Differential effects of mitochondrial Complex I inhibitors on production of reactive oxygen species

Abstract

We have investigated the production of reactive oxygen species (ROS) by Complex I in isolated open bovine heart submitochondrial membrane fragments during forward electron transfer in presence of NADH, by means of the probe 2′,7′-Dichlorodihydrofluorescein diacetate. ROS production by Complex I is strictly related to its inhibited state. Our results indicate that different Complex I inhibitors can be grouped into two classes: Class A inhibitors (Rotenone, Piericidin A and Rolliniastatin 1 and 2) increase ROS production; Class B inhibitors (Stigmatellin, Mucidin, Capsaicin and Coenzyme Q2) prevent ROS production also in the presence of Class A inhibitors. Addition of the hydrophilic Coenzyme Q1 as an electron acceptor potentiates the effect of Rotenone-like inhibitors in increasing ROS production, but has no effect in the presence of Stigmatellin-like inhibitors; the effect is not shared by more hydrophobic quinones such as decylubiquinone. This behaviour relates the prooxidant CoQ1 activity to a hydrophilic electron escape site. Moreover the two classes of Complex I inhibitors have an opposite effect on the increase of NADH–DCIP reduction induced by short chain quinones: only Class B inhibitors allow this increase, indicating the presence of a Rotenone-sensitive but Stigmatellin-insensitive semiquinone species in the active site of the enzyme. The presence of this semiquinone was also suggested by preliminary EPR data. The results suggest that electron transfer from the iron–sulphur clusters (N2) to Coenzyme Q occurs in two steps gated by two different conformations, the former being sensitive to Rotenone and the latter to Stigmatellin.

Sirt3 protects in vitro–fertilized mouse preimplantation embryos against oxidative stress–induced p53-mediated developmental arrest

“…This increase in fluorescent signals was abolished by treatment with the antioxidant N-acetyl-L-cysteine (NAC; Figure 2, C and E) and the mitochondrial complex III inhibitor stigmatellin (Figure 2, D and F), which also inhibits complex I at higher concentrations (33). Our observations indicate that intracellular ROS levels were increased by sirtinol, and excessive ROS generation by mitochondria appeared to be responsible for this phenomenon.…”

Hypoxia-cadmium interactions on rainbow trout (Oncorhynchus mykiss) mitochondrial bioenergetics: attenuation of hypoxia-induced proton leak by low doses of cadmium

“…Importantly, the impairment of ETC was directly confirmed by enzyme activity measurements that revealed a greatly reduced complex I activity (Fig. 4D) Irrespective of the cause, the quintessential effect of complex I inhibition is leakage of electrons from the ETC leading to increased production of ROS (Raha et al, 2000;Turrens, 2003;Galkin and Brandt, 2005;Shiva et al, 2007;Fato et al, 2009;Murphy, 2009), with oxidative damage of not only the enzyme itself but also other mitochondrial components. We therefore tested the hypothesis that inhibition of complex-I-driven state 3 respiration was mediated by oxidative damage following over-production of ROS after hypoxia/ reoxygenation.…”

Cells Lacking Rieske Iron-Sulfur Protein Have a Reactive Oxygen Species-Associated Decrease in Respiratory Complexes I and IV

“…Studies in submitochondrial particles suggested that rotenone inhibits the binding of coenzyme Q to its reduction site in CI, permitting the release of electrons directly to oxygen at the N2 center and forming superoxide radicals (24). The generation of free radicals by CIII is intimately linked to its catalytic mechanism.…”