Crystallographic determination of wild type, mutant and substrate-analogue inhibited structures of bacterial members of a family of superoxide dismutases : submitted as part of the requirements for the degree of Doctor of Philosophy, Institute of Fundamental Sciences, Chemistry, Massey University, New Zealand

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dc.contributor.authorOakley, Simon Hardie
dc.date.accessioned2009-10-19T01:41:26Z
dc.date.availableNO_RESTRICTIONen_US
dc.date.available2009-10-19T01:41:26Z
dc.date.issued2009
dc.description.abstractThe iron and manganese superoxide dismutases are a family of metallo-enzymes with highly conserved protein folds, active sites and dimer interfaces. They catalyse the elimination of the cytotoxic free radical superoxide to molecular oxygen and hydrogen peroxide by alternate reduction then oxidation of the activesite with the concomitant transfer of protons from the solvent. There are many key aspects of enzymatic function that lack a structural explanation. The focus of this study is on three crystal structures. The iron-substituted manganese superoxide dismutase from Escherichia coli complexed with azide, a substrate-mimicking inhibitor, was solved to 2.2 Å. This “wrong” metal form shows a binding pattern seen previously in the manganese superoxide dismutase from Thermus thermophilus. Wild-type manganese specific superoxide dismutase from the extremophile Deinococcus radiodurans was solved to 2.0 Å and has an active site reminiscent of other solved manganese superoxide dismutases despite a lack of product inhibition. The azide-inhibited manganese superoxide dismutase from Deinococcus radiodurans was determined to a resolution of 2.0 Å and showed binding of azide, and by inference superoxide, different to that seen in Thermus thermophilus, but reminiscent of that seen in azide-inhibited iron superoxide dismutases. These results indicate that the azide ion, and by inference superoxide, bind to the metal centre of manganese superoxide dismutases in two modes, and transition between the two modes may be entropy dependent. These structures, integrated with knowledge from other structures, known biochemistry and various spectra, provide insight into catalytic function. An outer-sphere mechanism of proton transfer that does not rely on through-peptide proton uptake is proposed and compared to a previously proposed inner-sphere mechanism. This is based on the observation that a water molecule moves into the active site of the manganese superoxide dismutase from Deinococcus radiodurans upon azide binding, providing a Grötthus pathway for rapid proton transfer to the active site from the bulk solvent. Also presented in this study are the partially refined structures of four point mutants (S82T, L83M, L133V, and M164L/L166V) of the manganese superoxide dismutase from Escherichia coli all solved to roughly 2 Å resolution, designed to investigate product inhibition which varies across species.en_US
dc.identifier.urihttp://hdl.handle.net/10179/1072
dc.language.isoenen_US
dc.publisherMassey Universityen_US
dc.rightsThe Authoren_US
dc.subjectCrystal structuresen_US
dc.subjectAzideen_US
dc.subjectCatalytic functionen_US
dc.subjectProduct inhibitionen_US
dc.subject.otherFields of Research::250000 Chemical Sciences::250300 Organic Chemistryen_US
dc.titleCrystallographic determination of wild type, mutant and substrate-analogue inhibited structures of bacterial members of a family of superoxide dismutases : submitted as part of the requirements for the degree of Doctor of Philosophy, Institute of Fundamental Sciences, Chemistry, Massey University, New Zealanden_US
dc.typeThesisen_US
massey.contributor.authorOakley, Simon Hardie
thesis.degree.disciplineChemistryen_US
thesis.degree.grantorMassey Universityen_US
thesis.degree.levelDoctoralen_US
thesis.degree.levelDoctoralen
thesis.degree.nameDoctor of Philosophy (Ph. D.)en_US
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