ROS production by cytochrome bc1: Its mechanism as inferred from the effects of heme b cofactor mutants.

Cytochrome bc1 is one of the enzymes of electron transport chain responsible for generation of reactive oxygen species (ROS). While ROS are considered to be products of side reactions of quinol oxidation site (Qo), molecular aspects of their generation remain unclear. One of them concerns significance of hemes b (bL and bH) redox potentials (Em) and properties on ROS generation by Qo. Here we addressed this question by examining ROS production in mutants of bacterial cytochrome bc1 that replaced one of the His ligand of either heme bL or bH with Lys or Asn. We observed that severe slowing down of electron flow by the Asn mutants induces similar effects on ROS production as inhibition by antimycin in the native cytochrome bc1 (WT). An increase in the Em of hemes b (either bL or bH) in Lys mutants does not exert major effect on the ROS production level, compared to WT. The experimental data were analyzed in the frame of a dynamic model to conclude that the observed ROS rates and levels reflect a combinatory effect of two factors: probability of heme bL being in the reduced state and probability of electron transfer from heme bL towards Qo. A significant contribution from short-circuits maintains the ROS levels at ~15 % in all tested forms. Overall, ROS production by cytochrome bc1 shows remarkably low susceptibility to changes in the Em of heme b cofactors, leaving significance of tuning the Em of hemes b as factor limiting superoxide production an open question.

ROS signaling capacity of cytochrome bc1: Opposing effects of adaptive and pathogenic mitochondrial mutations.

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.