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Title:Characterizations on bacterial photosynthesis and respiration with optical spectroscopy and magnetic resonance
Author(s):Sun, Chang
Director of Research:Gennis, Robert B
Doctoral Committee Chair(s):Gennis, Robert B
Doctoral Committee Member(s):Crofts, Antony R; Dikanov, Sergei A; Sligar, Stephen G
Department / Program:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Type II photosynthetic reaction center
LM dimer
bo3 oxidase
pulsed EPR
electron transfer
back reaction
ultrafast kinetics study
Selective electron nuclear double resonance (ENDOR)
Abstract:Photosynthesis and respiration are key energetic transductions. Both processes involve downhill electron transfers within transmembrane protein complexes. Here, I have examined different physical aspects of photosynthetic and respiratory electron transfer. For the photosynthesis section, I have carried out a detailed picosecond spectral and kinetics study (Chapter 4) on the electron transfers in the LM dimer of the reaction center from Rhodobacter sphaeroides. Compared to the intact reaction center with the H subunit, the LM dimer has similar electron transfer kinetics except for the forward electron transfer from HA- (bacteriopheophytin in the A branch) to QA (ubiquinone in the A branch) and back electron transfer from QA- to P+ (special pair of bacteriochlorophyll). Both electron transfers slows down by about 4 fold at room temperature. I have also examined the reactivity of the semiquinones in the LM dimer (Chapter 2). In accordance with previous reports, both semiquinones are less stable compared to their counterparts in the intact reaction center. Particularly, I have found that the oxidation of the QA- by the ferricyanide approaches diffusion limit at pH 5. Also, the QB semiquinone can be oxidized by oxygen which explains the very slow phase observed in the back reaction of the LM dimer when excess amount of ubiquinone-10 is present. To determine what kind of structural changes are responsible for the changes in electron transfer kinetics involving QA, the interactions between the QA- and the protein matrix in the LM dimer have been characterized with pulsed electron paramagnetic resonance (EPR, Chapter 3). Both nitrogen data and hydrogen data are consistent with elongated hydrogen bonds to the semiquinone. The binding conformation of QA- is heterogeneous in the LM dimer, with more flexibility on the O1 carbonyl side. The selective labeling of methoxy and methyl with 13C shows that the dihedral angles of methoxy substituents on the semiquinone ring are no longer fixed in the LM dimer, in accordance with the loosened semiquinone binding pocket. For the respiration section, the semiquinone pocket in the bo3 oxidase from Escherichia coli is examined with pulsed EPR (Chapter 5). To get a clear picture of the hydrogen bonded protons, I have fully deuterated the enzyme and successfully generated the semiquinone signal, with which the Davies ENDOR are carried out at Q-band (35 GHz). The higher frequency allows selective excitation of different orientations of semiquinone with respect to the external magnetic field. Our simulations on the ENDOR spectra reveal the Euler angles needed to transform the semiquinone g tensor to the proton hyperfine coupling tensor. It turns out the strongest coupled proton is almost perpendicular to the semiquinone plane, which supports the semiquinone being anionic. Molecular dynamics and density function theory calculations have also being carried out to provide a realistic model of semiquinone in bo3 oxidase.
Issue Date:2016-04-19
Rights Information:Copyright 2016 Chang Sun All rights reserved.
Date Available in IDEALS:2016-07-07
Date Deposited:2016-05

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