SummaryHydrogen peroxide (H2O2) is central to mitochondrial oxidative damage and redox signaling, but its roles are poorly understood due to the difficulty of measuring mitochondrial H2O2 in vivo. Here we report a ratiometric mass spectrometry probe approach to assess mitochondrial matrix H2O2 levels in vivo. The probe, MitoB, comprises a triphenylphosphonium (TPP) cation driving its accumulation within mitochondria, conjugated to an arylboronic acid that reacts with H2O2 to form a phenol, MitoP. Quantifying the MitoP/MitoB ratio by liquid chromatography-tandem mass spectrometry enabled measurement of a weighted average of mitochondrial H2O2 that predominantly reports on thoracic muscle mitochondria within living flies. There was an increase in mitochondrial H2O2 with age in flies, which was not coordinately altered by interventions that modulated life span. Our findings provide approaches to investigate mitochondrial ROS in vivo and suggest that while an increase in overall mitochondrial H2O2 correlates with aging, it may not be causative.

Complex I has reactive thiols on its surface that interact with the mitochondrial glutathione pool and are implicated in oxidative damage in many pathologies. However, the Cys residues and the thiol modifications involved are not known. Here we investigate complex I thiol modification within oxidatively stressed mammalian mitochondria, containing physiological levels of glutathione and glutaredoxin 2. In mitochondria incubated with the thiol oxidant diamide, complex I is only glutathionylated on the 75-kDa subunit. Of the 17 Cys residues on the 75-kDa subunit, 6 are not involved in iron-sulfur centers, making them plausible candidates for glutathionylation. Mass spectrometry of complex I from oxidatively stressed bovine heart mitochondria showed that only Cys-531 and Cys-704 were glutathionylated. The other four non-iron-sulfur center Cys residues remained as free thiols. Complex I glutathionylation also occurred in response to relatively mild oxidative stress caused by increased superoxide production from the respiratory chain. Although complex I glutathionylation within oxidatively stressed mitochondria correlated with loss of activity, it did not increase superoxide formation, and reversal of glutathionylation did not restore complex I activity. Comparison with the known structure of the 75-kDa ortholog Nqo3 from Thermus thermophilus complex I suggested that Cys-531 and Cys-704 are on the surface of mammalian complex I, exposed to the mitochondrial glutathione pool. These findings suggest that Cys-531 and Cys-704 may be important in preventing oxidative damage to complex I by reacting with free radicals and other damaging species, with subsequent glutathionylation recycling the thiyl radicals and sulfenic acids formed on the Cys residues back to free thiols.

The generation of mitochondrial superoxide (O2˙̄) by reverse electron transport (RET) at complex I causes oxidative damage in pathologies such as ischemia reperfusion injury, but also provides the precursor to H2O2 production in physiological mitochondrial redox signaling. Here, we quantified the factors that determine mitochondrial O2˙̄ production by RET in isolated heart mitochondria. Measuring mitochondrial H2O2 production at a range of proton-motive force (Δp) values and for several coenzyme Q (CoQ) and NADH pool redox states obtained with the uncoupler p-trifluoromethoxyphenylhydrazone, we show that O2˙̄ production by RET responds to changes in O2 concentration, the magnitude of Δp, and the redox states of the CoQ and NADH pools. Moreover, we determined how expressing the alternative oxidase from the tunicate Ciona intestinalis to oxidize the CoQ pool affected RET-mediated O2˙̄ production at complex I, underscoring the importance of the CoQ pool for mitochondrial O2˙̄ production by RET. An analysis of O2˙̄ production at complex I as a function of the thermodynamic forces driving RET at complex I revealed that many molecules that affect mitochondrial reactive oxygen species production do so by altering the overall thermodynamic driving forces of RET, rather than by directly acting on complex I. These findings clarify the factors controlling RET-mediated mitochondrial O2˙̄ production in both pathological and physiological conditions. We conclude that O2˙̄ production by RET is highly responsive to small changes in Δp and the CoQ redox state, indicating that complex I RET represents a major mode of mitochondrial redox signaling.

Nitric oxide (NO • ) competitively inhibits oxygen consumption by mitochondria at cytochrome c oxidase and S-nitrosates thiol proteins. We developed mitochondria-targeted S-nitrosothiols (MitoSNOs) that selectively modulate and protect mitochondrial function. The exemplar MitoSNO1, produced by covalently linking an Snitrosothiol to the lipophilic triphenylphosphonium cation, was rapidly and extensively accumulated within mitochondria, driven by the membrane potential, where it generated NO • and Snitrosated thiol proteins. MitoSNO1-induced NO • production reversibly inhibited respiration at cytochrome c oxidase and increased extracellular oxygen concentration under hypoxic conditions. MitoSNO1 also caused vasorelaxation due to its NO • generation. Infusion of MitoSNO1 during reperfusion was protective against heart ischemia-reperfusion injury, consistent with a functional modification of mitochondrial proteins, such as complex I, following S-nitrosation. These results support the idea that selectively targeting NO • donors to mitochondria is an effective strategy to reversibly modulate respiration and to protect mitochondria against ischemia-reperfusion injury.nitric oxide ͉ S-nitrosation

