Chemical Modification of the Rieske Protein from Thermus thermophilus Using Diethyl Pyrocarbonate Modifies Ligating Histidine 154 and Reduces the [2Fe-2S] Cluster

摘要

Rieske proteins are a class of electron transport proteins that are intricately involved in respiratorynand photosynthetic processes. One unique property of Rieske proteins is that the reduction potential isnpH-dependent. The ionizable groups responding to changes in pH have recently been shown to be the twonhistidine residues that ligate the [2Fe-2S] cluster. To probe the chemical reactivity toward and the accessibilitynof the ligating histidines to small molecules, akin to the substrate quinol and the inhibitor stigmatellin, thenThermus thermophilus Rieske protein was reacted with diethyl pyrocarbonate (DEPC) over a range ofnpH values. The modification was followed by UV-visible, circular dichroism, and EPR spectroscopies andnthe end product analyzed by mass spectrometry. The ligating His154, as well as the two nonligating histidinesnand surface-exposed lysines, were modified. Interestingly, modification of the protein by DEPC was alsonfound to reduce the metal cluster. The ability to control the redox state was examined by the addition ofnoxidants and reductants and removal of the DEPC-histidine adduct by sodium hydroxide. Characterizationnof the DEPC-modified Rieske protein, which remains redox active, offers a probe to analyze the effects ofnsmallmolecules that inhibit the function of the bc1 complex and that have also been shown to interact with thenligating histidines of the Rieske [2Fe-2S] cluster in crystal structures of the complex.