Rieske protein from Thermus thermophilus:: 15N NMR titration study demonstrates the role of iron-ligated histidines in the pH dependence of the reduction potential

A unique feature of Rieske proteins is the pH dependence of their reduction potentials. It has been proposed that protonation of the Nε2 atoms of the two histidine rings ligated to the iron-sulfur cluster is coupled with cluster reduction (electron transfer). We have incorporated [15Nδ1, 15Nε2]-histidine into the Rieske protein from Thermus thermophilis and have used 15N NMR spectroscopy to determine the pKa values of the histidine residues in the oxidized state of the protein. As expected from studies of a Rieske-type ferredoxin, the signals from the 15Nδ1 atoms directly bound to iron were too broad to be detected, but broad signals could be detected from the 15Nε2 atom of each of the ligated histidine rings. We measured the chemical shifts of these signals as a function of pH between pH 6 and pH 12 and fitted them to theoretical titration curves. The results yielded well-separated pKa values for the two histidines (7.46 and 9.24), with Hill coefficients close to unity. The pKa values are in excellent agreement with values predicted from the pH dependence of the reduction potentials (7.85 and 9.65). Copyright © 2006 American Chemical Society.