Mitochondrial reactive oxygen species (ROS) can be either detrimental or beneficial depending on the amount, duration, and location of their production. Mitochondrial complex I is a component of the electron transport chain and transfers electrons from NADH to ubiquinone. Complex I is also a source of ROS production. Under certain thermodynamic conditions, electron transfer can reverse direction and reduce oxygen at complex I to generate ROS. Conditions that favor this reverse electron transport (RET) include highly reduced ubiquinone pools, high mitochondrial membrane potential, and accumulated metabolic substrates. Historically, complex I RET was associated with pathological conditions, causing oxidative stress. However, recent evidence suggests that ROS generation by complex I RET contributes to signaling events in cells and organisms. Collectively, these studies demonstrate that the impact of complex I RET, either beneficial or detrimental, can be determined by the timing and quantity of ROS production. In this article we review the role of site-specific ROS production at complex I in the contexts of pathology and physiologic signaling.

The goal of the present study was to elucidate the modulatory effects of cadmium (Cd) on hypoxia/reoxygenation-induced mitochondrial dysfunction in light of the limited understanding of the mechanisms of multiple stressor interactions in aquatic organisms. Rainbow trout (Oncorhynchus mykiss) liver mitochondria were isolated and energized with complex I substrates (malate-glutamate), and exposed to hypoxia (0>P O 2 <2 Torr) for 0-60 min followed by reoxygenation and measurement of coupled and uncoupled respiration and complex I enzyme activity. Thereafter, 5 min hypoxia was used to probe interactions with Cd (0-20 μmol l) and to test the hypothesis that deleterious effects of hypoxia/reoxygenation on mitochondria were mediated by reactive oxygen species (ROS). Hypoxia/reoxygenation inhibited state 3 and uncoupler-stimulated (state 3u) respiration while concomitantly stimulating states 4 and 4 ol (proton leak) respiration, thus reducing phosphorylation and coupling efficiencies. Low doses of Cd (≤5 μmol l −1 ) reduced, while higher doses enhanced, hypoxia-stimulated proton leak. This was in contrast to the monotonic enhancement by Cd of hypoxia/reoxygenationinduced reductions of state 3 respiration, phosphorylation efficiency and coupling. Mitochondrial complex I activity was inhibited by hypoxia/reoxygenation, hence confirming the impairment of at least one component of the electron transport chain (ETC) in rainbow trout mitochondria. Similar to the effect on state 4 and proton leak, low doses of Cd partially reversed the hypoxia/reoxygenation-induced complex I activity inhibition. The ROS scavenger and sulfhydryl group donor N-acetylcysteine, administrated immediately prior to hypoxia exposure, reduced hypoxia/reoxygenation-stimulated proton leak without rescuing the inhibited state 3 respiration, suggesting that hypoxia/reoxygenation influences distinct aspects of mitochondria via different mechanisms. Our results indicate that hypoxia/reoxygenation impairs the ETC and sensitizes mitochondria to Cd via mechanisms that involve, at least in part, ROS. Moreover, we provide, for the first time in fish, evidence for a hormetic effect of Cd on mitochondrial bioenergetics -the attenuation of hypoxia/reoxygenation-stimulated proton leak and partial rescue of complex I inhibition by low Cd doses.

Fluorescent proteins can generate reactive oxygen species (ROS) upon absorption of photons via type I and II photosensitization mechanisms. The red fluorescent proteins KillerRed and SuperNova are phototoxic proteins engineered to generate ROS and are used in a variety of biological applications. However, their relative quantum yields and rates of ROS production are unclear, which has limited the interpretation of their effects when used in biological systems. We cloned and purified KillerRed, SuperNova, and mCherry -a related red fluorescent protein not typically considered a photosensitizer -and measured the superoxide (O 2•-) and singlet oxygen( 1 O 2 ) quantum yields with irradiation at 561 nm. The formation of the O 2 •--specific product 2hydroxyethidium (2-OHE + ) was quantified via HPLC separation with fluorescence detection.Relative to a reference photosensitizer, Rose Bengal, the O 2 •quantum yield (ΦO 2 •-) of SuperNova was determined to be 1.5×10 −3 , KillerRed was 0.97×10 −3 , and mCherry 1.2×10 −3 . At an excitation fluence of 916.5 J/cm 2 and matched absorption at 561 nm, SuperNova, KillerRed and mCherry made 3.81, 2.38 and 1.65 μM O 2 •-/min, respectively. Using the probe Singlet OxygenSensor Green (SOSG), we ascertained the 1 O 2 quantum yield (Φ 1 O 2 ) for SuperNova to be 22.0×10 −3 , KillerRed 7.6×10 −3 , and mCherry 5.7×10 −3 . These photosensitization characteristics of SuperNova, KillerRed and mCherry improve our understanding of fluorescent proteins and are pertinent for refining their use as tools to advance our knowledge of redox biology.

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Alzheimer’s disease (AD) is a devastating progressive neurodegenerative disease characterized by neuronal dysfunction, and decreased memory and cognitive function. Iron is critical for neuronal activity, neurotransmitter biosynthesis, and energy homeostasis. Iron accumulation occurs in AD and results in neuronal dysfunction through activation of multifactorial mechanisms. Mitochondria generate energy and iron is a key co-factor required for: (1) ATP production by the electron transport chain, (2) heme protein biosynthesis and (3) iron-sulfur cluster formation. Disruptions in iron homeostasis result in mitochondrial dysfunction and energetic failure. Ferroptosis, a non-apoptotic iron-dependent form of cell death mediated by uncontrolled accumulation of reactive oxygen species and lipid peroxidation, is associated with AD and other neurodegenerative diseases. AD pathogenesis is complex with multiple diverse interacting players including Aβ-plaque formation, phosphorylated tau, and redox stress. Unfortunately, clinical trials in AD based on targeting these canonical hallmarks have been largely unsuccessful. Here, we review evidence linking iron dysregulation to AD and the potential for targeting ferroptosis as a therapeutic intervention for AD.

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The osmorespiratory compromise is a physiological trade-off between the characteristics of the gill that promote respiratory gas-exchange and those that limit passive fluxes of ions and water with the environment. In hypoxia, changes in gill blood flow patterns and functional surface area that increase gas transfer can promote an exacerbation in ion and water fluxes. Our goal was to determine whether the osmorespiratory compromise is flexible, depending on environmental salinity (fresh, isosmotic and sea water) and oxygen levels (hypoxia) in euryhaline killifish, Fundulus heteroclitus. Plasma ion concentrations were minimally affected by hypoxia, indicating a maintenance of osmoregulatory homeostasis. In FW-killifish, hypoxia exposure reduced branchial Na+/K+-ATPase and NEM-sensitive-ATPase activities, as well as diffusive water flux rates. Unidirectional Na+ influx and Na+ efflux decreased during hypoxia in FW, but net Na+ flux remained unchanged. Net loss rates of Cl−, K+ and ammonia were also attenuated in hypoxia, suggesting both transcellular and paracellular reductions in permeability. These reductions appeared to be regulated phenomena as fluxes were restored immediately in normoxia. Na+ flux rates increased during hypoxia in 11 ppt, but decreased in 35 ppt, the latter suggesting a similar response to hypoxia as in FW. In summary, FW- and SW-killifish experience a reduction in gill permeability, as seen in other hypoxia-tolerant species. Fish acclimated to isosmotic salinity increased Na+ influx and efflux rates, as well as paracellular permeability in hypoxia, responses in accord with the predictions of the classic osmorespiratory compromise.