Spectroscopic and Electrochemical Analysis of the Rieske Iron-Sulfur Protein of the Rhodobacter Sphaeroides Bc1 Complex
Kolling, Derrick R.
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https://hdl.handle.net/2142/85442
Description
Title
Spectroscopic and Electrochemical Analysis of the Rieske Iron-Sulfur Protein of the Rhodobacter Sphaeroides Bc1 Complex
Author(s)
Kolling, Derrick R.
Issue Date
2005
Doctoral Committee Chair(s)
Crofts, Antony R.
Department of Study
Biophysics and Computational Biology
Discipline
Biophysics and Computational Biology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biophysics, General
Language
eng
Abstract
The Rieske iron-sulfur protein (ISP) subunit of Rhodobacter sphaeroides cytochrome bc1 complex was isolated and then investigated using molecular spectroscopies (high-resolution electron paramagnetic resonance and resonance Raman), protein-film voltammetry, and x-ray crystallography. Major accomplishments include the following: (1) developing protocols for the isolation of ISP and its water-soluble head domain via alkaline treatment and proteolysis, respectively; (2) characterizing the electrochemical and protolytic properties of the ISP with protein-film cyclic voltammetry: Eacid = 308 +/- 3 mV, Ealk = -134 +/- 6 mV, pKox1 = 7.6 +/- 0.1 mV, pKox2 = 9.6 +/- 0.1 mV, and pKred1,2 = 12.4 +/- 0.4 mV; (3) determining the crystallographic structure of WT ISF, at 1.2 A resolution, and Y156F ISF, at 1.5 A; (4) gaining insight into the physical nature of the interaction of the hydroxyl groups from the amino acid side chains of serine 154 and tyrosine 156 with the S1 inorganic sulfur of the [2Fe-2S] cluster and the cysteine 129 thiolate, respectively; and (5) obtaining isotropic and anisotropic hyperfine coupling constants for 11 protons in the local environment of the [2Fe-2S] cluster, three of which were assigned using comparisons with other systems and theoretical calculations. Work done supports the hypothesis that the redox potential of Rieske iron-sulfur proteins are tuned by hydrogen bonds to the inorganic and terminal sulfurs. Electrochemical measurements showed that the elimination of the H-bond between tyrosine 156 and the thiolate of cysteine 129 (using the mutant strain Y156F) resulted in an E m drop of ∼50 mV, and the elimination of the H-bond between serine 154 and the cluster resulted in an Em drop of ∼120 mV. Using resonance Raman spectroscopy to explore the oxidized [2Fe-2S] cluster, it was shown that Tyr156-OetaH plays a small role in establishing the geometry of the cluster, whereas Ser154-OgammaH does not play a role at all. Information about the structure of the reduced Rieske [2Fe-2S] cluster was obtained from hyperfine sublevel correlation spectroscopy and can be extrapolated to the cytochrome bc1 complex to determine if structural perturbation of the [2Fe-2S] cluster or its local environment occurs upon exposure to different Qo-site-binding occupants.
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