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Title:Biochemical characterization of a-type heme-copper oxidases in escherichia coli, bacillus subtilis and thermus thermophilus
Author(s):Ding, Ziqiao
Director of Research:Gennis, Robert G
Doctoral Committee Chair(s):Gennis, Robert G
Doctoral Committee Member(s):Lu, Yi; Crofts, Antony R.; Kalsotra, Auinash
Department / Program:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Electron transport chain
Heme copper oxidase
A-type heme-copper oxidases
Cytochrome bo3 ubiquinol oxidase
Cytochrome aa3-600 menaquinol oxidase
Cytochrome caa3 oxidase
A2-type heme copper oxidase
Tyrosine serine pair(YS)
Glutamic acid
Steady state oxidase activity
Proton pumping
Electron paramagnetic resonance
Menasemiquinone, MK4
Quinone binding site
Tyrosyl radical
Homologous recombination
X-ray crystallography
Quinol oxidase
Escherichia coli
Bacillus subtilis
Thermus thermophilus
Abstract:Heme-copper oxidases (HCOs) couple the free energy of oxygen reduction and translocate protons across membrane to generate a proton electrochemical gradient, which was used to produce ATP by ATP synthase. Based on the sequences and structures of core subunits, they are classified into 3 types. A-type oxidases are by far the most abundant including mitochondrial cytochrome c oxidase and its close homologs. Over the years, great efforts have been invested to study the function of A-type oxidases. A highly conserved glutamic acid residue has been proved to be the branch point for proton translocation, delivering protons either to active site for oxygen reduction or to proton loading site for pumping. However, a growing number of oxidases that lack this key glutamate and instead are replaced by a tyrosine-serine (YS) pair in proximity have been discovered and classified as A2-type oxidase. Several A2-type members have been isolated and shown their capability of proton pumping. Thermus thermophilus cytochrome caa3 oxidase, a member of A2 subtype, was successfully expressed, purified and characterized by site-directed mutagenesis, especially on YS pair as well as around the region of glutamate. It is suggested that the conserved YS pair in A2-type is a functional substitute to glutamate in A1-type. In addition, mutations of D proton channel exhibit different phenotype from that of A1-type, presumably due to a different mechanism. The quinol oxidases in A-type HCOs are relatively well characterized. Previous studies have implicated the four residues (Arg71, Asp75, His98, and Gln101) involved in ubiquinone semiquinone radical stabilization at quinone binding site in E. coli cytochrome bo3 oxidase. Similar result was reported on cytochrome aa3-600 oxidase in B. subtilis, a close homolog to cytochrome bo3 that uses menaquinone instead of ubiquinone. Recently, in vivo experiments have demonstrated that cytochrome bo3 could also use menaquinol as substrate. Therefore, with 14N and 15N labeling, the interaction between menaquinone semiquinone radical and quinone binding site was examined using EPR spectroscopy. Overall, the hyperfine coupling shows difference between ubiquinone and menaquinone semiquinone with quinone binding site in cytochrome bo3, suggesting different binding geometry. Three nitrogens were indicated interacting with menaquinone semiquinone with the strongest interaction from presumably Nε of R71. Separate approach was used to study quinone binding site in quinol oxidases. We managed to overexpress and purify recombinant B. subtilis cytochrome aa3-600 utilizing a new method for membrane protein expression in B. subtilis. The enzyme was subjected to crystallography and preliminary structure revealed several new features that were not observed in cytochrome bo3 structure around quinone binding site. Finally, this approach has proved to be successful in overexpression of B. subtilis succinate dehydrogenase (SQR). Reduced cytochrome bo3 oxidase was studied spectroscopically treated with H2O2 or O2 in attempt to study amino acid radicals related to active site catalysis. By site-directed mutagenesis, a stable tyrosyl radical assigned on Y173 was observed in reaction with oxygen in YII184F bo3 mutant using EPR spectroscopy. This is the first time that a tyrosyl radical has been shown generated by O2. Moreover, we proposed a proton coupled electron transfer mechanism (PCET) causes this long-living tyrosyl radical as well as its migration.
Issue Date:2018-12-05
Rights Information:Copyright 2018 Ziqiao Ding
Date Available in IDEALS:2019-02-08
Date Deposited:2018-12

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