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

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

Electron Transfer Mechanism of the Rieske Protein from <i>Thermus thermophilus</i> from Solution Nuclear Magnetic Resonance Investigations

We report nuclear magnetic resonance (NMR) data indicating that the Rieske protein from the cytochrome <i>bc</i> complex of <i>Thermus thermophilus</i> (<i>Tt</i>Rp) undergoes modest redox-state-dependent and ligand-dependent conformational changes. To test models concerning the mechanism by which <i>Tt</i>Rp transfers between different sites on the complex, we monitored ¹H, ¹⁵N, and ¹³C NMR signals as a function of the redox state and molar ratio of added ligand. Our studies of full-length <i>Tt</i>Rp were conducted in the presence of dodecyl phosphocholine micelles to solvate the membrane anchor of the protein and the hydrophobic tail of the ligand (hydroubiquinone). NMR data indicated that hydroubiquinone binds to <i>Tt</i>Rp and stabilizes an altered protein conformation. We utilized a truncated form of the Rieske protein lacking the membrane anchor (trunc-<i>Tt</i>Rp) to investigate redox-state-dependent conformational changes. Local chemical shift perturbations suggested possible conformational changes at prolyl residues. Detailed investigations showed that all observable prolyl residues of oxidized trunc-<i>Tt</i>Rp have <i>trans</i> peptide bond configurations but that two of these peptide bonds (Cys151–Pro152 and Gly169–Pro170 located near the iron–sulfur cluster) become <i>cis</i> in the reduced protein. Changes in the chemical shifts of backbone signals provided evidence of redox-state- and ligand-dependent conformational changes localized near the iron–sulfur cluster. These structural changes may alter interactions between the Rieske protein and the cytochrome <i>b</i> and <i>c</i> sites and provide part of the driving force for movement of the Rieske protein between these two sites

tPA level in the peritoneum fluid.

<p>tPA level in the peritoneum fluid.</p

PAI-I level in the peritoneum fluid.

<p>PAI-I level in the peritoneum fluid.</p

Intra-abdominal adhesion score.

<p>Intra-abdominal adhesion score.</p