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 and pH-dependent reduction potentials. 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.
Three mutants of the Rieske protein from T. thermophilus have been produced and characterized: Y158F, L135A, and G156S. 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.
We use two different version of the ‘unmodified' protein. The full-length wild type (FL-WT) is the protein characterized in the literature previously1, and the truncated wild type (tWT) which has the N-terminal 9 amino acids and the C-terminal 8 amino acids removed. Truncation of the protein allowed crystallization at low pH.
Structural Studies
tWT, Y158F, and L135A crystallize under a new condition (pH 6.5, PEG 8000, 1mM Pr3+). Data to ~2.0 Å has been collected for tWT and L135A at the UT Health Science Center in collaboration with the laboratory of John Hart. The tWT structure is being refined, with current R =24% and Rfree = 28%. Pr3+ ions were necessary as an additive, and several unique binding sites on the protein are observed. The oxophilic Pr3+ ions bind to surface Glu and Asp residues. Crystals of L135A unexpectedly fall into a different space group and refinement of the structure will begin shortly.
G156S had been crystallized under a different condition and data was collected. The data showed the presence of the putative hydrogen bond to the cluster, but unfortunately the data was not of publishable quality. Efforts are now directed toward finding conditions to crystallize the oxidized protein (see below).
Spectroscopic studies
A unique feature of G156S is that it appears to be purified as partially reduced. EPR spectra, obtained in collaboration with Dr. David Britt, UC Davis, have shown that the as isolated protein has an EPR spectrum, indicating that it is at least partially reduced. Preliminary oxidation studies show that incubation with K3Mo(CN)8 for 24 hours alters the UV-Visible spectrum such that is has the same shape as other oxidized Rieske proteins.
The pH dependence of the UV-Visible spectra of Y158F, L135A, and G156S have been recorded and compared to FL-WT and tWT. The FL-WT and tWT Rieske 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. The midpoint of the curve will be an observed pKa of the protein. In the case of the Rieske protein, this should be an average of the two pKa's reported for the FL-WT, as determined from the pH-dependent reduction potential. In the FL-WT and tWT spectra, the midpoints of the curves are pH 8.8, which is the average of the two reported pKa's (7.85 and 9.65) 1. A pH-dependence of the UV-Visible spectra is also observed for Y158F and L135A. The observed pKa's for these mutants are 9.4 and 9.2 respectively. These lower observed pKa's correlate to a lower reduction potential, which is predicted based previous reports in the literature. G156S as isolated does not show a pH-dependent UV-Visible spectrum. This is consistent with the protein being at least partially reduced. A reduced Rieske protein has a much higher pKa, presumably shifting the curve to higher pH values than can be measured reliably using this technique.
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. 1 Y158F also shows a decrease in potential by ~100 mV consistent with previous reports in the literature. (c.f.2)
References
(1) Zu, Y.; Fee, J. A.; Hirst, J. Journal of the American Chemical Society 2001, 123, 9906-9907.
(2) Leggate, E. J.; Hirst, J. Biochemistry 2005, 44, 7048-7058.
