Discrimination between two possible reaction sequences that create potential risk of generation of deleterious radicals by cytochrome bc₁. Implications for the mechanism of superoxide production.
In addition to its bioenergetic function of building up proton motive force, cytochrome bc₁ can be a source of superoxide. One-electron reduction of oxygen is believed to occur from semiquinone (SQ(o)) formed at the quinone oxidation/reduction Q(o) site (Q(o)) as a result of single-electron oxidation of quinol by the iron-sulfur cluster (FeS) (semiforward mechanism) or single-electron reduction of quinone by heme b(L) (semireverse mechanism). It is hotly debated which mechanism plays a major role in the overall production of superoxide as experimental data supporting either reaction exist. To evaluate a contribution of each of the mechanisms we first measured superoxide production under a broad range of conditions using the mutants of cytochrome bc₁ that severely impeded the oxidation of FeS by cytochrome c₁, changed density of FeS around Q(o) by interfering with its movement, or combined these two effects together. We then compared the amount of generated superoxide with mathematical models describing either semiforward or semireverse mechanism framed within a scheme assuming competition between the internal reactions at Q(o) and the leakage of electrons on oxygen. We found that only the model of semireverse mechanism correctly reproduced the experimentally measured decrease in ROS for the FeS motion mutants and increase in ROS for the mutants with oxidation of FeS impaired. This strongly suggests that this mechanism dominates in setting steady-state levels of SQ(o) that present a risk of generation of superoxide by cytochrome bc₁. Isolation of this reaction sequence from multiplicity of possible reactions at Q(o) helps to better understand conditions under which complex III might contribute to ROS generation in vivo.
Sarewicz
Marcin
M
Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland.
Borek
Arkadiusz
A
Cieluch
Ewelina
E
Swierczek
Monika
M
Osyczka
Artur
A
D004578:Electron Spin Resonance Spectroscopy; D014450:Electron Transport Complex III / Q000737:chemistry* / Q000378:metabolism*; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000201:enzymology*; D013455:Sulfur / Q000737:chemistry / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*
D007506:Iron-Sulfur Proteins; D017382:Reactive Oxygen Species; D013481:Superoxides; D013455:Sulfur; D007501:Iron; D014450:Electron Transport Complex III