Reports: GB3
43999-GB3 Modulation of Reduction Potentials of 2Fe-2S Iron Sulfur Clusters
Background
The goal of the project is to understand how iron sulfur proteins modulate reduction potential in order to perform specific functions. The family of Rieske and Rieske-type proteins is an ideal model to study, since it encompasses several proteins that have a wide range of reduction potentials (-150 mV to +475 mV) in a common fold. The Rieske proteins tend to have the higher reduction potentials, and have a pH-dependent reduction potential. The research plan is to produce a series of mutants using the Rieske protein from Thermus thermophilus to evaluate the effect of the mutation on the reduction potential and to characterize each mutant using spectroscopic and structural techniques. In addition, we also aim to establish if the ligating histidines to the [2Fe-2S] cluster become deprotonated as pH is raised. Several mutants of the Rieske protein from T. thermophilus have been produced and characterized: Y158F, L135A, G156S, and L135E. Y158F is designed to remove a hydrogen bond from the Sg of cysteine to the cluster in order to evaluate the effect of the removal on the reduction potential. L135A is designed to test how increasing the solvent exposure of one histidine ligand affects potential. G156S is a novel mutants, which is designed to add a single hydrogen bond to the S* atom within the cluster. L135E is designed to add a negative charge adjacent to the cluster. The gene for L135R has been produced, and this mutant will add a positive charge adjacent to the cluster.
Structural Studies
Y158F, and L135A crystallize under the same condition as the wild type and data to ~2.0 Å has been collected for L135A and to 1.85 Å for Y158 F at the UT Health Science Center in collaboration with the laboratory of John Hart. L135A crystals unexpectedly fall into a different space group and refinement of the structure is underway. The Y158F refinement will begin shortly.
Spectroscopic studies
The pH dependence of the UV-Visible spectra of Y158F, L135A, and L135E have been accomplished. The proteins have an absorption at 458 nm which shifts to 436 nm and increases in intensity as a function of pH. A plot of the change in absorbance or molar absorptivity at 436 nm produces a sigmoidal curve that can be fit, as was performed for the truncated wild type (1). The Y158F, L135A and L135E show the expected increase in pKa values that occur with a decrease in reduction potential.
Cicular dichroism is also being used to probe the Rieske protein. The CD spectrum in the visible wavelengths is pH-dependent (1) and the pH-dependent CD spectra of Y158F and L135E have been completed. L135A is underway.
Reduction potential measurements
A collaboration has been established with Sean Elliot of Boston University to measure the reduction potentials of each of these proteins. Preliminary measurements indicate that the FL-WT and tWT have low pH potentials corresponding to those of the protein previously reported (2). Y158F also shows a decrease in potential by ~65 mV consistent with previous reports in the literature and L135A decreased by 27 mV (c.f.(3)).
Chemical modification of the Rieske protein
One fundamental question about the Rieske protein is if the ligating histidines become deprotonated as pH is raised. All of the changes observed in the spectroscopic studies described above are consistent with histidine deprotonation. However, there is no direct evidence. This question is important since it addresses how the Rieske protein functions to carry both an electron and a proton across the membrane.
To address this To address this question directly, chemical modification of the histidines by diethyl pyrocarbonate (DEPC) can be tested at varying pH. DEPC will add C(O)OCH2CH3 to the Nitrogen of a histidine to form a carbamoyl if the Nitrogen is deprotonated (4). Thus, if the pKa values determined for the protein do reflect deprotonation of the histidine ligands, then chemical modification should not occur, or occur very slowly at lower pH and should increase in rate and extent of modification as pH is raised. Early work with the bc1 complex of bovine heart mitochondria showed inhibition when treated with DEPC and hinted at the possible modification of the ligating histidines (5, 6). The reaction can be monitored by UV-Visible spectroscopy, circular dichroism, and the products analyzed by mass spectrometry. The Mass spectrometry is performed in collaboration with Sue Weintraub at the University of Texas Health Science Center San Antonio. Preliminary data indicate that Histidine 154 (one of the ligating histidines) is modified, even at moderate pH. Modification of several lysines is also observed. This result provides some of the most direct evidence that the histidine ligands become deprotonated. Continuing work will establish if Histidine 134 becomes deprotonated as well.
References
1. Konkle, M. E., Muellner, S. K., Schwander, A. L., Dicus, M. M., Pokhrel, R., Britt, R. D., Taylor, A. B., and Hunsicker-Wang, L. M. (2009) Effect of pH on the Rieske Protein from Thermus Thermophilus: A Spectroscopic and Structural Analysis. Biochemistry.
2. Zu, Y., Fee, J. A., and Hirst, J. (2001) Complete Thermodynamic Characterization of Reduction and Protonation of the Bc1-Type Rieske [2Fe-2S] Center of Thermus Thermophilus. J. Am. Chem. Soc. 123, 9906-9907.
3. Leggate, E. J., and Hirst, J. (2005) Roles of the Disulfide Bond and Adjacent Residues in Determining the Reduction Potentials and Stabilities of Respiratory-Type Rieske Clusters. Biochemistry. 44, 7048-7058.
4. Safarian, S., Moosavi-Movahedi, A. A., Hosseinkhani, S., Xia, Z., Habibi-Rezaei, M., Hosseini, G., Soerenson, C., and Sheibani, N. (2003) The Structural and Functional Studies of His119 and His 12 in RNase A Via Chemical Modification. J. Prot. Chem. 22, 643-654.
5. Yagi, T., Vik, S. B., and Hatefi, Y. (1982) Reversible Inhibition of the Mitochondrial Ubiquinol-Cytochrome c Oxidoreductase Complex (Comple III) by Ethoxyformic Anhydride. Biochemistry. 21, 4777-4782.
6. Ohnishi, T., Meinhardt, S. W., von Jagow, G., Yagi, T., and Hatefi, Y. (1994) Effect of Ethoxyformic Anhydride on the Rieske Iron-Sulfur Protein of Bovine Heart Ubiquinole: Cytochrome c Oxidoreductase. FEBS Lett. 353, 103-107.
