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.

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.

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.

C. elegans react to metabolic distress caused by mismatches in oxygen and energy status via distinct behavioral responses. At the molecular level, these responses are coordinated by under-characterized, redox-sensitive processes, thought to initiate in mitochondria. Complex I of the electron transport chain is a major site of reactive oxygen species (ROS) production and is canonically associated with oxidative damage following hypoxic exposure. Here, we use a combination of optogenetics and CRISPR/Cas9-mediated genome editing to exert spatiotemporal control over ROS production. We demonstrate a photo-locomotory remodeling of avoidance behavior by local ROS production due to the reversible oxidation of a single thiol on the complex I subunit NDUF-2.1. Reversible thiol oxidation at this site is necessary and sufficient for the behavioral response to hypoxia, does not respond to ROS produced at more distal sites, and protects against lethal hypoxic exposure. Molecular modeling suggests that oxidation at this thiol residue alters the ability for NDUF-2.1 to coordinate electron transfer to coenzyme Q by destabilizing the Q-binding pocket, causing decreased complex I activity. Overall, site-specific ROS production regulates behavioral responses and these findings provide a mechanistic target to suppress the detrimental effects of hypoxia.

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