Theses and Dissertations at Montana State University (MSU)
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Item A molecular, structural, and cellular multiple-level study aimed at understanding the unique reaction catalyzed by the last enzyme in the heme-biosynthesis pathway of gram-positive bacteria, coproheme decarboxylase (CHDC)(Montana State University - Bozeman, College of Letters & Science, 2018) Celis Luna, Arianna I.; Chairperson, Graduate Committee: Jennifer DuBois; Bennett R. Streit, Garrett C. Moraski, Timothy D. Lash, Gudrun S. Lukat-Rodgers, Kenton R. Rodgers and Jennifer L. DuBois were co-authors of the article, 'Unusual peroxide-dependent, heme-transforming reaction catalyzed by hemQ' in the journal 'Biochemistry' which is contained within this thesis.; George H. Gauss, Bennett R. Streit, Krista Shisler, Garrett C. Moraski, Kenton R. Rodgers, Gudrun S. Lukat-Rodgers, John W. Peters and Jennifer L. DuBois were co-authors of the article, 'A structure-based mechanism for oxidative decarboxylation reactions mediated by amino acids and heme propionates in coproheme decarboxylase (hemQ)' in the journal 'Journal of the American Chemical Society' which is contained within this thesis.; Dissertation contains two articles of which Arianna I. Celis Luna is not the main author.Heme b is one of nature's most ancient and versatile co-factors and is essential for aerobic life. As such, heme b is synthesized by almost every living organism and plays a major role in bacterial virulence. A pathway for heme b biosynthesis, which is unique to some of the most primitive gram-positive bacteria including many important pathogens, was recently discovered. This pathway, now known as the coproprophyrin-dependent (CPD) branch, ends in a step catalyzed by an unusual enzyme known alternately as coproheme decarboxylase (ChdC) or HemQ. This research aimed to understand ChdC function at the molecular, structural, and cellular levels. Using the ChdC enzyme from Staphylococcus aureus (SaChdC) and a variety of biochemical and analytical tools (conventional and stopped-flow UV-Vis spectroscopy, resonance Raman, HPLC, LC-MS, site-directed mutagenesis, EPR, and X-ray crystallography), the work presented here describes how the coproheme substrate is accommodated in the SaChdC active site and poised for reactivity. The cumulative results show that ChdC catalyzes the oxidative decarboxylation of coproheme III to generate heme b in a sequential and clock-wise fashion, generating harderoheme III in the process. This reaction is H 2 O 2-dependent and the mechanism involves the formation of the high-valent Fe(IV) intermediate (Compound I) and a tyrosine radical (Tyr °). The coproheme-bound ChdC structure revealed a helical-loop that is flexible and moves in towards the active site in the presence of substrate. This loop is hypothesized to act as an 'active site gate' which mediates substrate entry and product egress. Due to the cytotoxicity of heme and its porphyrin precursors, we proposed that the metabolite flux in this pathway is controlled by transient protein-protein interactions. Using the UV-Vis characteristics of porphyrins and phenotype characterization of the deltachdC knock-out strain of S. aureus complemented with ChdC point mutants, we present preliminary evidence for an interaction between ChdC the preceding enzyme of the pathway, CpfC. The same approaches also implicated potential interactions between ChdC and an unidentified heme-chaperone, which delivers heme to its final cellular destination. We propose that this chaperone is HemW. Experiments to test this hypothesis are outlined. This work elucidates yet different way that nature has equipped cells to perform radical chemistry in order to accomplish essential molecule transformations, such as that of decarboxylation and the simultaneous generation of CO 2, and emphasizes the importance of substrate/product post-catalysis cellular trafficking.Item Characterization of integral membrane proteins using mass spectrometry : bacteriorhodopsin, rhodopsin, and neutrophil cytochrome b558(Montana State University - Bozeman, College of Letters & Science, 1998) Barnidge, David RobertItem Function of non-heme-binding domains of the Staphylococcus aureus IsdB protein in heme assimilation from methemoglobin(Montana State University - Bozeman, College of Agriculture, 2014) Li, Dengfeng; Chairperson, Graduate Committee: Benfang LeiAs a hemoglobin acceptor, IsdB rapidly and efficiently acquires heme from methemoglobin (metHb) in the heme acquisition pathway of Staphylococcus aureus. The pathway of heme assimilation in S. aureus involving IsdB has been established; however, the mechanism of rapid and efficient heme assimilation of metHb heme by IsdB remains unclear. IsdB consists of five major domains: the N-terminal (ND), NEAr Transporter 1 (N1), middle (MD), heme binding NEAr Transporter 2 (N2), and C-terminal (CD) domains. The goal of this study is to elucidate the roles of these IsdB domains in the metHb-to-IsdB heme transfer reaction. Deletion of the CD region does not alter the kinetics and equilibrium of the reaction. Sequential deletions of ND and N1 of ND-N1-MD-N2 progressively reduce heme transfer rates but have no effect on the reaction equilibrium. Further deletion of MD decreases the efficiency of heme transfer from metHb to N2. The MD domain reduces heme dissociation from holo-N2 and drives the metHb/N2 reaction to the formation of holo-N2. ND-N1-MD and N2 fragments, but not ND-N1, MD, and N2, reconstitute the rapid metHb/IsdB reaction, indicating an MD/N2 interaction. Analyses of MD, N2, and MD-N2 mixture by size exclusion chromatography support an interaction between MD and N2. These results indicate that ND-N1 and MD domains critically contribute to the kinetics and equilibrium of the metHb-to-IsdB heme transfer reaction, respectively. The results also suggest that CD functions as a spacer to position IsdB in the cell wall envelope for heme relay through the cell wall. These findings support a mechanism of direct extraction of metHb heme by IsdB that involves the four structural domains of IsdB.