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Title:Quinol activity of cytochrome aa3-600 menaquinol oxidase from Bacillus subtilis characterized by biochemical and EPR methods
Author(s):Yi, Sophia Myongsin
Director of Research:Gennis, Robert B
Doctoral Committee Chair(s):Gennis, Robert B
Doctoral Committee Member(s):Lu, Yi; Crofts, Antony R; van der Donk, Wilfred A
Department / Program:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):cytochrome aa3-600
vitamin K2
Abstract:The membrane protein cytochrome (cyt) aa3-600 menaquinol oxidase from B. subtilis is uniquely a heme-copper oxygen reductase that uses substrate menaquinol instead of cytochrome c. The cyt aa3-600 accepts electrons from menaquinol (or Vitamin K2) and channels them one-at-a-time to low-spin heme A and finally to the binuclear center where oxygen is reduced to water. In order to characterize the protein-quinone interaction, cyt aa3-600 has been cloned with a His-tag, expressed and purified from the native system. One equivalent of quinone is found to strongly associate with isolated protein. The enzyme stabilizes a menasemiquinone radical (SQ) at a high affinity site (Qh) that is important for catalysis. The molecular details of the stabilized SQ revealed by 1D-, 2D- X-band 14N, 15N electron spin echo envelope modulation (ESEEM) experiments indicate that a nitrogen having the largest hyperfine interaction (14A ~ 0.9 MHz) likely belongs to side chain of Arg. In addition, 1H, 2H ESEEM spectra show two strongly coupled exchangeable protons with anisotropic components (|T| = 5.6 and 2.9 MHz) that form H-bonds with the SQ. The H-bond interactions involve protonated nitrogen of Arg70 and acidic side chain of Asp74. These residues face one carbonyl group of the SQ ring structure and orchestrate an asymmetric H-bonding around the quinone head group. The Qh-site of cyt aa3-600 contains conserved residues Arg70, Asp74, His94 and Glu97 that are considered functionally important. Several mutations have been introduced at the Qh-site. Among the Qh-site mutants, the R70H mutant, showing a strong quinol oxidation activity, was able to generate menasemiquinone radical that was examined further by advanced X- and Q-band EPR. The bound SQ of the R70H mutant exhibits a strong isotropic hyperfine coupling (14A ~ 2.0 MHz) with a hydrogen bonded nitrogen. This nitrogen originates from a histidine side chain, based on its quadrupole coupling constant, e2qQ/h = 1.44 MHz, typical for protonated imidazole nitrogen atoms. In the wild type cyt aa3-600, the SQ is instead hydrogen bonded with N from the Arg70 side chain. Analysis of the 2D- X-band 1H, 2H ESEEM spectra shows that the mutation also changes the number and strength of the hydrogen bonds between the SQ and the surrounding protein. Despite the alteration in the immediate environment of the SQ, the R70H mutant remains catalytically active. These findings are in contrast to the equivalent mutation in the close homologue, cytochrome bo3 ubiquinol oxidase from E. coli, where the R71H mutation eliminates function. The current work on cyt aa3-600 and its mutants suggests that one-site quinone model describes the specific activity values better than the two-site model that has been advocated, thus far. None of the Qh-site mutants in cyt aa3-600 is able to eliminate quinol oxidase activity, except for Asp74 mutants that show structural disturbance of low-spin heme A. In the past, the corresponding Qh-site mutants in cyt bo3 have shown complete loss of activity, but new evidence suggests that some of the quinol activity can be restored with menaquinol-based substrate, a molecule having low midpoint potential. The differences between the protein-quinol interactions of the bo3-type ubiquinol oxidase and the aa3-600 menaquinol oxidase appear to be based on the reactivity of the substrate quinol and the asymmetric H-bonding pattern imposed by the protein structure. A possible mechanism of quinol oxidation is proposed, where the Glu97 in cyt aa3-600 replaced by Gln101 in homologous cytochrome bo3 ubiquinol oxidase of E. coli is considered to play a significant role. Overall, the evidence leads to structural similarity of the Qh-sites found in cyt bo3 and cyt aa3-600, and cyt bo3 is remarkable in being able to accommodate both ubiquinol and menaquinol as substrates.
Issue Date:2015-12-07
Rights Information:Copyright 2015 Sophia M. Yi
Date Available in IDEALS:2016-07-07
Date Deposited:2016-05

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