Teaching the fundamentals of electron transfer reactions in mitochondria and the production and detection of reactive oxygen species

Abstract

Mitochondria fulfill a number of biological functions which inherently depend on ATP and O2(-•)/H2O2 production. Both ATP and O2(-•)/H2O2 are generated by electron transfer reactions. ATP is the product of oxidative phosphorylation whereas O2(-•) is generated by singlet electron reduction of di-oxygen (O2). O2(-•) is then rapidly dismutated by superoxide dismutase (SOD) producing H2O2. O2(-•)/H2O2 were once viewed as unfortunately by-products of aerobic respiration. This characterization is fitting considering over production of O2(-•)/H2O2 by mitochondria is associated with range of pathological conditions and aging. However, O2(-•)/H2O2 are only dangerous in large quantities. If produced in a controlled fashion and maintained at a low concentration, cells can benefit greatly from the redox properties of O2(-•)/H2O2. Indeed, low rates of O2(-•)/H2O2 production are required for intrinsic mitochondrial signaling (e.g. modulation of mitochondrial processes) and communication with the rest of the cell. O2(-•)/H2O2 levels are kept in check by anti-oxidant defense systems that sequester O2(-•)/H2O2 with extreme efficiency. Given the importance of O2(-•)/H2O2 in cellular function, it is imperative to consider how mitochondria produce O2(-•)/H2O2 and how O2(-•)/H2O2 genesis is regulated in conjunction with fluctuations in nutritional and redox states. Here, I discuss the fundamentals of electron transfer reactions in mitochondria and emerging knowledge on the 11 potential sources of mitochondrial O2(-•)/H2O2 in tandem with their significance in contributing to overall O2(-•)/H2O2 emission in health and disease. The potential for classifying these different sites in isopotential groups, which is essentially defined by the redox properties of electron donator involved in O2(-•)/H2O2 production, as originally suggested by Brand and colleagues is also surveyed in detail. In addition, redox signaling mechanisms that control O2(-•)/H2O2 genesis from these sites are discussed. Finally, the current methodologies utilized for measuring O2(-•)/H2O2 in isolated mitochondria, cell culture and in vivo are reviewed.

Keywords: Bioenergetics; Mitochondria; Reactive oxygen species; Redox signaling.

Graphical abstract

Fig. 1

Reduction of O₂ to H₂O and its free radical intermediates (A) Lewis structures for molecular oxygen (O₂) and its singlet electron derivatives superoxide (O₂^•−), hydrogen peroxide (H₂O₂), and hydroxyl radical (OH^•). (B) The step wise reduction of O₂ to H₂O during aerobic respiration. The standard redox potential for reduction of each intermediate is also shown (reproduced and modified from [23]). Irradiation-mediated cleavage of H₂O which produces OH^• accounts for the damaging effects of radiation therapy.

Fig. 2

The oxidative Krebs cycle. The Krebs cycle is the most central metabolic pathway for all life on Earth since it yields the necessary carbon intermediates required for either ATP formation or genesis of biomolecules like amino acids, lipids, and nucleotides. Different sources of carbon including glucose (and other monosaccharides), fatty acids, and amino acids are converted into common intermediates acetyl-CoA, oxaloacetate, and 2-oxoglutarate, Krebs cycle intermediates that are then systematically stripped of electrons (oxidized) to yield electron carriers NADH and succinate which are then oxidized by the respiratory chain for ATP production. Note that enzymes Idh and Gdh can utilize either NAD⁺ or NADP⁺ as cofactors which is isozyme dependent. Irreversible steps of the Krebs cycle involve enzymes that couple decarboxylation to electron movement. For clarity key metabolic cascades involved in the conversion of glucose and fatty acids into key intermediates, pyruvate and acetyl-CoA, have been omitted. Pdh; pyruvate dehydrogenase, PC; pyruvate carboxylase, CS; citrate synthase, Acn; aconitase, Idh; isocitrate dehydrogenase, Odh; 2-oxoglutarate dehydrogenase, Scs; succinyl-CoA synthetase, Sdh; succinate dehydrogenase, Fum; fumarase, Mdh; malate dehydrogenase, Gcl; glutaminase, Gdh; glutamate dehydrogenase, At; aminotransferase, Aat; aspartate aminotransferase, Cpt; carnitine palmitoyltransferase, 2-OG; 2-oxoglutarate, MIM; mitochondrial inner membrane, IMS; intermembrane space.

