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Since 1981, Gordon Research Conferences have been held on the topic of Oxygen Radicals on a biennial basis, to highlight and discuss the latest cutting edge research in this area. Since the first meeting, one special feature of this conference has been the awarding of the so-called Iron Bolt, an award that started in jest but has gained increasing reputation over the years. Since no written documentation exists for this Iron Bolt award, this perspective serves to overview the history of this unusual award, and highlights various experiences of previous winners of this "prestigious" award and other interesting anecdotes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The genetically encoded probes roGFP2-Orp1 and Grx1-roGFP2 have been designed to be selectively oxidized by hydrogen peroxide (H 2 O 2 ) and glutathione disulfide (GSSG), respectively. Both probes have demonstrated such selectivity in a broad variety of systems and conditions. In this study, we systematically compared the in vitro response of roGFP2, roGFP2-Orp1 and Grx1-roGFP2 to increasing amounts of various oxidant species that may also occur in biological settings. We conclude that the previously established oxidant selectivity is highly robust and likely to be maintained under most physiological conditions. Yet, we also find that hypochlorous acid, known to be produced in the phagocyte respiratory burst, can lead to non-selective oxidation of roGFP2-based probes at concentrations ≥2µM, in vitro. Further, we confirm that polysulfides trigger direct roGFP2 responses. A side-by-side comparison of all three probes can be used to reveal micromolar amounts of hypochlorous acid or polysulfides., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The transcription factor nuclear factor-κB (NF-κB) mediates expression of key genes involved in innate immunity and inflammation. NF-κB activation has been repeatedly reported to be modulated by hydrogen peroxide (H 2 O 2 ). Here, we show that the NF-κB-activating signaling adapter myeloid differentiation primary response gene 88 (MyD88) is highly sensitive to oxidation by H 2 O 2 and may be redox-regulated in its function, thus facilitating an influence of H 2 O 2 on the NF-κB signaling pathway. Upon oxidation, MyD88 forms distinct disulfide-linked conjugates which are reduced by the MyD88-interacting oxidoreductase nucleoredoxin (Nrx). MyD88 cysteine residues functionally modulate MyD88-dependent NF-κB activation, suggesting a link between MyD88 thiol oxidation state and immune signaling., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Most studies investigating the influence of H2O2 on cells in culture apply nonphysiological concentrations over nonphysiological time periods (i.e., a one-time bolus that is metabolized in minutes). As an alternative, the glucose oxidase/catalase (GOX/CAT) system allows application of physiologically relevant H2O2 concentrations (300nM-10µM) over physiologically relevant time periods (up to 24h). Recent findings suggest that bolus and GOX/CAT treatments can lead to opposing cellular responses, thus warranting a quantitative comparison between the two approaches. First, we established a reaction-diffusion model that can predict the behavior of the GOX/CAT system with spatiotemporal resolution, thus aiding selection of optimal experimental conditions for its application. Measurements of H2O2 concentration in the cellular supernatant with the luminol/hypochlorite system were consistent with the predictions of the model. Second, we compared the impact of bolus and GOX/CAT treatments on cytosolic H2O2 levels over time. Intracellular H2O2 was monitored by the response of the thiol peroxidase Prx2 and the H2O2 sensor roGFP2-Orp1. We found that Prx2 rapidly and reversibly responds to submicromolar H2O2 levels and accurately reflects kinetic competition with cellular catalase. Our measurements reveal fundamental differences in the dynamic response of cellular H2O2 concentrations following either bolus or GOX/CAT treatments. Thus, different, or even opposing, biological outcomes from differing means of H2O2 delivery may be expected. Cellular responses induced by bolus treatment may not occur under GOX/CAT conditions, and vice versa., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Redox biochemistry plays an important role in a wide range of cellular events. However, investigation of cellular redox processes is complicated by the large number of cellular redox couples, which are often not in equilibrium with one another and can vary significantly between subcellular compartments and cell types. Further, it is becoming increasingly clear that different redox systems convey different biological information; thus it makes little sense to talk of an overall "cellular redox state". To gain a more differentiated understanding of cellular redox biology, quantitative, redox couple-specific, in vivo measurements are necessary. Unfortunately our ability to investigate specific redox couples or redox-reactive molecules with the necessary degree of spatiotemporal resolution is very limited. The development of genetically encoded redox biosensors offers a promising new way to investigate redox biology. Recently developed redox-sensitive green fluorescent proteins (roGFPs), genetically fused to redox-active proteins, allow rapid equilibration of the roGFP moiety with a specific redox couple. Two probes based on this principle are now available: Grx1-roGFP2 for the measurement of glutathione redox potential (E(GSH)) and roGFP2-Orp1 for measuring changes in H(2)O(2) concentration. Here we provide a detailed protocol for the use of these probes in both yeast and mammalian systems using either plate-reader- or microscopy-based measurements., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Reactive oxygen species (ROS) are released at the mitochondrial inner membrane by the electron transport chain (ETC). Increasing evidence suggests that mitochondrial H2O2 acts as a signaling molecule and participates in the (feedback) regulation of mitochondrial activity and turnover. It seems likely that key mitochondrial components contain redox-sensitive thiols that help to adapt protein function to changes in electron flow. However, the identity of most redox-regulated mitochondrial proteins remains to be defined. Thioredoxin 2 (Trx2) is the major protein-thiol-reducing oxidoreductase in the mitochondrial matrix. We used in situ mechanism-based kinetic trapping to identify disulfide-exchange interactions of Trx2 within functional mitochondria of intact cells. Mass spectrometry successfully identified known and suspected Trx2 target proteins and, in addition, revealed a set of new candidate target proteins. Our results suggest that the mitochondrial protein biosynthesis machinery is a major target of ETC-derived ROS. In particular, we identified mitochondrial methionyl-tRNA synthetase (mtMetRS) as one of the most prominent Trx2 target proteins. We show that an increase in ETC-derived oxidants leads to an increase in mtMetRS oxidation in intact cells. In conclusion, we find that in situ kinetic trapping provides starting points for future functional studies of intramitochondrial redox regulation., (Copyright © 2011 Elsevier Inc. All rights reserved.)