The net emission of hydrogen peroxide (H2O2) from mitochondria results from the balance between reactive oxygen species (ROS) continuously generated in the respiratory chain and ROS scavenging. The relative contribution of the two major antioxidant systems in the mitochondrial matrix, glutathione (GSH) and thioredoxin (Trx), has not been assessed. In this paper, we examine this key question via combined experimental and theoretical approaches, using isolated heart mitochondria from mouse, rat, and guinea pig.As compared with untreated control mitochondria, selective inhibition of Trx reductase with auranofin along with depletion of GSH with 2,4-dinitrochlorobenzene led to a species-dependent increase in H2O2 emission flux of 17, 11, and 6 fold in state 4 and 15, 7, and 8 fold in state 3 for mouse, rat, and guinea pig mitochondria, respectively. The maximal H2O2 emission as a percentage of the total O2 consumption flux was 11%/2.3% for mouse in states 4 and 3 followed by 2%/0.25% and 0.74%/0.29% in the rat and guinea pig, respectively.A minimal computational model accounting for the kinetics of GSH/Trx systems was developed and was able to simulate increase in H2O2 emission fluxes when both scavenging systems were inhibited separately or together. Model simulations suggest that GSH/Trx systems act in concert. When the scavenging capacity of either one of them saturates during H2O2 overload, they relieve each other until complete saturation, when maximal ROS emission occurs.Quantitatively, these results converge on the idea that GSH/Trx scavenging systems in mitochondria are both essential for keeping minimal levels of H2O2 emission, especially during state 3 respiration, when the energetic output is maximal. This suggests that the very low levels of H2O2 emission observed during forward electron transport in the respiratory chain are a result of the well-orchestrated actions of the two antioxidant systems working continuously to offset ROS production.

To understand the mechanisms involved in the control and regulation of mitochondrial reactive oxygen species (ROS) levels, a two-compartment computational mitochondrial energetic-redox (ME-R) model accounting for energetic, redox, and ROS metabolisms is presented. The ME-R model incorporates four main redox couples (NADH/NAD(+), NADPH/NADP(+), GSH/GSSG, Trx(SH)(2)/TrxSS). Scavenging systems-glutathione, thioredoxin, superoxide dismutase, catalase-are distributed in mitochondrial matrix and extra-matrix compartments, and transport between compartments of ROS species (superoxide: O(2)(⋅-), hydrogen peroxide: H(2)O(2)), and GSH is also taken into account. Model simulations are compared with experimental data obtained from isolated heart mitochondria. The ME-R model is able to simulate: i), the shape and order of magnitude of H(2)O(2) emission and dose-response kinetics observed after treatment with inhibitors of the GSH or Trx scavenging systems and ii), steady and transient behavior of ΔΨ(m) and NADH after single or repetitive pulses of substrate- or uncoupler-elicited energetic-redox transitions. The dynamics of the redox environment in both compartments is analyzed with the model following substrate addition. The ME-R model represents a useful computational tool for exploring ROS dynamics, the role of compartmentation in the modulation of the redox environment, and how redox regulation participates in the control of mitochondrial function.

Living systems exhibit non-randomly organized biochemical, physiological, and behavioral processes that follow distinctive patterns. In particular, animal behavior displays both fractal dynamics and periodic rhythms yet the relationship between these two dynamic regimens remain unexplored. Herein we studied locomotor time series of visually isolated Japanese quails sampled every 0.5 s during 6.5 days (>106 data points). These high-resolution, week-long, time series enabled simultaneous evaluation of ultradian rhythms as well as fractal organization according to six different analytical methods that included Power Spectrum, Enright, Empirical Mode Decomposition, Wavelet, and Detrended Fluctuation analyses. Time series analyses showed that all birds exhibit circadian rhythms. Although interindividual differences were detected, animals presented ultradian behavioral rhythms of 12, 8, 6, 4.8, 4 h and/or lower and, irrespective of visual isolation, synchronization between these ultradian rhythms was observed. Moreover, all birds presented similar overall fractal dynamics (for scales ∼30 s to >4.4 h). This is the first demonstration that avian behavior presents fractal organization that predominates at shorter time scales and coexists with synchronized ultradian rhythms. This chronobiological pattern is advantageous for keeping the organism’s endogenous rhythms in phase with internal and environmental periodicities, notably the feeding, light-dark and sleep-wake cycles.