The Rieske protein component of the cytochrome bc complex contains a [2Fe-2S] cluster ligated by two cysteines and two histidines. We report here the pK(a) values of each of the imidazole rings of the two ligating histidines (His134 and His154) in the oxidized and reduced states of the Rieske protein from Thermus thermophilus (TtRp) as determined by NMR spectroscopy. Knowledge of these pK(a) values is of critical interest because of their pertinence to the mechanism of electron and proton transfer in the bifurcated Q-cycle. Although we earlier had observed the pH dependence of a (15)N NMR signal from each of the two ligand histidines in oxidized TtRp (Lin, I. J.; Chen, Y.; Fee, J. A.; Song, J.; Westler, W. M.; Markley, J. L. J. Am. Chem. Soc. 2006, 128, 10672-10673), the strong paramagnetism of the [2Fe-2S] cluster prevented the assignment of these signals by conventional methods. Our approach here was to take advantage of the unique histidine-leucine (His134-Leu135) sequence and to use residue-selective labeling to establish a key sequence-specific assignment, which was then extended. Analysis of the pH dependence of assigned (13)C', (13)C(alpha), and (15)N(epsilon2) signals from the two histidine cluster ligands led to unambiguous assignment of the pK(a) values of oxidized and reduced TtRp. The results showed that the pK(a) of His134 changes from 9.1 in oxidized to approximately 12.3 in reduced TtRp, whereas the pK(a) of His154 changes from 7.4 in oxidized to approximately 12.6 in reduced TtRp. This establishes His154, which is close to the quinone when the Rieske protein is in the cytochrome b site, as the residue experiencing the remarkable redox-dependent pK(a) shift. Secondary structural analysis of oxidized and reduced TtRp based upon our extensive chemical shift assignments rules out a large conformational change between the oxidized and reduced states. Therefore, TtRp likely translocates between the cytochrome b and cytochrome c sites by passive diffusion. Our results are most consistent with a mechanism involving the coupled transfer of an electron and transfer of the proton across the hydrogen bond between the hydroquinone and His154 at the cytochrome b site.