The Rieske protein is an iron-sulfur protein found in Complex III of the electron transport chain and is responsible for electron transfer within the complex. Upon deprotonation, the histidine ligands ligated to the [2Fe-2S] cluster can act as nucleophiles toward chemical modifiers such as diethylpyrocarbonate (DEPC), leading to the labeling of the histidine ligands in a reversible reaction. Modification of the active-site histidine ligands can be followed using voltammetry. Wild type (WT) and H134C truncated Rieske protein constructs from <i>Thermus thermophilus</i> were treated with varying excesses of DEPC at pH 7, pH 6, and pH 5 and monitored via cyclic voltammetry (CV) and square wave voltammetry (SWV) over time at room temperature. Upon modification of histidine ligands, new signals appeared in the voltammetry at higher potentials than for the unmodified protein, revealing the modification of one or both histidine ligands in WT Rieske. As conditions became more acidic, the amount of excess DEPC required to push the reaction forward increased. Notably, wild-type Rieske protein is reactive with DEPC even under mildly acidic conditions where histidine deprotonation is not favored. The H134C mutant of Rieske protein was also examined. Its potential is substantially lower than that of wild-type (-240 mV vs. +155 mV vs. SHE at pH 7) and also shifts upward upon modification of its single active site histidine.

Chemical modification of the Rieske protein

ACS Spring 2022

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