Fig. 3

The electron transport chain and oxidative phosphorylation. Following the genesis of NADH or succinate by the Krebs cycle, both electron carriers are oxidized by complex I (NADH:ubiquinone oxidoreductase) and complex II (succinate:ubiquinone oxidoreductase or succinate dehydrogenase). Oxidation of NADH by complex I yields NAD which returns to the Krebs cycle and two electrons which reduce FMN and are then systematically passed through 7–8 Fe−S clusters to the quinone binding site reducing Q to QH₂. Due to the large difference in E°′ between NADH and Q, electron transfer induces changes in the membrane module of complex I resulting in the pumping of four protons into the IMS. Similarly succinate oxidation by complex II yields fumaric acid and the liberated electrons are passed through FAD and three Fe–S clusters reducing Q to QH₂. Note that complex II is a Krebs cycle enzyme providing a direct link between Krebs cycle flux and electron transfer in the respiratory chain. Complex II is also embedded in the MIM but is not a transmembrane protein. Unlike complexes I, III, and IV, complex II does not couple electron transfer to proton translocation into the IMS. This is attributed to the low Gibbs free energy change for electron transfer in complex II to Q (refer to text for E°′ values for FAD and Q). Complex II also harbors a heme (b₅₆₀) which is thought to be involved in electron recycling. QH₂ is then oxidized by complex III in the Q_o (quinone outer membrane) binding site resulting in the transfer of one electron through the Rieske Fe−S protein and cytochrome C₁ to cytochrome C. Note that C₁ and C can only accept one electron at a time. This generates a semiquinone radical in the Q_o site. The second electron is then passed through heme groups b_L and b_H and utilized to re-reduce Q in the Q_i (quinone inner membrane) binding site. This is referred to as the Q-cycle and is required to recycle electrons during aerobic respiration. Electron movement from QH₂ to C is coupled to the transfer of two protons into the IMS. C then binds subunit II on complex IV where electrons are passed systematically through two copper moieties and two heme groups (a and a₃) resulting in the reduction of oxygen to water at subunit I. For two electrons transferred only one oxygen atom is reduced which also requires an input of two protons. This process is coupled to the pumping of two additional protons into the IMS. Considering, that the the full reduction of di-oxygen (O₂) requires four electrons that means four protons are actually pumped into the IMS (for every two electrons from NADH or succinate two protons are pumped out). Electron transfer and proton pumping creates are transmembrane potential of protons called the protonmotive force (pmf) which is utilized by complex V to drive ATP synthesis. Note that proton re-uptake by complex V is coupled to the rotation of the stalk and F₁ portion of complex V resulting in the biosynthesis of ATP. P; positive side; IMS and N; negative side; matrix.

Fig. 4

Anti-oxidant defense systems in mitochondria: Mitochondria can be a major source of reactive oxygen species (ROS) and production which depends on the metabolic state and redox poise of mitochondria. Metabolic state refers to the efficiency of electron transfer from nutrients to O₂ whereas redox poise is associated with the anti-oxidant capacity, maintenance of a reductive environment by reduced glutathione (GSH; normally in the mitochondrial matrix, the glutathione pool is highly reduced with the ratio of 2GSH to GSSG (2GSH/GSSG) ~100 giving E°′=−320 mV) and the redox state of anti-oxidant enzymes Prx and Trx. The proximal ROS O₂^−• is dismutated rapidly by MnSOD or Cu/ZnSOD in the matrix or intermembrane space, respectively, to H₂O₂ which is then used for signaling via oxidation of protein cysteine thiols (see Fig. 8 for thiol based reactions with oxidants and reductants). Note that H₂O₂ can also diffuse passively from one side of the mitochondrial membrane to the next with the aid of aquaporins (AQP). H₂O₂ levels are continuously monitored by endogenous anti-oxidant systems. The far most efficient systems utilized to quench H₂O₂ are 1. Glutathione peroxidase (GPx)/glutathione reductase (GR) and 2. Peroxiredoxin (Prx)/thioredoxin (Trx)/Thioredoxin Reductase (TR) system. Mitochondria contain two GPx and two Prx isozymes; GPx1 and GPx4; Prx3 and Prx5. Although all four enzymes quench H₂O₂, GPx1 and Prx3 have a higher affinity for H2O2 while GPx4 and Prx5 metabolize lipid hydroperoxides more efficiently . Systems 1 and 2 are supported by system 3 which produces NADPH, the reductive power required to rejuvenate anti-oxidant systems after a round of H₂O₂ sequestration. Note that NADPH is either generated from the metabolism of isocitrate, malate, or glucose-6-phosphate by isocitrate dehydrogenase (Idh), malic enzyme (ME), or glucose-6-phosphate dehydrogenase (G6pd) or via conversion of NADH into NADPH by energy liberating transhydrogenase (Elth).

