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NMR Investigations of the Rieske Protein from Thermus thermophilus Support a Coupled Proton and Electron Transfer Mechanism.

Authors :
Kuang-Lung Hsueh
Westler, William M.
Markley, John L.
Source :
Journal of the American Chemical Society. 6/16/2010, Vol. 132 Issue 23, p7908-7918. 11p.
Publication Year :
2010

Abstract

The Rieske protein component of the cytochrome bccomplex contains a [2Fe-2S] cluster ligated by two cysteines and two histidines. We report here the PKa 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 Therm us thermophilus (TtRp) as determined by NMR spectroscopy. Knowledge of these PKa values is of critical interest because of their pertinence to the mechanism of electron and proton transfer in the bifurcated O-cycle. Although we earlier had observed the pH dependence of a 15N 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 (Hisi 34-Leul 35) 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, 13Cα, and 15Nϵ2 signals from the two histidine cluster ligands led to unambiguous assignment of the PKa values of oxidized and reduced TtRp. The results showed that the pKa of His134 changes from 9.1 in oxidized to ∼12.3 in reduced TtRp, whereas the pKa of His154 changes from 7.4 in oxidized to ∼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 pKa 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, TtAp 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. [ABSTRACT FROM AUTHOR]

Details

Language :
English
ISSN :
00027863
Volume :
132
Issue :
23
Database :
Academic Search Index
Journal :
Journal of the American Chemical Society
Publication Type :
Academic Journal
Accession number :
51796625
Full Text :
https://doi.org/10.1021/ja1026387