Establishing the Role of Rieske Protein Reduction Potential in the Formation of Reactive Oxygen Species in Complex III

Abstract

The factors that modulate the reduction potential of complex III's catalytic Rieske metalloprotein subunit (RP) have been the subject of much study. During the bifurcated Q‐cycle, RP transports one electron from quinol to cytochrome c1. Quinol's other electron is passed from heme bL to heme bH, which reduces a quinone bound at the QN site. If the Q‐cycle is disrupted, electrons may interact with molecular oxygen to produce reactive oxygen species (ROS). For example, if RP's reduction potential is too low, it may never accept quinol's electron or do so inefficiently. The latter scenario may shift Q‐cycle equilibria against RP movement towards cytochrome c1, instead favoring RP electron release and interaction with molecular oxygen to produce ROS. Thus, we propose that a mismatch in reduction potential between RP and quinol may serve as a possible source of ROS generated from complex III. Understanding the sources of ROS in the electron transport chain may lead to better treatments for diseases that arise from high levels of ROS.We are investigating this hypothesis by (1) measuring the reduction potential of isolated Saccharomyces cerevisiae (yeast) RP expressed in Escherichia coli (E. coli) cells (ScRieske) and (2) measuring the ROS produced by intact yeast complex III expressed in yeast cells (ScComplexIII). We created a wild‐type ScComplexIII yeast strain (JPJ1‐RIP1) by restoring respiratory function to an anaerobic RP‐deletion strain (JPJ1) via complementation with a RP expression vector. Now, we are measuring JPJ1‐RIP1 and JPJ1 ROS production by fluorometry using dichlorofluorescein (DCF) as a ROS indicator. We have also expressed and purified wild‐type ScRieske in E. coli and have characterized its structure using circular dichroism (CD) and UV‐visible spectroscopy. Furthermore, we have probed the electronics of ScRieske's [2Fe‐2S] cluster via chemical modification experiments with diethyl pyrocarbonate (DEPC). Lastly, we have obtained E. coli and JPJ1‐RIP1 expression vectors for ScRieske mutants Y185F, S183A, and S183T. Ultimately, we will use our ROS assay to quantify ROS production of ScRieske mutants with modulated reduction potentials to study the relationship between RP reduction potential and ROS production in complex III.Support or Funding InformationTrinity Chemistry and Neuroscience Departments, Welch Foundation, The San Antonio Area Foundation, 2017 Murchison Summer Research Fellowship, 2017 ASBMB Undergraduate Research AwardThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.