Regulation of Mitochondrial Hydrogen Peroxide Availability by Protein S-glutathionylation

Abstract

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.

Keywords: bioenergetics; glutathionylation; hydrogen peroxide; mitochondria; redox signaling.

Figure 1

The addition and removal of glutathione to and from target proteins is a rapid event that occurs in response to spatiotemporal fluctuations in GSH and GSSG availability. Cell nutrient metabolism and electron release from various enzymes (e.g., flavin-dependent dehydrogenases) causes the rapid production of H₂O₂ and its clearance by glutathione peroxidases (GPX). This results in the oxidation of the glutathione pool and a decrease in the GSH/GSSG ratio. The forward glutathionylation reaction is driven by glutaredoxins (GRX1; cytoplasm and intermembrane space, GRX2; matrix) in response to glutathione pool oxidation. Production of NADPH by the same nutrient oxidizing pathways that generate H₂O₂ is used by glutathione reductases (GR) to reduce the disulfide bridge in GSSG, reforming GSH and restoring the reducing power of the glutathione pool. This activates the deglutathionylase activities of glutaredoxins, resulting in the removal of GSH from a target protein. Figure was generated with Biorender.com, accessed on 1 June 2022.

Figure 2

Reversible glutathionylation reactions in mitochondria are intimately linked to oxido-reduction reactions that drive nutrient oxidation and NADPH production. Oxidation of nutrients to produce NADH, either by the Krebs cycle or other non-Krebs cycle reactions (e.g., amino acid catabolism or fatty acid oxidation), fuels electron transfer and the creation of a transmembrane potential of protons. Nutrients can also be oxidized directly by the electron transport chain. Electrons are used to reduce O₂ at the end of the chain to H₂O and the protonmotive force (PMF) induces the phosphorylation of ADP by complex V. Electrons originating from these nutrients can also react with O₂ in several flavin-dependent dehydrogenases and the electron transferring chain to generate H₂O₂. Note that sites that generate ROS in mitochondria produce a mixture of O₂^•− and H₂O₂, but the latter form dominates over the former. Any residual O₂^−- formed is converted to H₂O₂ by SOD. Clearance of H₂O₂ oxidizes the glutathione pool driving protein S-glutathionylation. The same electron transferring pathways that generate H₂O₂ also generate NADPH, a reducing equivalent required for antioxidant defenses. This is achieved by the mitochondrial redox buffer sentinel, nicotinamide nucleotide transhydrogenase (NNT). The proton gradient yielded from nutrient metabolism is tapped by NNT to drive the transfer of a hydride from NADH to NADP⁺. The NADPH generated is used to reduce the glutathione pool, restoring antioxidant defenses and inducing deglutathionylation.

Figure 3

Reversible glutathionylation reactions are integral for controlling the production of H₂O₂ by complex I and the electron transport chain. (A). Forward electron flow from the Krebs cycle to complex I through the production of NADH (generated by Krebs cycle flux or oxidation of nutrients by non-Krebs cycle enzymes) drives the genesis of both ATP and H₂O₂, which is dependent on electron transfer reactions through the respiratory chain. H₂O₂ is used in mitochondria-to-cell signaling through direct oxidation of protein thiols or oxidation of redox networks (e.g., glutathione and the reversible glutathionylation of cell proteins). Oxidation of the same redox networks feeds back on sites for H₂O₂ production in the respiratory chain resulting in glutathionylation. This inhibits H₂O₂ production, decreasing oxidant genesis by mitochondria and promoting the reactivation of mitochondrial redox networks to maintain antioxidant defenses. This mechanism also desensitizes H₂O₂ signals emanating from mitochondria during the metabolism of nutrients that generate NADH, which is oxidized by complex I. Blockage of complex I through the glutathionylation of the NDUFS1 subunit inhibits NADH oxidation by preventing electron flow to flavin mononucleotide (FMN), the main site for H₂O₂ production in complex I. This mechanism for inhibition of oxidant production also prevents H₂O₂ by sites downstream from complex I, such as complex III. (B). Over oxidation or prolonged oxidation of glutathione pools results in the extended glutathionylation of target proteins in mitochondria. This can result in increased H₂O₂ production of the respiratory chain. Increased H₂O₂ production occurs after prolonged glutathionylation of NDUFS1 subunit in complex I. Although this inhibits NADH-mediated H₂O₂ genesis, this also promotes ROS production through the oxidation of nutrients that by-pass the Krebs cycle and donate electrons directly to the UQ pool. Bioenergetics that favor reverse electron transfer (e.g., over reduction of electron donating/accepting centers in the respiratory chain and a polarized mitochondrial inner membrane) augments H₂O₂ by complex I. Aberrant or prolonged glutathionylation of complex I under these conditions also activates H₂O₂ genesis during forward electron transfer to complex III. This prolonged glutathionylation of complex I and the subsequent increase in H₂O₂ genesis is likely associated with the oxidative distress observed in many pathologies such as heart disease, development of cataracts, and age-related sarcopenia.

Figure 3

Reversible glutathionylation reactions are integral for controlling the production of H₂O₂ by complex I and the electron transport chain. (A). Forward electron flow from the Krebs cycle to complex I through the production of NADH (generated by Krebs cycle flux or oxidation of nutrients by non-Krebs cycle enzymes) drives the genesis of both ATP and H₂O₂, which is dependent on electron transfer reactions through the respiratory chain. H₂O₂ is used in mitochondria-to-cell signaling through direct oxidation of protein thiols or oxidation of redox networks (e.g., glutathione and the reversible glutathionylation of cell proteins). Oxidation of the same redox networks feeds back on sites for H₂O₂ production in the respiratory chain resulting in glutathionylation. This inhibits H₂O₂ production, decreasing oxidant genesis by mitochondria and promoting the reactivation of mitochondrial redox networks to maintain antioxidant defenses. This mechanism also desensitizes H₂O₂ signals emanating from mitochondria during the metabolism of nutrients that generate NADH, which is oxidized by complex I. Blockage of complex I through the glutathionylation of the NDUFS1 subunit inhibits NADH oxidation by preventing electron flow to flavin mononucleotide (FMN), the main site for H₂O₂ production in complex I. This mechanism for inhibition of oxidant production also prevents H₂O₂ by sites downstream from complex I, such as complex III. (B). Over oxidation or prolonged oxidation of glutathione pools results in the extended glutathionylation of target proteins in mitochondria. This can result in increased H₂O₂ production of the respiratory chain. Increased H₂O₂ production occurs after prolonged glutathionylation of NDUFS1 subunit in complex I. Although this inhibits NADH-mediated H₂O₂ genesis, this also promotes ROS production through the oxidation of nutrients that by-pass the Krebs cycle and donate electrons directly to the UQ pool. Bioenergetics that favor reverse electron transfer (e.g., over reduction of electron donating/accepting centers in the respiratory chain and a polarized mitochondrial inner membrane) augments H₂O₂ by complex I. Aberrant or prolonged glutathionylation of complex I under these conditions also activates H₂O₂ genesis during forward electron transfer to complex III. This prolonged glutathionylation of complex I and the subsequent increase in H₂O₂ genesis is likely associated with the oxidative distress observed in many pathologies such as heart disease, development of cataracts, and age-related sarcopenia.