Mitochondrial transition ROS spike (mTRS) results from coordinated activities of complex I and nicotinamide nucleotide transhydrogenase

Abstract

Mitochondria exhibit suppressed ATP production, membrane potential (∆Ψ) polarization and reactive oxygen species (ROS) bursts during some cellular metabolic transitions. Although mitochondrial ROS release is influenced by ∆Ψ and respiratory state, the relationship between these properties remains controversial primarily because they have not been measured simultaneously. We developed a multiparametric method for probing mitochondrial function that allowed precise characterization of the temporal relationship between ROS, ∆Ψ and respiration. We uncovered a previously unknown spontaneous ROS spike - termed mitochondrial transition ROS spike (mTRS) - associated with re-polarization of ∆Ψ that occurs at the transition between mitochondrial energy states. Pharmacological inhibition of complex CI (CI), nicotinamide nucleotide transhydrogenase (NNT) and antioxidant system significantly decreased the ability of mitochondria to exhibit mTRS. NADH levels followed a similar trend to that of ROS during the mTRS, providing a link between CI and NNT in mTRS regulation. We show that (i) mTRS is enhanced by simultaneous activation of CI and complex II (CII); (ii) CI is the principal origin of mTRS; (iii) NNT regulates mTRS via NADH- and ∆Ψ-dependent mechanisms; (iv) mTRS is not a pH spike; and (v), mTRS changes in amplitude under stress conditions and its occurrence can be a signature of mitochondrial health. Collectively, we uncovered and characterized the biophysical properties and mechanisms of mTRS, and propose it as a potential diagnostic tool for CI-related dysfunctions, and as a biomarker of mitochondrial functional integrity.

Use the Protonmotive Force: Mitochondrial Uncoupling and Reactive Oxygen Species

“…Nevertheless, the capability of ROS production at both low and high rates of ETC activity highlights the complex nature of ROS production under different conditions. Recently, simultaneous measurement of oxygen consumption, Δψ m and ROS production showed that at low Δψ m and high ETC activity, ROS levels were low, but a transient ROS increase occurred when switching to a state of low ETC activity [50]. In summary, at both low ETC flux (high pmf) and high ETC flux (low pmf) there is potential for ROS production, but under most circumstances, increasing ETC flux decreases ROS (Fig.…”

“…Accelerated ETC activity increases the volume of electrons flowing, which could increase the probability of ROS production [40]. Experimentally, ROS formation at high ETC rates can be observed when ROS scavenging pathways are overwhelmed [50], but under physiologic conditions these pathways maintain ROS homeostasis [40]. Further, it has been recently shown that ROS levels in cells are more dependent upon rates of mitochondrial ROS production than they are to cellular ROS scavenging rates [51].…”

“…Therefore, ROS production by RET is very sensitive to proton leak decreasing the ΔpH [37, 43]. In terms of Δψ m , when transitioning from high to low ETC activities (low to high Δψ m ) a transient production of ROS occurs at complex I [50].…”

“…Mitochondria are central hubs for sensing certain types of mild to moderate stress, and signal to initiate appropriate cellular responses. Since mitochondrial pmf and/or ROS production are interdependent [50], perturbations to either can induce retrograde stress signaling cascades in a process termed mitohormesis [24, 138]. For instance, modulation of mitochondrial ROS production may activate redox sensitive transcription factors such as nuclear factor (erythroid-like 2) related factor (NRF2), a master regulator of the cellular antioxidant response [139, 140].…”

Redox-Driven Signaling: 2-Oxo Acid Dehydrogenase Complexes as Sensors and Transmitters of Metabolic Imbalance

“…Both the free (15,131,207) and enzyme-bound states of reduced flavins (78,130,131) are known to produce ROS on oxidation with molecular oxygen. Importance of the NADH/NAD + ratio for E3-catalyzed ROS production links the ROS generated by the 2-oxo acid dehydrogenase complexes at a high NADH to the NADHdependent production of NADPH by nicotinamide nucleotide transhydrogenase, a protein of the inner mitochondrial membrane, which is crucial for redox homeostasis (137,163,171). Obviously, in vivo utilization of NADH in the transhydrogenase reaction would not only increase the disulfide reductant NADPH but also decrease NADH available for the backward side reaction of E3 yielding ROS.…”