MitoNEET is a mitochondrial [2Fe-2S]-containing protein known for its involvement in cellular metabolism, iron regulation, and oxidative stress. The protein has been associated with diseases ranging from diabetes to Parkinson's disease, prompting the development of compounds designed to target mitoNEET selectively. Unfortunately, drug development is limited due to a lack of mechanistic understanding of how mitoNEET integrates into pathophysiological processes, and biological compounds that govern mitoNEET function are still ill-defined. We demonstrate an oxygen-dependent reaction with biological thiols catalyzed by mitoNEET. Specifically, we observed that mitoNEET converts L-cysteine to cystine. Finally, we showed that reduced glutathione (L-GSH) regulates the reactivity of two lipid-derived biomarkers of oxidative stress, 4-HNE and 4-ONE, towards mitoNEET. We found that exposure to L-GSH before treatment with either of the electrophilic aldehydes prevents the formation of a covalently linked mitoNEET dimer. Meanwhile, adding L-GSH after electrophile treatment recovers mitoNEET from the 4-HNE induced modification but not from the modification induced by 4-ONE. These results indicate a possible role for mitoNEET in thiol-mediated oxidative stress and may facilitate the development of drugs designed to modulate mitoNEET activity to improve pathophysiological states.

Tumor cell proliferation requires sufficient metabolic flux through the pentose phosphate pathway to meet the demand for biosynthetic precursors and to increase protection against oxidative stress which in turn requires an upregulation of substrate flow through glycolysis. This metabolic poise is often coupled with a shift in ATP production from mitochondrial OXPHOS to substrate-level phosphorylation. Despite major advances that were facilitated by using tumor-derived cell lines in research areas spanning from membrane to cytoskeletal biology, this distorted metabolic profile limits their impact as a model in physiology and toxicology. Substitution of glucose with galactose in the cell culture medium has been demonstrated to shift ATP production from substrate-level phosphorylation to mitochondrial OXPHOS. This increase in oxygen utilization is coupled to a global metabolic reorganization with potential impacts on macromolecule biosynthesis and cellular redox homeostasis, but a comprehensive analysis on the effects of sugar substitution in tumor-derived cells is still missing. To address this gap in knowledge we performed transcriptomic and metabolomic analyses on human hepatocellular carcinoma (HepG2) cells adapted to either glucose or galactose as the aldohexose source. We observed a shift toward oxidative metabolism in all primary metabolic pathways at both transcriptomic and metabolomic levels. We also observed a decrease in nicotinamide dinucleotide (NAD(P)) levels and subcellular NAD+-to-NADH ratios in cells cultured with galactose compared with glucose control cells. Our results suggest that galactose reduces both glycolytic and biosynthetic flux and restores a metabolic poise in HepG2 cells that closely reflects the metabolic state observed in primary hepatocytes.

MitoNEET is a mitochondrial [2Fe‐2S] protein known for its involvement in mitochondrial bioenergetics, iron metabolism, and oxidative stress. The protein has been extensively characterized at the biochemical level since its discovery in 2004, but the mechanisms of physiological function(s) remain unresolved. Two observations seem especially relevant for understanding the physiological function of mitoNEET: (1) under anaerobic conditions in vitro thiol compounds such as glutathione (GSH) and cysteine (Cys) reduce the 2Fe‐2S cluster from the Fe+3Fe+3 to the Fe3+Fe2+ state, and (2) mitoNEET expression levels increase in cells when the glutathione redox balance is shifted towards the oxidized state. We hypothesize that mitoNEET’s in vivo function can be elucidated by bridging these two observations and postulate that mitoNEET is an enzyme responsible for the oxidation of thiol groups on either low molecular‐weight compounds or cysteine residues on target proteins involved in redox signaling. To test this hypothesis, we monitored thiol oxidation by following oxygen consumption rates and demonstrate that mitoNEET accelerates thiol oxidation of cysteine and glutathione (GSH). Four distinct experiments comparing the reactivity of Fe3+ in the form of FeCl3 to mitoNEET demonstrate that the observed thiol oxidation by mitoNEET was not due to free Fe3+contamination. These observations were: (1) rates of cysteine oxidation (8 mM) catalyzed by mitoNEET (1.5 mM) or Fe3+ (1.5 mM) show different optima in the pH range 6.4 to 9.4 and the mitoNEET‐catalyzed reaction is 137% faster at pH 8.4 than the rate observed for Fe3+ alone. (2) Oxidation rates of the nonphysiological compound, n‐acetylcysteine (NAC) by mitoNEET are 181% higher compared to Fe3+ at a pH of 9.0. (3) MitoNEET‐mediated glutathione oxidation remained unaffected by addition of cysteine while oxidation catalyzed by free Fe3+ decreased after addition of cysteine. (4) Dimedone, a probe used to inhibit thiol oxidation from sulfenic acid to sulfinic and sulfonic acid, was 25% more effective at inhibiting cysteine oxidation when free Fe3+ was the catalyst compared to mitoNEET. These results support our hypothesis that mitoNEET possesses an active site which functions to oxidize biological thiols. While the in vivo substrate is currently unknown, we have narrowed down potential candidates to small thiol containing compounds and/or cysteine residues on specific target proteins. We are currently investigating how the enzymatic activity may be regulated by endogenous compounds and through pharmacological compounds targeting mitoNEET.Support or Funding InformationThis work was supported by NSF CHE‐1806266 to M.A.M and M.E.K and UofL Mentored Undergraduate Research and Creative Activities Grant to R.A.S.