Fig. 5

Overview of the 11 different sources for O₂^−•/H₂O₂ production. Mitochondrial ATP and O₂^−•/H₂O₂ are intimately linked by electron transfer from nutrients to di-oxygen (O₂). Nutrients (glucose, fatty acids, amino acids) are enzymatically converted to common intermediates (acetyl-CoA, oxaloacetate, pyruvate) which enter the Krebs cycle to undergo further oxidation. Metabolite oxidation is coupled to the evolution of carbon dioxide (CO₂) and the production of NADH and succinate which are then oxidized by complexes I and II respectively. Electron flow through the respiratory complexes through ubiquinone (Q) and cytochrome C (C) and the reduction of O₂ to H₂O is coupled to the formation of a transmembrane potential of proteins across the mitochondrial inner membrane (MIM) which is then utilized to drive ATP synthesis by complex V. ATP is then transported out of mitochondria in exchange for ADP by ATP:ADP exchanger (ANT). The proton gradient can also be mildly uncoupled by uncoupling proteins (UCP) 2 and 3 which are utilized to control O₂^−•/H₂O₂ production. Electron transfer flavoprotein oxidoreductase (ETFQO), dihydroorotate dehydrogenase (Dhodh), proline dehydrogenase (Prodh), succinate:quinone reductase (SQR), sn-glycerol-3-phosphate dehydrogenase (G3PDH) can also feed electrons into the Q pool following oxidation of their cognate substrates. Red stars indicate that 11 potential sources of O₂^−•/H₂O₂. Dotted lines represent flow of electrons. Bold dotted lines indicate flow of protons (H⁺). (1) Citrate synthase, (2) aconitase, (3) NAD(P)⁺-isocitrate dehydrogenase, (4) 2-oxoglutarate dehydrogenase, (5) succinyl-CoA synthase, (6) fumarase, (7) malate dehydrogenase, (8) pyruvate dehydrogenase, (9) pyruvate carboxylase, (10) branched chain keto acid dehydrogenase.

Fig. 6

Modulation of Pdh by allosteric regulation and phosphorylation. The enzymatic products acetyl-CoA and NADH serve as allosteric inhibitors whereas NAD⁺, ADP, and CoASH serve as activators of Pdh activity. Thus, Pdh efficiency is reliant on the oxidation of NADH by complex I, condensation of acetyl-CoA with oxaloacetate, and turnover of ATP in the cell. Note that these allosteric modulators also control pyruvate dehydrogenase kinase (Pdk) and pyruvate dehydrogenase phosphatase (Pdp) which phosphorylate and dephosphorylate, respectively, the E₁ subunit to modulate Pdh activity. Phosphorylation inhibits Pdh activity whereas dephosphorylation has the opposite effect. In addition, hormonal signaling cascades like insulin signaling also play a part in modulating Pdh activity in response to whole body changes in nutrition and energy state. Odh is also modulated by allosteric regulators, calcium, and phosphorylation in a similar manner.

