial. Here we discovered that complex I-encoding mtDNA mutations are sufficient to remodel the tumour immune landscape and therapeutic resistance to immune checkpoint blockade. Using mtDNA base editing technology4 we engineered recurrent truncating mutations in the mtDNA-encoded complex I gene, Mt-Nd5, into murine models of melanoma. Mechanistically, these mutations promoted utilisation of pyruvate as a terminal electron acceptor and increased glycolytic flux without major effects on oxygen consumption, driven by an over-reduced NAD pool and NADH shuttling between GAPDH and MDH1, mediating a Warburg-like metabolic shift. In turn, without modifying tumour growth, this altered cancer cell-intrinsic metabolism reshaped the tumour microenvironment in both mice and humans, promoting an anti-tumour immune response characterised by loss of resident neutrophils. This subsequently sensitised tumours bearing high mtDNA mutant heteroplasmy to immune checkpoint blockade, with phenocopy of key metabolic changes being sufficient to mediate this effect. Strikingly, patient lesions bearing >50% mtDNA mutation heteroplasmy also demonstrated a >2.5-fold improved response rate to checkpoint inhibitor blockade. Taken together these data nominate mtDNA mutations as functional regulators of cancer metabolism and tumour biology, with potential for therapeutic exploitation and treatment stratification.

In Silico Modeling of the Mitochondrial Pumping Complexes with Markov State Models.

The mechanism of proton pumping by the mitochondrial electron transport chain complexes is still enigmatic after decades of research. Recently, there has been interest in in silico Markov state models to model the mitochondrial pumping complexes at the microscopic level, and this chapter describes the methods of constructing and simulating such models.

The proton pumping mechanism of the bc1 complex.

The bc1 complex is a proton pump of the mitochondrial electron transport chain which transfers electrons from ubiquinol to cytochrome c. It operates via the modified Q cycle in which the two electrons from oxidation of ubiquinol at the Qo center are bifurcated such that the first electron is passed to Cytc via an iron sulfur center and c1 whereas the second electron is passed across the membrane by bL and bH to reduce ubiquinone at the Qi center. Proton pumping occurs because oxidation of ubiquinol at the Qo center releases protons to the P-side and reduction of ubiquinone at the Qi center takes up protons from the N-side. However, the mechanisms which prevent the thermodynamically more favorable short circuit reactions and so ensure precise bifurcation and proton pumping are not known. Here we use statistical thermodynamics to show that reaction steps that originate from high energy states cannot support high flux even when they have large rate constants. We show how the chemistry of ubiquinol oxidation and the structure of the Qo site can result in free energy profiles that naturally suppress flux through the short circuit pathways while allowing high rates of bifurcation. These predictions are confirmed through in-silico simulations using a Markov state model.

Thermodynamic efficiency, reversibility, and degree of coupling in energy conservation by the mitochondrial respiratory chain.

The protonmotive mitochondrial respiratory chain, comprising complexes I, III and IV, transduces free energy of the electron transfer reactions to an electrochemical proton gradient across the inner mitochondrial membrane. This gradient is used to drive synthesis of ATP and ion and metabolite transport. The efficiency of energy conversion is of interest from a physiological point of view, since the energy transduction mechanisms differ fundamentally between the three complexes. Here, we have chosen actively phosphorylating mitochondria as the focus of analysis. For all three complexes we find that the thermodynamic efficiency is about 80-90% and that the degree of coupling between the redox and proton translocation reactions is very high during active ATP synthesis. However, when net ATP synthesis stops at a high ATP/ADP.Pi ratio, and mitochondria reach "State 4" with an elevated proton gradient, the degree of coupling drops substantially. The mechanistic cause and the physiological implications of this effect are discussed.

Measuring the functionality of the mitochondrial pumping complexes with multi-wavelength spectroscopy.

The proton pumps of the mitochondrial electron transport chain (ETC) convert redox energy into the proton motive force (ΔP), which is subsequently used by the ATP synthase to regenerate ATP. The limited available redox free energy requires the proton pumps to operate close to equilibrium in order to maintain a high ΔP, which in turn is needed to maintain a high phosphorylation potential. Current biochemical assays measure complex activities far from equilibrium and so shed little light on their function under physiological conditions. Here we combine absorption spectroscopy of the ETC hemes, NADH fluorescence spectroscopy and oxygen consumption to simultaneously measure the redox potential of the intermediate redox pools, the components of ΔP and the electron flux in RAW 264.7 mouse macrophages. We confirm that complex I and III operate near equilibrium and quantify the linear relationship between flux and disequilibrium as a metric of their function under physiological conditions. In addition, we quantify the dependence of complex IV turnover on ΔP and the redox potential of cytochrome c to determine the complex IV driving force and find that the turnover is proportional to this driving force. This form of quantification is a more relevant metric of ETC function than standard biochemical assays and can be used to study the effect of mutations in either mitochondrial or nuclear genome affecting mitochondrial function, post-translation changes, different subunit isoforms, as well as the effect of pharmaceuticals on ETC function.