Executive summary – Box 1: In brief: Mitochondria and bioblasts .......................................... 2 1. Introduction ............................................................................................................................................................................. 8 2. Coupling states and rates in mitochondrial preparations ................................................................................... 8 2.1. Cellular and mitochondrial respiration ................................................................................................................. 8 2.1.1. Aerobic and anaerobic catabolism and ATP turnover Consortium Communication 2 of 44 Gnaiger E et al ― MitoEAGLE Task Group (2020) Bioenerg Commun 2020.1 2.1.2. Specification of biochemical dose and exposure 2.2. Mitochondrial preparations .................................................................................................................................... 10 2.3. Electron transfer pathways ..................................................................................................................................... 11 2.4. Respiratory coupling control .................................................................................................................................. 12 2.4.1. Coupling 2.4.2. Phosphorylation P» and P»/O2 ratio 2.4.3. Uncoupling 2.5. Coupling states and respiratory rates ................................................................................................................. 13 2.5.1. LEAK state 2.5.2. OXPHOS state 2.5.3. Electron transfer state 2.5.4. ROX state 2.5.5. Quantitative relations 2.5.6. The steady state 2.6. Classical terminology for isolated mitochondria ............................................................................................ 19 2.6.1. – 2.6.5. State 1 – State 5 2.7. Control and regulation .............................................................................................................................................. 21 3. What is a rate? – Box 2: Metabolic flows and fluxes: vectoral, vectorial, and scalar ............................. 21 4. Normalization of rate per sample................................................................................................................................. 23 4.1. Flow: per object ............................................................................................................................................................ 23 4.1.1. Count concentration 4.1.2. Flow per single object 4.2. Size-specific flux: per sample size .......................................................................................................................... 25 4.2.1. Mass concentration 4.2.2. Size-specific flux 4.3. Marker-specific flux: per mitochondrial content ............................................................................................. 26 4.3.1. Mitochondrial concentration and mitochondrial density 4.3.2. mt-Marker-specific flux 5. Normalization of rate per system ................................................................................................................................ 28 5.1. Flow: per chamber ...................................................................................................................................................... 28 5.2. Flux: per chamber volume ....................................................................................................................................... 28 5.2.1. System-specific flux 5.2.2. Advancement per volume 6. Conversion of units ............................................................................................................................................................ 30 7. Conclusions – Box 3: Recommendations for studies with mitochondrial preparations ...................... 31 References ............................................................................................................................................................................. 36 Authors (MitoEAGLE Task Group) – Author contributions .............................................................................. 41 Acknowledgements – Competing financial interests – Correspondence

Nutrient-deprivation autophagy factor-1 (NAF-1, miner1; gene cisd2) is part of the [2Fe-2S]-containing protein family which includes mitoNEET (gene cisd1) and MiNT (miner2; gene cisd3). These proteins are redox active and are thought to play an important role in cellular energy homeostasis with NAF-1 playing a critical role in calcium regulation and aging. To date, no studies have investigated potential ligand interaction with NAF-1. Here we show that the thiazolidinediones pioglitazone and rosiglitazone along with the mitoNEET ligand, NL-1, bind to NAF-1 with low micromolar affinities. Further, we show that overexpression of NAF-1 in hepatocellular carcinoma (HepG2) cells reduces inhibition of mitochondrial respiration by pioglitazone. Our findings support the need for further efforts of the rational design of selective NAF-1 ligands.

As the knowledge base and importance of mitochondrial physiology to human health expands, the necessity for harmonizing the terminologyconcerning mitochondrial respiratory states and rates has become increasingly apparent. Thechemiosmotic theoryestablishes the mechanism of energy transformationandcoupling in oxidative phosphorylation. Theunifying concept of the protonmotive force providestheframeworkfordeveloping a consistent theoretical foundation ofmitochondrial physiology and bioenergetics.We followguidelines of the International Union of Pure and Applied Chemistry(IUPAC)onterminology inphysical chemistry, extended by considerationsofopen systems and thermodynamicsof irreversible processes.Theconcept-driven constructive terminology incorporates the meaning of each quantity and alignsconcepts and symbols withthe nomenclature of classicalbioenergetics. We endeavour to provide a balanced view ofmitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes.Uniform standards for evaluation of respiratory states and rates will ultimatelycontribute to reproducibility between laboratories and thussupport the development of databases of mitochondrial respiratory function in species, tissues, and cells.Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery.

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

Clarity of concepts and consistency of nomenclature are trademarks of the quality of a research field across its specializations, facilitating transdisciplinary communication and education. As research and knowledge on mitochondrial physiology expand, the necessity for harmonization of nomenclature on mitochondrial respiratory states and rates has become apparent. Peter Mitchell’s concept of the protonmotive force establishes the link between the electric and chemical components of energy transformation and coupling in oxidative phosphorylation. This unifying concept provides the framework for developing a consistent terminology on mitochondrial physiology and bioenergetics. We follow IUPAC guidelines on general terms of physical chemistry, extended by concepts of open systems and irreversible thermodynamics. We align the nomenclature of classical bioenergetics on respiratory states with a concept-driven constructive terminology to address the meaning of each respiratory state. Hence we focus primarily on the conceptual ‘why’ along with clarification of the experimental ‘how’. The capacity of oxidative phosphorylation, OXPHOS, provides diagnostic reference values and is, therefore, measured at kinetically saturating concentrations of ADP, inorganic phosphate and fuel substrates. The contribution of intrinsically uncoupled oxygen consumption is most easily studied by arresting phosphorylation, when oxygen consumption compensates mainly for the proton leak, and the corresponding states are collectively classified as LEAK states. The oxidative capacity of the electron transfer system, ETS, reveals the limitation of OXPHOS capacity mediated by the phosphorylation system. Experimental standards for evaluation of respiratory coupling states must be followed for the development of databases of mitochondrial respiratory function in diverse physiological and experimental conditions and organisms. This page contains no comments