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 (Q_o), molecular aspects of their generation remain unclear. One of them concerns significance of hemes b (b_L and b_H) redox potentials (E_m) and properties on ROS generation by Q_o. 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 b_L or b_H 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 E_m of hemes b (either b_L or b_H) 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 b_L being in the reduced state and probability of electron transfer from heme b_L towards Q_o. 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 E_m of heme b cofactors, leaving significance of tuning the E_m of hemes b as factor limiting superoxide production an open question.