Electron transfer between cytochrome c and the binuclear center of cytochrome oxidase.

The Minnaert model, which can account for the reaction kinetics between cytochrome oxidase and cytochrome c (Cytc), has been used to justify equal binding rate constants for reduced and oxidized Cytc. Here we extend the model beyond reversible binding of Cytc and its irreversible oxidation to include CuA, heme a and the oxidation cycle of the binuclear center. The model reproduces the experimental reduction of CuA and heme a during turnover and the low population of the ferryl and ferrous heme a3. It predicts that the off rate constants for reduced and oxidized Cytc can be unequal and that the non-Nernst response of CuA and heme a is due to disequilibrium between free Cytc and CuA rather than redox anticooperativity.

The dependence of brain mitochondria reactive oxygen species production on oxygen level is linear, except when inhibited by antimycin A.

Reactive oxygen species (ROS) are by-products of physiological mitochondrial metabolism that are involved in several cellular signaling pathways as well as tissue injury and pathophysiological processes, including brain ischemia/reperfusion injury. The mitochondrial respiratory chain is considered a major source of ROS; however, there is little agreement on how ROS release depends on oxygen concentration. The rate of H2 O2 release by intact brain mitochondria was measured with an Amplex UltraRed assay using a high-resolution respirometer (Oroboros) equipped with a fluorescent optical module and a system of controlled gas flow for varying the oxygen concentration. Three types of substrates were used: malate and pyruvate, succinate and glutamate, succinate alone or glycerol 3-phosphate. For the first time we determined that, with any substrate used in the absence of inhibitors, H2 O2 release by respiring brain mitochondria is linearly dependent on the oxygen concentration. We found that the highest rate of H2 O2 release occurs in conditions of reverse electron transfer when mitochondria oxidize succinate or glycerol 3-phosphate. H2 O2 production by complex III is significant only in the presence of antimycin A and, in this case, the oxygen dependence manifested mixed (linear and hyperbolic) kinetics. We also demonstrated that complex II in brain mitochondria could contribute to ROS generation even in the absence of its substrate succinate when the quinone pool is reduced by glycerol 3-phosphate. Our results underscore the critical importance of reverse electron transfer in the brain, where a significant amount of succinate can be accumulated during ischemia providing a backflow of electrons to complex I at the early stages of reperfusion. Our study also demonstrates that ROS generation in brain mitochondria is lower under hypoxic conditions than in normoxia. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Spectral components of detergent-solubilized bovine cytochrome oxidase.

Cytochrome oxidase is the terminal oxidase of the mitochondrial electron transport chain and pumps 4 protons per oxygen reduced to water. Spectral shifts in the α-band of heme a have been observed in multiple studies and these shifts have the potential to shed light on the proton pumping intermediates. Previously we found that heme a had two spectral components in the α-band during redox titrations in living RAW 264.7 mouse macrophage cells, the classical 605 nm form and a blue-shifted 602 nm form. To confirm these spectral changes were not an artifact due to the complex milieu of the living cell, redox titrations were performed in the isolated detergent-solubilized bovine enzyme from both the Soret- and α-band using precise multiwavelength spectroscopy. This data verified the presence of the 602 nm form in the α-band, revealed a similar shift of heme a in the Soret-band and ruled out the reversal of calcium binding as the origin of the blue shift. The 602 nm form was found to be stabilized at high pH or by binding of azide, which is known to blue shift the α-band of heme a. Azide also stabilized the 602 nm form in the living cells. It is concluded there is a form of cytochrome oxidase in which heme a undergoes a blue shift to a 602 nm form and that redox titrations can be successfully performed in living cells where the oxidase operates in its authentic environment and in the presence of a proton motive force.

Fumarate Hydratase Loss Causes Combined Respiratory Chain Defects.

