Chemical Modification of the Rieske Protein fromThermus thermophilusUsing Diethyl Pyrocarbonate Modifies Ligating Histidine 154 and Reduces the [2Fe-2S] Cluster

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Abstract

Rieske proteins are a class of electron transport proteins that are intricately involved in respiratory and photosynthetic processes. One unique property of Rieske proteins is that the reduction potential is pH-dependent. The ionizable groups responding to changes in pH have recently been shown to be the two histidine residues that ligate the [2Fe-2S] cluster. To probe the chemical reactivity toward and the accessibility of the ligating histidines to small molecules, akin to the substrate quinol and the inhibitor stigmatellin, the Thermus thermophilus Rieske protein was reacted with diethyl pyrocarbonate (DEPC) over a range of pH values. The modification was followed by UV-visible, circular dichroism, and EPR spectroscopies and the end product analyzed by mass spectrometry. The ligating His154, as well as the two nonligating histidines and surface-exposed lysines, were modified. Interestingly, modification of the protein by DEPC was also found to reduce the metal cluster. The ability to control the redox state was examined by the addition of oxidants and reductants and removal of the DEPC-histidine adduct by sodium hydroxide. Characterization of the DEPC-modified Rieske protein, which remains redox active, offers a probe to analyze the effects of small molecules that inhibit the function of the bc(1) complex and that have also been shown to interact with the ligating histidines of the Rieske [2Fe-2S] cluster in crystal structures of the complex.

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