Cytochrome bc1 is part of the mitochondrial respiratory chain and serves to provide electrons to cytochrome c. The Rieske domain of cytochrome bc1 contains a [2Fe-2S] cluster that accepts electrons from the quinol pool and then relays each electron over to cytochrome c1 for delivery to cytochrome c. However, the reduction potential observed for the Rieske protein varies greatly from one organism to another. We have examined a soluble version of the Rieske protein from T. thermophilus using direct electrochemistry. We have looked at the effects of mutations introducing or removing charges at varying distances from the [2Fe-2S] cluster, for distances ranging from 5 Å to 20 Å. For example, positively charged arginine and lysine residues can be replaced by neutral alanines or negatively charged glutamates. Voltammetric methods show that the effects of these mutations correlate with distance and charge. Positive charges help stabilize the reduced protein and give higher reduction potentials. The more distant mutation sites have smaller effects, and high ionic strength shields the active site from the influence of distant charges. We have also found that multiple mutations can have additive effects. However, the influence of a carboxylate side chain is lost when it is protonated at low pH. Introducing a neutral polar group, such as glutamine, close to the active site (L135Q) will raise the potential relative to wild type, though not as high as does a positively charged group (L135K), and the anionic carboxylate mutant (L135E) lowers the potential relative to that higher starting point.

Protein charge effects on Rieske protein reduction potentials

253rd National Meeting (2017)

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