The biophysical and spectroscopic characterization of a diheme enzyme and putative phosphatase partner from Burkholderia thailandensis
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Using a bioinformatics approach to explore the oxidation chemistry of peroxidases in microorganisms, I generated a sequence similarity network (SSN) of proteins that fall under the umbrella of diheme peroxidases. Bacterial cytochrome c peroxidases (bCCPs) are responsible for the electrochemical conversion of H2O2 to water by use of two c-type heme prosthetic groups. Despite structural similarity to bCCPs, the diheme enzyme MauG from P. denitrificans does not catalytically convert H2O2 to water. MauG, part of the mau operon, instead utilizes H2O2 to generate a highly unusual bis-Fe(IV) intermediate for the biosynthesis of tryptophan trpytophanyl quinone (TTQ), the cofactor required for methylamine dehydrogenase (MADH). The results within this thesis provide evidence for unreported diheme proteins conserved in all strains of Burkholderia, a gram-negative bacterium with implications in cystic fibrosis. Sharing sequence similarity to MauG, the Burkholderia orthologs separate into two classes referred to as Class A and Class B, but genomic analysis reveals a lack of a mau operon for all species. Instead these diheme enzymes are highly conserved downstream of a putative phosphatase protein partner. The work presented in this thesis expands our knowledge of the peroxidatic chemistry of heme containing proteins in microbes. The results for the biochemical and biophysical characterization of Class A enzyme BthA and Class B diheme enzyme BthB from non-pathogenic B. thailandensis prove BthA and BthB are dual functioning peroxidases, capable of both H2O2 reduction and formation of the same bis-Fe(IV) intermediate proposed for MauG. Mutational analysis of the six-coordinate heme ligand of BthA, Tyr463, addresses the electrochemical and spectroscopic properties important in the dual functionality, as well as provides evidence for a role of BthA in O2 reactivity. Furthermore, the biochemical characterization of PhosA, the putative protein partner of BthA based on genomic analysis, confirms presence of a dimetal Fe-Zn center reminiscent of purple acid phosphatases, and reactivity toward nucleotide diphosphates. While studies to understand the biological role of BthA and PhosA are still underway, results from the biophysical characterization of both proteins supports a role of the Class A system in unprecedented chemistry.