Cytochrome bc1, also known as mitochondrial complex III, is considered to be one of the important producers of reactive oxygen species (ROS) in living organisms. Under physiological conditions, a certain level of ROS produced by mitochondrial electron transport chain (ETC) might be beneficial and take part in cellular signaling. However, elevated levels of ROS might exhibit negative effects, resulting in cellular damage. It is well known that inhibiting the electron flow within mitochondrial complex III leads to high production of ROS. However, superoxide production by cytochrome bc1 in a non-inhibited system remained controversial. Here, we propose a novel method for ROS detection in ETC hybrid system in solution comprising bacterial cytochrome bc1 and mitochondrial complex IV. We clearly show that non-inhibited cytochrome bc1 generates ROS and that adaptive and pathogenic mitochondrial mutations suppress and enhance ROS production, respectively. We also noted that cytochrome bc1 produces ROS in a rate-dependent manner and that the mechanism of ROS generation changes according to the rate of operation of the enzyme. This dependency has not yet been reported, but seems to be crucial when discussing ROS signaling originating from mitochondria.
Keywords: Amplex red; Cytochrome bc(1); Mitochondrial mutations; Reactive oxygen species; Superoxide anion radical; aa(3) oxidase.
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