The Catalytic Mechanism of SoxAX Cytochromes: Insights from Site-directed Mutagenesis, Spectroscopy and Enzyme Kinetics

James Kilmartin (2011). The Catalytic Mechanism of SoxAX Cytochromes: Insights from Site-directed Mutagenesis, Spectroscopy and Enzyme Kinetics PhD Thesis, School of Chemistry & Molecular Biosciences, The University of Queensland.

       
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Author James Kilmartin
Thesis Title The Catalytic Mechanism of SoxAX Cytochromes: Insights from Site-directed Mutagenesis, Spectroscopy and Enzyme Kinetics
School, Centre or Institute School of Chemistry & Molecular Biosciences
Institution The University of Queensland
Publication date 2011-11
Thesis type PhD Thesis
Supervisor Assoc. Prof. Mark Riley
Dr. Ulrike Kappler
Total pages 166
Total colour pages 43
Total black and white pages 123
Language eng
Subjects 030201 Bioinorganic Chemistry
030406 Proteins and Peptides
Abstract/Summary The SoxAX cytochromes are part of a multi-enzyme complex that catalyses the oxidation of reduced sulfur compounds. The diheme SoxAX cytochrome from Starkeya novella is a critical component in the bacterial sulfur oxidation pathway and catalyses the covalent attachment of thiosulfate to the SoxYZ carrier protein. The highly unusual His/Cys ligated heme group found within the SoxA subunit is believed to be the active site of SoxAX, however, the extremely low SoxA heme redox potential and the presence of a Cu(II) redox centre raised many questions regarding the role of the SoxA heme and Cys236 in the reaction mechanism of SoxAX. In this study we substituted Cys236 with other well-characterised heme ligands (methionine and histidine), to create different heme ligand environments with which to investigate the proposed function of Cys236. The substituted SoxAX proteins were spectroscopically analysed by near infra red magnetic circular dichroism (NIR-MCD) and electronic paramagnetic resonance (EPR) spectroscopy in order to fully characterise the ligand sphere surrounding the SoxA heme before determining the kinetic parameters of each using the recently developed reduced glutathione (GSH) assay system. The initial characterisation of the SoxA heme ligand environment involved UV-vis absorption and NIR-MCD spectroscopy, as well as optical heme redox titrations, and the results corresponded to what was expected based on the literature. The exception was SoxAXC236A; the presence of a high spin SoxA heme group could not be confirmed based solely on these results. The use of EPR increased the resolution of our ligand field characterisation and demonstrated multiple low spin heme species for SoxAXC236M and SoxAXC236H, as well as the presence of a small amount of high spin heme. Analysis of the SoxAXC236M protein crystal structure revealed variability in the bond lengths between the axial ligands and the Fe3+ ion centre. It can be concluded that the heme heterogeneity observed in the EPR spectra arises from multiple conformations of the heme ligands. Unlike the other Cys236 substituted proteins, the EPR spectrum of SoxAXC236A revealed virtually no high spin heme signal intensity, instead a low spin species was identified indicating a new heme ligand has coordinated to the SoxA heme. Taken together, the EPR data for SoxAXWT and the Cys236 substituted proteins demonstrates an inherent flexibility in the local SoxA heme ligand environment, which may be an important property in the reaction mechanism of SoxAX. The creation of Cys236 substituted proteins allowed, for the first time, an opportunity to test whether properties of the SoxA such as the heme redox potential or the nature of the heme ligands are critical for the SoxAX reaction mechanism. Utilising a recently developed assay system that uses reduced glutathione as a substrate, we have demonstrated neither the heme redox potential nor the nature of the heme ligands of the SoxA heme has any significant impact on the substrate binding affinity. However, a noticeable decrease in the kcat values indicates that Cys236 is necessary for efficient enzymatic turnover. It is possible that this is due to the Cys236 interacting with the substrate and requires the conformational flexibility demonstrated in the EPR spectroscopy. The results then demonstrate that the substitution of Cys236 with other heme ligating residues has no significant affect on the glutathione binding affinity and is further evidence strengthening the hypothesis that the Cu(II) site is the active site of SoxAX.
Keyword SoxAX
Cytochromes
Spectroscopy
Mutagenesis
Kinetics
Copper
Additional Notes Colour page numbers: 13, 22, 48, 50, 53-57, 70, 72, 75-76, 78-80, 96, 100-101, 103, 108-109, 115-116, 121, 124, 131-132, 147-151, 153-154, 157-160, 163-166.

 
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Created: Thu, 28 Jun 2012, 13:57:00 EST by Mr James Kilmartin on behalf of Library - Information Access Service