Production of superoxide and hydrogen peroxide in the mitochondrial matrix is dominated by site IQ of complex I in diverse cell lines

Abstract

Understanding how mitochondria contribute to cellular oxidative stress and drive signaling and disease is critical, but quantitative assessment is difficult. Our previous studies of cultured C2C12 cells used inhibitors of specific sites of superoxide and hydrogen peroxide production to show that mitochondria generate about half of the hydrogen peroxide released by the cells, and site I Q of respiratory complex I produces up to two thirds of the superoxide and hydrogen peroxide generated in the mitochondrial matrix. Here, we used the same approach to measure the engagement of these sites in seven diverse cell lines to determine whether this pattern is specific to C2C12 cells, or more general. These diverse cell lines covered primary, immortalized, and cancerous cells, from seven tissues (liver, cervix, lung, skin, neuron, heart, bone) of three species (human, rat, mouse). The rate of appearance of hydrogen peroxide in the extracellular medium spanned a 30-fold range from HeLa cancer cells (3 pmol/min/mg protein) to AML12 liver cells (84 pmol/min/mg protein). The mean contribution of identified mitochondrial sites to this extracellular hydrogen peroxide signal was 30 ± 7% SD; the mean contribution of NADPH oxidases was 60 ± 14%. The relative contributions of different sites in the mitochondrial electron transport chain were broadly similar in all seven cell types (and similar to published results for C2C12 cells). 70 ± 4% of identified superoxide/hydrogen peroxide generation in the mitochondrial matrix was from site I Q ; 30 ± 4% was from site III Qo . We conclude that although absolute rates vary considerably, the relative contributions of different sources of hydrogen peroxide production are similar in nine diverse cell types under unstressed conditions in vitro . Identified mitochondrial sites account for one third of total cellular hydrogen peroxide production (half each from sites I Q and III Qo ); in the mitochondrial matrix the majority (two thirds) of superoxide/hydrogen peroxide is from site I Q .

NADH/NAD+ Redox Imbalance and Diabetic Kidney Disease

“…There are at least 11 mitochondrial sites that are involved in superoxide generation [ 147 ]. Therefore, overall approaches of dismutating superoxide could alleviate DKD [ 148 ].…”

“…Typical examples of these small molecules are S1QELs and S3QELs [ 145 , 149 ], which do not interfere with the process of oxidative phosphorylation or ATP production [ 144 ]. S1QELs acts at the site I Q of complex I [ 145 , 147 ] while S3QELs acts at the Q site of complex III [ 150 , 151 ]. The usefulness of these suppressors in combating oxidative stress has been tested in certain experimental systems [ 151 , 152 , 153 , 154 ].…”

Miro1-mediated mitochondrial positioning supports subcellular redox status

“…ROS are produced from several cellular sources including enzyme specific production of ROS by NADPH oxidases (NOXs) and from mitochondria during oxidative phosphorylation [ 30 , 31 ]. Approximately 0.15%–2% of the total electron flow through the mitochondrial electron transport chain (ETC) supports generation of mitochondrial ROS (mROS) [ 32 ], accounting for ~30% of cell derived extracellular H 2 O 2 [ 33 ]. ETC complexes are arranged spatially in a hierarchy of redox potential, although the transfer of electrons between complexes is not confined to a closed system, as thermodynamically all ETC complexes can effectively reduce molecular oxygen [ 34 ].…”

S3QELs protect against diet‐induced intestinal barrier dysfunction

“…The identification of S1QELs, which are specific small‐molecule Suppressors of site I Q Electron Leak, and S3QELs, which are specific small‐molecule Suppressors of site III Qo Electron Leak, offers precise tools to identify and prevent superoxide/hydrogen peroxide production by complex I or III and its downstream effects without interfering with other sites (Brand et al, 2016 ; Orr et al, 2015 ). Studies using these compounds have established that sites I Q and III Qo not only have the highest capacity of all mitochondrial sites to produce superoxide/hydrogen peroxide in vitro, but also that they are the main contributors of superoxide in the mitochondrial matrix in several cell lines (Brand, 2016 ; Fang et al, 2020 ; Wong et al, 2019 ). These tools enable investigation of the contributions and importance of superoxide/hydrogen peroxide production by mitochondrial sites I Q and III Qo in pathologies and physiology (Brand, 2020 ; Watson et al, 2019 ).…”