Fundamental investigations into two oxygen-sensitive metalloenzymes from the obligate anaerobe Pyrococcus furiosus
Dillard, Bret Donald
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The objective of this research was to gain a more complete understanding of the enzymatic activities of two oxygen-sensitive enzymes from Pyrococcus furiosus. When working with these enzymes from this obligate anaerobe, it is necessary that all manipulations be carried out in an anaerobic environment in order to maintain the stability of both the metal cofactors and the proteins. The first aim was to show that proline dehydrogenase 1 (PDH1) contains an iron-sulfur cluster and to determine its role in catalysis. The cluster was not evident in the published crystal structure reported by others due to the fact that the protein was prepared aerobically. Using anaerobically purified PDH1, it was shown in this work by electron paramagnetic resonance and inductively coupled plasma spectroscopic analyses that it contains a [2Fe-2S] cluster and that it is reduced by proline, the natural substrate. It is also shown that the cluster transfers electrons to an artificial electron acceptor, resorufin, only when the enzyme is maintained in an anoxic environment. In addition, when the cluster is present in the enzyme, its affinity for proline increases by two orders of magnitude. The second aim of this research was to determine the structure of the iron-containing enzyme rubrerythrin in its native form in both the reduced and oxidized states. The enzyme precipitates when exposed to oxygen or to an excess of its substrate, hydrogen peroxide. In order to maintain its enzymatic activity, the enzyme must be maintained in an anaerobic environment. For crystallization trials, two approaches were merged to make an efficient tool for screening and optimizing the crystallization in an anaerobic environment. The crystal structure of rubrerythrin was shown to contain three iron atoms per monomer and its unique feature of domain swapping was confirmed. The structure of the oxidized form gave great insights into both the catalytic mechanism and the cause of the precipitation of the enzyme upon oxidation.