Fig. 7

Hypothetical mechanism for the regulation of Odh and Pdh by reversible S-glutathionylation is required to modulate mitochondrial O₂^−•/H₂O₂. Odh and Pdh couple oxidation of 2-oxoglutarate or pyruvate to formation of either acetyl-CoA or succinyl-CoA and the production of NADH. O₂^−•/H₂O₂ production is minimal due to the rapid oxidation of NADH to rejuvenate NAD pools. PHASE 1, ACCUMULATION: NADH oxidation slows most likely due to a decrease in complex I activity resulting in an increase in NADH levels. This prompts increased O₂^−•/H₂O₂ production by the E₃ subunit of either enzyme. Substrate oxidation also slows diminishing NADH formation. PHASE 2, OXIDATION: vicinal thiols (-SH) on E₂ subunit are oxidized by the increase in H₂O₂ levels yielding highly reactive sulfenic acids (-SOH) deactivating the enzyme complex. Although O₂^−•/H₂O₂ emission is decreased by this modification the –SOH renders the enzyme complex amenable to irreversible oxidation. PHASE 3, PROTECTION: sulfenic acids are modified by S-glutathionylation by conjugation to glutathione (GSH), a reaction potentially catalyzed by glutathione S-transferase (GST). This effectively protects Odh and Pdh from further oxidation. Enzyme remains inactive during this phase of regulation which also prevents O₂^−•/H₂O₂ production. PHASE 4, RECOVERY: the glutathionyl moiety is removed potentially by glutaredoxin-2 (Grx2) yielding a fully active enzyme complex. Odh and Pdh are now fully active coupling substrate oxidation to NADH formation. Grx2-mediated deglutathionylation generates GSSG which is reduced back to GSH by glutathione reductase and NADPH.

Fig. 8

Regulation of protein function by S-glutathionylation. Redox signaling refers to the control of protein function via site specific oxidation of protein cysteine thiols in response to redox fluctuations in the surrounding cellular environment. Although there are a number of redox modifications that are known to modulate cellular protein functions , S-glutathionylation and the formation of protein glutathione mixed disulfides is highly specific, sensitive to redox fluctuations and mediated enzymatically and thus the most relevant redox modification. The reactivity of a thiol towards glutathione depends on its ability to ionize and form a reactive thiolate anion. Ionization is heavily influenced by the chemistry of the surrounding protein environment. In the presence of sufficient quantities of H₂O₂, the thiolate anion nucleophilically attacks H₂O₂ yielding an oxidized sulfur residue (SO⁻; sulfenic acid). If H₂O₂ is in high enough amounts, the sulfenic acid can be further oxidized to sulfinic (SO₂H) and sulfonic acid (SO₃H). Note that sulfinic acid can be reduced back to a thiolate by the action of sulfiredoxin (Srx) which requires ATP. Sulfonic acids, however, are an irreversible type of oxidation associated with oxidative stress. Proteins can either be S-glutathionylated at the level of the thiolate or sulfenic acid. In terms of the former, S-glutathionylation is driven by glutaredoxin-2 in mitochondria (Grx2) or glutaredoxin-1 (Grx1) in the cytosol and intermembrane space. S-glutathionylation can be quickly reversed by Grx. Importantly, protein S-glutathionylation is highly sensitive to fluctuations in reduced and oxidized glutathione levels and the circumstances by which a protein is S-glutathionylated varies according to the type of protein and the environment surrounding the protein cysteine thiol (reviewed in [6,12,23]). In terms of the latter, S-glutathionylation of sulfenic acids is required to protect cysteine thiols from further oxidation when H₂O₂ is at higher concentrations.

Fig. 9

Hypothetical mechanism for the modulation of O₂^−•/H₂O₂ by complex III via S-glutathionylation controlled proton leaks. Complex III systematically oxidizes QH₂ donating one electron at a time to cytochrome C. The resulting QH^−• is recycled via the Q-cycle for another round of oxidation. Fully oxidized Q returns to the Q pool to be reduced by complex I, II, or other electron donors (see Fig. 1). PHASE 1, POLARIZATION: electrochemical transmembrane potential of protons (ΔΨ_m) increasing membrane polarity which limits QH₂ and QH^−● oxidation. QH^−● accumulates in the Q_o site augmenting mitochondrial O₂^−•/H₂O₂ production. PHASE 2, ACTIVATION: the rise in mitochondrial O₂^−•/H₂O₂ production and ΔΨ_m results in the deglutathionylation and activation of UCP2 and UCP3. Note that UCP2 and UCP3 are expressed in different tissues and activation can have different physiological consequences. Deglutathionylation is mediated by low µM increase in H₂O₂. The reaction is catalyzed by an as of yet unidentified enzyme . Activation induces mild uncoupling of the mitochondrial inner membrane decreasing protonic pressure on complex III thus limiting O₂^−•/H₂O₂ production. PHASE 3, CONJUGATION: the decrease O₂^−•/H₂O₂ production and ΔΨ_m results in the reglutathionylation and deactivation of UCP2 and UCP3. The reaction is catalyzed by Grx2. PHASE 4, RESTORATION: with the ΔΨ_m brought back down QH₂ oxidation by complex III resumes with efficient recovery of electrons from QH^−● in the Q-cycle. Note that in this diagram ANT was omitted for clarity however; as indicated in the text it also plays an important role in inducible proton leaks and is targeted for S-glutathionylation.

