Fatty acid oxidation drives mitochondrial hydrogen peroxide production by α-ketoglutarate dehydrogenase.

In the present study, we examined the mitochondrial hydrogen peroxide (mH2O2) generating capacity of α-ketoglutarate dehydrogenase (KGDH) and compared it to components of the electron transport chain using liver mitochondria isolated from male and female C57BL6N mice. We show for the first time there are some sex dimorphisms in the production of mH2O2 by electron transport chain complexes I and III when mitochondria are fueled with different substrates. However, in our investigations into these sex effects, we made the unexpected and compelling discovery that 1) KGDH serves as a major mH2O2 supplier in male and female liver mitochondria and 2) KGDH can form mH2O2 when liver mitochondria are energized with fatty acids but only when malate is used to prime the Krebs cycle. Surprisingly, 2-keto-3-methylvaleric acid (KMV), a site-specific inhibitor for KGDH, nearly abolished mH2O2 generation in both male and female liver mitochondria oxidizing palmitoyl-carnitine. KMV inhibited mH2O2 production in liver mitochondria from male and female mice oxidizing myristoyl-, octanoyl-, or butyryl-carnitine as well. S1QEL 1.1 (S1) and S3QEL 2 (S3), compounds that inhibit reactive oxygen species generation by complexes I and III, respectively, without interfering with OxPhos and respiration, had a negligible effect on the rate of mH2O2 production when pyruvate or acyl-carnitines were used as fuels. However, inclusion of KMV in reaction mixtures containing S1 and/or S3 almost abolished mH2O2 generation. Together, our findings suggest KGDH is the main mH2O2 generator in liver mitochondria, even when fatty acids are used as fuel.

Coenzyme Q10 and nicotinamide nucleotide transhydrogenase: Sentinels for mitochondrial hydrogen peroxide signaling.

Mitochondria use hydrogen peroxide (H2O2) as a mitokine for cell communication. H2O2 output for signaling depends on its rate of production and degradation, both of which are strongly affected by the redox state of the coenzyme Q10 (CoQ) pool and NADPH availability. Here, we propose the CoQ pool and nicotinamide nucleotide transhydrogenase (NNT) have evolved to be central modalities for mitochondrial H2O2 signaling. Both factors play opposing yet equally important roles in dictating H2O2 availability because they are connected to one another by two central parameters in bioenergetics: electron supply and Δp. The CoQ pool is the central point of convergence for electrons from various dehydrogenases and the electron transport chain (ETC). The increase in Δp creates a significant amount of protonic backpressure on mitochondria to promote H2O2 genesis through CoQ pool reduction. These same factors also drive the activity of NNT, which uses electrons and the Δp to eliminate H2O2. In this way, electron supply and the magnitude of the Δp manifests as a redox connection between the two sentinels, CoQ and NNT, which serve as opposing yet equally important forces required for budgeting H2O2. Taken together, CoQ and NNT are sentinels linked through mitochondrial bioenergetics to manage H2O2 availability for interorganelle and intercellular redox signaling.

S-nitroso-glutathione (GSNO) inhibits hydrogen peroxide production by alpha-ketoglutarate dehydrogenase: An investigation into sex and diet effects.

Pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (KGDH) are vital sources of hydrogen peroxide (H2O2) and key sites for redox regulation. Here, we report KGDH is more sensitive to inhibition by S-nitroso-glutathione (GSNO) when compared to PDH and deactivation of both enzymes by nitro modification is affected by sex and diet. Liver mitochondria from male C57BL/6 N mice displayed a robust inhibition of H2O2 production after exposure to 500-2000 µM GSNO. H2O2 genesis by PDH was not significantly affected by GSNO. Purified KGDH of porcine heart origin displayed a ∼82% decrease in H2O2 generating activity at 500 µM GSNO, which was mirrored by a decrease in NADH production. By contrast, H2O2- and NADH-producing activity of purified PDH was only minimally affected by an incubation in 500 µM GSNO. Incubations in GSNO had no significant effect on the H2O2-generating activity of KGDH and PDH in female liver mitochondria when compared to samples collected from males, which was attributed to higher GSNO reductase (GSNOR) activity. High fat feeding augmented the GSNO-mediated inhibition of KGDH in liver mitochondria from male mice. Exposure of male mice to a HFD also resulted in a significant decrease in the GSNO-mediated inhibition of H2O2 genesis by PDH, an effect not observed in mice fed a control-matched diet (CD). Female mice displayed higher resistance to the GSNO-induced inhibition of H2O2 production, regardless of being fed a CD or HFD. However, exposure to a HFD did result in a small but significant decrease in H2O2 production by KGDH and PDH when female liver mitochondria were treated with GSNO. Although, the effect was less when compared to their male counterparts. Collectively, we show for the first time GSNO deactivates H2O2 production by α-keto acid dehydrogenases and we demonstrate that sex and diet are determinants for the nitro-inhibition of both KGDH and PDH.

High-throughput design of bacterial anti-sense RNAs using CAREng.

Summary: Short RNA (sRNA) modulation of gene expression is an increasingly popular tool for bacterial functional genomics. Antisense pairing between an sRNA and a target messenger RNA results in post-transcriptional down-regulation of a specific gene and can thus be used both for investigating individual gene function and for large-scale genetic screens. sRNAs have several advantages over knockout libraries in studies of gene function, including inducibility, the capacity to interrogate essential genes and easy portability to multiple genetic backgrounds. High-throughput, systematic design of antisense RNAs will increase the efficiency and repeatability of sRNA screens. To this end, we present CAREng, the Computer-Automated sRNA Engineer. CAREng designs antisense RNAs for all coding sequences in a given genome, while checking for potential off-targets. Availability and implementation: CAREng is available as a Python script and through a web portal (https://caren.carleton.ca). Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Regulation of Mitochondrial Hydrogen Peroxide Availability by Protein S-glutathionylation.

BACKGROUND: It has been four decades since protein S-glutathionylation was proposed to serve as a regulator of cell metabolism. Since then, this redox-sensitive covalent modification has been identified as a cell-wide signaling platform required for embryonic development and regulation of many physiological functions. SCOPE OF THE REVIEW: Mitochondria use hydrogen peroxide (H2O2) as a second messenger, but its availability must be controlled to prevent oxidative distress and promote changes in cell behavior in response to stimuli. Experimental data favor the function of protein S-glutathionylation as a feedback loop for the inhibition of mitochondrial H2O2 production. MAJOR CONCLUSIONS: The glutathione pool redox state is linked to the availability of H2O2, making glutathionylation an ideal mechanism for preventing oxidative distress whilst playing a part in desensitizing mitochondrial redox signals. GENERAL SIGNIFICANCE: The biological significance of glutathionylation is rooted in redox status communication. The present review critically evaluates the experimental evidence supporting its role in negating mitochondrial H2O2 production for cell signaling and prevention of electrophilic stress.