NMR Investigations of the Rieske Protein from <i>Thermus thermophilus</i> Support a Coupled Proton and Electron Transfer Mechanism

Abstract

The Rieske protein component of the cytochrome <i>bc</i> complex contains a [2Fe−2S] cluster ligated by two cysteines and two histidines. We report here the p<i>K</i>_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 <i>Thermus thermophilus</i> (<i>Tt</i>Rp) as determined by NMR spectroscopy. Knowledge of these p<i>K</i>_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 ¹⁵N NMR signal from each of the two ligand histidines in oxidized <i>Tt</i>Rp (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 ¹³C′, ¹³C^α, and ¹⁵N^ε2 signals from the two histidine cluster ligands led to unambiguous assignment of the p<i>K</i>_a values of oxidized and reduced <i>Tt</i>Rp. The results showed that the p<i>K</i>_a of His134 changes from 9.1 in oxidized to ∼12.3 in reduced <i>Tt</i>Rp, whereas the p<i>K</i>_a of His154 changes from 7.4 in oxidized to ∼12.6 in reduced <i>Tt</i>Rp. This establishes His154, which is close to the quinone when the Rieske protein is in the cytochrome <i>b</i> site, as the residue experiencing the remarkable redox-dependent p<i>K</i>_a shift. Secondary structural analysis of oxidized and reduced <i>Tt</i>Rp based upon our extensive chemical shift assignments rules out a large conformational change between the oxidized and reduced states. Therefore, <i>Tt</i>Rp likely translocates between the cytochrome <i>b</i> and cytochrome <i>c</i> 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 <i>b</i> site