Fig. 10

Proposed mechanism for the reaction of MitoSOX with O₂^•− (modified from [121] and [120]). MitoSOX is composed of hydroethidine which reacts with O₂^•− and triphenylphosphonium (R-group) which prompts accumulation in mitochondria (1). The structure is univalently reduced by one electron generate a chemical structure that resonates between a hydroethidine radical with the lone electron delocalized to throughout the ring (2a) and an amide radical (2b). Electron delocalization between the ring and amide aids in stabilizing this reactive intermediate. This is followed by an interaction with O₂^•− which results in the production of a perhydroxyl intermediate (3) which is then dehydrated to yield a carbonyl derivative (4) that is then protonated to generate 2-hydroxy-ethidine or 2-hydroxy-MitoSOX (5).

Fig. 11

cpYFP is not a mitochondrial O₂^•− indicator but pH detector. Cyclic permuted YFP (cpYFP) gene can be tagged with a mitochondrial localization sequence (MLS) and stably expressed in cells or animal tissues. Note that cpYFP gene can also be placed under control of tissue specific promoters to allow for site specific expression of cpYFP. Following gene transcription and translation the MLS selectively targets cpYFP for uptake and accumulation in the matrix of mitochondria where cpYFP detects transient changes in mitochondrial metabolism and physiology. Although cpYFP has been utilized in numerous studies to measure “stochastic” changes in mitochondrial O₂^•− emission the mechanism by which it does so remains elusive but also seems chemically improbable. Rather, given the features of cpYFP and its sensitivity to changes in matrix pH it is far more likely that cpYFP serves as a protein-based pH sensor akin to SyPHer. As indicated in the text, it has been well documented that transient shifts in cpYFP fluorescence are sensitive to changes in the concentration of H⁺ in the matrix. In addition, shifts in cpYFP fluorescence correlate strongly with changes in Δμ_m as indicated by fluctuations in TMRE fluorescence.

Fig. 12

Detection of mitochondrial H₂O₂ with Amplex Red and MitoB. a. In the presence of H₂O₂, horseradish peroxidase catalyzes the oxidation of nonfluorescent Amplex red forming fluorescent product resorufin. The fluorescent signal is directly proportion to the amount of H₂O₂ present in the sample. H₂O₂ can be generated within mitochondria following dismutation of O₂^•− which is exported by aquaporins for detection. Alternatively H₂O₂ can be produced outside the matrix environment if O₂^•− is produced in the intermembrane space. To ensure maximal conversion of O₂^•− to H₂O₂, exogenous SOD can be added which provides a proxy measure of both O₂^•−/H₂O₂. Control reactions may include addition of exogenous catalase. Note that presence of endogenous antioxidant system such as GSH/Gpx/GR and Prx/Trx/TR quench H₂O₂. Thus, Gpx or Trx inhibitors can be utilized to afford accurate H₂O₂ quantification. b. The arylboronic acid moiety is tagged with a triphenylphosphonium ion (CH₂−P⁺(Ph)₃) group which prompts matrix accumulation of the detector allowing for more accurate quantification of matrix H₂O₂. Following its interaction with H₂O₂ with MitoB, the corresponding phenol can be detected by fluorescence. Alternatively, the tissue can be isolated and MitoP and MitoB levels can be subjected to LC-MS/MS analysis. Detection of MitoP and MitoB levels by LC-MS/MS and calculation of the MitoP/MitoB ratio provides a highly quantitative measure of matrix H₂O₂ levels.

Fig. 13

Protein-based mitochondrial H₂O₂ detectors. Three separate protein based probes can be employed to detect mitochondrial H₂O₂ levels, HyPer (OxyR-cpYFP) and roGFP2-Orp1 which directly detect fluctuations in H₂O₂ and Grx1-roGFP2 which indirectly detects H₂O₂ via interactions with GSSG (produced by glutathione peroxidase which catalyzes the sequestration of H₂O₂ oxidizing two GSH generating GSSG). Mitochondrial targeting of the different probes can be achieved by tagging the protein gene sequence with a mitochondrial localization signal (MLS).