Fumarate hydratase (FH) is an enzyme of the tricarboxylic acid (TCA) cycle mutated in hereditary and sporadic cancers. Despite recent advances in understanding its role in tumorigenesis, the effects of FH loss on mitochondrial metabolism are still unclear. Here, we used mouse and human cell lines to assess mitochondrial function of FH-deficient cells. We found that human and mouse FH-deficient cells exhibit decreased respiration, accompanied by a varying degree of dysfunction of respiratory chain (RC) complex I and II. Moreover, we show that fumarate induces succination of key components of the iron-sulfur cluster biogenesis family of proteins, leading to defects in the biogenesis of iron-sulfur clusters that affect complex I function. We also demonstrate that suppression of complex II activity is caused by product inhibition due to fumarate accumulation. Overall, our work provides evidence that the loss of a single TCA cycle enzyme is sufficient to cause combined RC activity dysfunction.

Modelling the free energy profile of the mitochondrial ADP/ATP carrier.

The mitochondrial ADP/ATP carrier catalyses the equimolar exchange of adenosine di- and tri-phosphates. It operates by an alternating access mechanism in which a single substrate-binding site is made available either to the mitochondrial matrix or the intermembrane space through conformational changes. These changes are prevented in the absence of substrate by a large energy barrier due to the need for sequential disruption and formation of a matrix and cytoplasmic salt bridge network that are located on either side of the central cavity. In analogy to enzyme catalysis, substrate lowers the energy barrier by binding tighter in the intermediate state. Here we provide an in-silico kinetic model that captures the free energy profile of these conformational changes and treats the carrier as a nanomachine moving stochastically from the matrix to cytoplasmic conformation under the influence of thermal energy. The model reproduces the dependency of experimentally determined kcat and KM values on the cytoplasmic network strength with good quantitative accuracy, implying that it captures the transport mechanism and can provide a framework to understand the structure-function relationships of this class of transporter. The results show that maximum transport occurs when the interaction energies of the cytoplasmic network, matrix network and substrate binding are approximately equal such that the energy barrier is minimized. Consequently, the model predicts that there will be other interactions in addition to those of the cytoplasmic network that stabilise the matrix conformation of the ADP/ATP carrier.

The transport mechanism of the mitochondrial ADP/ATP carrier.

The mitochondrial ADP/ATP carrier imports ADP from the cytosol and exports ATP from the mitochondrial matrix, which are key transport steps for oxidative phosphorylation in eukaryotic organisms. The transport protein belongs to the mitochondrial carrier family, a large transporter family in the inner membrane of mitochondria. It is one of the best studied members of the family and serves as a paradigm for the molecular mechanism of mitochondrial carriers. Structurally, the carrier consists of three homologous domains, each composed of two transmembrane α-helices linked with a loop and short α-helix on the matrix side. The transporter cycles between a cytoplasmic and matrix state in which a central substrate binding site is alternately accessible to these compartments for binding of ADP or ATP. On both the cytoplasmic and matrix side of the carrier are networks consisting of three salt bridges each. In the cytoplasmic state, the matrix salt bridge network is formed and the cytoplasmic network is disrupted, opening the central substrate binding site to the intermembrane space and cytosol, whereas the converse occurs in the matrix state. In the transport cycle, tighter substrate binding in the intermediate states allows the interconversion of conformations by lowering the energy barrier for disruption and formation of these networks, opening and closing the carrier to either side of the membrane in an alternating way. Conversion between cytoplasmic and matrix states might require the simultaneous rotation of three domains around a central translocation pathway, constituting a unique mechanism among transport proteins. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.

Formation of a cytoplasmic salt bridge network in the matrix state is a fundamental step in the transport mechanism of the mitochondrial ADP/ATP carrier.

Mitochondrial ADP/ATP carriers catalyze the equimolar exchange of ADP and ATP across the mitochondrial inner membrane. Structurally, they consist of three homologous domains with a single substrate binding site. They alternate between a cytoplasmic and matrix state in which the binding site is accessible to these compartments for binding of ADP or ATP. It has been proposed that cycling between states occurs by disruption and formation of a matrix and cytoplasmic salt bridge network in an alternating way, but formation of the latter has not been shown experimentally. Here, we show that state-dependent formation of the cytoplasmic salt bridge network can be demonstrated by measuring the effect of mutations on the thermal stability of detergent-solubilized carriers locked in a specific state. For this purpose, mutations were made to increase or decrease the overall interaction energy of the cytoplasmic network. When locked in the cytoplasmic state by the inhibitor carboxyatractyloside, the thermostabilities of the mutant and wild-type carriers were similar, but when locked in the matrix state by the inhibitor bongkrekic acid, they correlated with the predicted interaction energy of the cytoplasmic network, demonstrating its formation. Changing the interaction energy of the cytoplasmic network also had a profound effect on the kinetics of transport, indicating that formation of the network is a key step in the transport cycle. These results are consistent with a unique alternating access mechanism that involves the simultaneous rotation of the three domains around a central translocation pathway.