Mitochondria-targeted molecules comprising the lipophilic TPP (triphenylphosphonium) cation covalently linked to a hydrophobic bioactive moiety are used to modify and probe mitochondria in cells and in vivo. However, it is unclear how hydrophobicity affects the rate and extent of their uptake into mitochondria within cells, making it difficult to interpret experiments because their intracellular concentration in different compartments is uncertain. To address this issue, we compared the uptake into both isolated mitochondria and mitochondria within cells of two hydrophobic TPP derivatives, [3H]MitoQ (mitoquinone) and [3H]DecylTPP, with the more hydrophilic TPP cation [3H]TPMP (methyltriphenylphosphonium). Uptake of MitoQ by mitochondria and cells was described by the Nernst equation and was approximately 5-fold greater than that for TPMP, as a result of its greater binding within the mitochondrial matrix. DecylTPP was also taken up extensively by cells, indicating that increased hydrophobicity enhanced uptake. Both MitoQ and DecylTPP were taken up very rapidly into cells, reaching a steady state within 15 min, compared with approximately 8 h for TPMP. This far faster uptake was the result of the increased rate of passage of hydrophobic TPP molecules through the plasma membrane. Within cells MitoQ was predominantly located within mitochondria, where it was rapidly reduced to the ubiquinol form, consistent with its protective effects in cells and in vivo being due to the ubiquinol antioxidant. The strong influence of hydrophobicity on TPP cation uptake into mitochondria within cells facilitates the rational design of mitochondria-targeted compounds to report on and modify mitochondrial function in vivo.

No abstract

Remodeling of the plant cell surface occurs during the establishment of cell polarity, cellular differentiation, and organ development. This report demonstrates the existence of multiple glycosylphosphatidylinositol (GPI)‐anchored proteins in the model plant Arabidopsis. Using two‐dimensional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), we also show that GPI‐anchored proteins are a relatively abundant class of protein and that they are present at the plant plasma membrane. Furthermore, some of these proteins are released into the extracellular matrix. At least one of these is an arabinogalactan protein (AGP), a class of proteins known to be associated with cellular differentiation. Analysis of the amino acid sequences of two novel AGP‐like proteins from Arabidopsis predicts that these proteins contain consensus signals for GPI‐anchor addition. These findings support a model where GPI‐anchored proteins are involved in the generation of specialized cell surfaces and extracellular signaling molecules.

Reactive oxygen species (ROS) produced by the mitochondrial respiratory chain can be a redox signal, but whether they affect mitochondrial function is unclear. Here we show that low levels of ROS from the respiratory chain under physiological conditions reversibly modify the thiol redox state of mitochondrial proteins involved in fatty acid and carbohydrate metabolism. As these thiol modifications were specific and occurred without bulk thiol changes, we first had to develop a sensitive technique to identify the small number of proteins modified by endogenous ROS. In this technique, redox difference gel electrophoresis, control, and redox-challenged samples are labeled with different thiol-reactive fluorescent tags and then separated on the same two-dimensional gel, enabling the sensitive detection of thiol redox modifications by changes in the relative fluorescence of the two tags within a single protein spot, followed by protein identification by mass spectrometry. Thiol redox modification affected enzyme activity, suggesting that the reversible modification of enzyme activity by ROS from the respiratory chain may be an important and unexplored mode of mitochondrial redox signaling.Reactive oxygen and nitrogen species (ROS 2 and RNS) are used by mammalian cells as signaling molecules for redox regulation (1, 2). Examples are hydrogen peroxide (H 2 O 2 ) and nitric oxide (NO), which are weak oxidants that can cross membranes and react with defined targets (2, 3). Modifications to protein thiols by ROS/RNS are an important aspect of redox signal transduction (2, 4, 5). These thiol modifications include oxidation to sulfenic acids, intra-and intermolecular disulfides, glutathionylation, sulfenyl-amide linkages, and S-nitrosation (6 -9). As many of these modifications can be reversed through interaction with glutathione or thioredoxin, protein thiol redox state can respond to the redox environment (10, 11). The mitochondrial respiratory chain is the major source of ROS in most cells (12,13) and is involved in redox signaling (14). The proximity of mitochondrial protein thiols to the major ROS source and the high matrix pH (ϳ8.0) make modification of mitochondrial protein thiols by ROS more likely than for cytosolic thiols (15). Some mitochondrial proteins have shown ROS-dependent thiol oxidation:peroxiredoxin III (PrxIII) (16), NADP ϩ -dependent isocitrate dehydrogenase (17), and complex I (18, 19). The role of protein thiol modification in mitochondrial redox regulation, however, remains unclear, in large part because of the difficulty of identifying mitochondrial thiol proteins responsive to the low ROS fluxes found in vivo.To explore the role of mitochondrial protein thiols in redox signaling, we developed a sensitive proteomic approach to identify thiol proteins that are modified by low levels of endogenous ROS. We adapted the difference gel electrophoresis (DIGE) technique (20, 21) labeling protein thiols in control and redox-challenged samples with two different fluorescent dyes containing thiol-reactive mal...