Novel methods for measuring the mitochondrial membrane potential.

The mitochondrial membrane potential is a critical parameter for understanding mitochondrial function, but it is challenging to quantitate with current methodologies which are based on the accumulation of cation indicators. Recently we have introduced a new methodology based on the redox poise of the b-hemes of the bc 1 complex. Here we describe the thermodynamic framework and algorithms necessary to calculate the membrane potential from the measured oxidation states of the b-hemes.

MNRR1 (formerly CHCHD2) is a bi-organellar regulator of mitochondrial metabolism.

Our understanding of stress-associated regulatory mechanisms for mitochondria remains incomplete. We now report a new regulator of mitochondrial metabolism, the coiled-coil-helix-coiled-coil-helix domain-containing protein 2 (CHCHD2) which, based on the functionality described here, is renamed MNRR1 (Mitochondria Nuclear Retrograde Regulator 1). It functions in a novel way by acting in two cellular compartments, mitochondria and nucleus. In normally growing cells most MNRR1 is located in mitochondria; during stress most MNRR1 is now located in the nucleus. MNRR1 is imported to the mitochondrial intermembrane space by a Mia40-mediated pathway, where it binds to cytochrome c oxidase (COX). This association is required for full COX activity. Decreased MNRR1 levels produce widespread dysfunction including reduced COX activity, membrane potential, and growth rate, and increased reactive oxygen species and mitochondrial fragmentation. In the nucleus, MNRR1 acts as a transcription factor, one of whose targets is the COX subunit 4 isoform, COX4I2, which is transcriptionally stimulated by hypoxia. This MNRR1-mediated stress response may provide an important survival mechanism for cells under conditions of oxidative or hypoxic stress, both in the acute phase by altering mitochondrial oxygen utilization and in the chronic phase by promoting COX remodeling.

Mammalian complex I pumps 4 protons per 2 electrons at high and physiological proton motive force in living cells.

Mitochondrial complex I couples electron transfer between matrix NADH and inner-membrane ubiquinone to the pumping of protons against a proton motive force. The accepted proton pumping stoichiometry was 4 protons per 2 electrons transferred (4H(+)/2e(-)) but it has been suggested that stoichiometry may be 3H(+)/2e(-) based on the identification of only 3 proton pumping units in the crystal structure and a revision of the previous experimental data. Measurement of proton pumping stoichiometry is challenging because, even in isolated mitochondria, it is difficult to measure the proton motive force while simultaneously measuring the redox potentials of the NADH/NAD(+) and ubiquinol/ubiquinone pools. Here we employ a new method to quantify the proton motive force in living cells from the redox poise of the bc(1) complex measured using multiwavelength cell spectroscopy and show that the correct stoichiometry for complex I is 4H(+)/2e(-) in mouse and human cells at high and physiological proton motive force.

Acute mitochondrial inhibition by mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) 1/2 inhibitors regulates proliferation.

The Ras-MEK1/2-ERK1/2 kinase signaling pathway regulates proliferation, survival, and differentiation and, because it is often aberrant in tumors, is a popular target for small molecule inhibition. A novel metabolic analysis that measures the real-time oxidation state of NAD(H) and the hemes of the electron transport chain and oxygen consumption within intact, living cells found that structurally distinct MEK1/2 inhibitors had an immediate, dose-dependent effect on mitochondrial metabolism. The inhibitors U0126, MIIC and PD98059 caused NAD(H) reduction, heme oxidation, and decreased oxygen consumption, characteristic of complex I inhibition. PD198306, an orally active MEK1/2 inhibitor, acted as an uncoupler. Each MEK1/2 inhibitor depleted phosphorylated ERK1/2 and inhibited proliferation, but the most robust antiproliferative effects always correlated with the metabolic failure which followed mitochondrial inhibition rather than inhibition of MEK1/2. This warrants rethinking the role of ERK1/2 in proliferation and emphasizes the importance of mitochondrial function in this process.