Oxidative stress is the imbalance of oxidants and reductants; it affects different parts of the organisms. In methane-producing archaea, Methanosarcina acetivorans, a rubrerythrin-like protein methanogen rubrerythrin, is involved in the oxidative defense of the microorganism. It is a 45 kDa homodimer protein in the buffer solution. ICPMS and EXAFS analyses confirmed the presence of iron and zinc in this protein's structure. EPR confirmed the iron (Fe3+) of the rubredoxin center with the characteristic peak in the low field having a 9.35 g value and a diiron center peak in the high field region with a characteristic 2.01 g value. A BioSAXS analysis was conducted to investigate the structural changes in a mRbr under different conditions. The results revealed that the protein undergoes substantial structural modifications when exposed to either oxidation or reduction. When treated with H2O2, the protein sample displayed an Rg of 47Ao and a Dmax of 167 Ao, indicating a larger size than the reduced state. Conversely, when subjected to Na2S2O4, the protein sample had an Rg of 45 Ao and a Dmax of 45 Ao.

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Methanogens, members of the domain Archaea, are unique in their ability to reduce carbon substrates to methane. Coenzyme M (CoM) is required in all methanogenic pathways. The biosynthesis of this coenzyme has been well studied in Class I Methanogens, but in Class II Methanogens, such as Methanosarcina acetivorans, little is known. The first step in the biosynthetic pathway might be catalyzed by cysteate synthase (CS), which converts phosphoserine to cysteate by the addition of sulfite. The 46 kDa enzyme was successfully purified from inclusion bodies and characterized. The identity of the product was confirmed by liquid chromatography-mass spectrometry (LC-MS) results as well as by derivatization of the reaction product coupled with high pressure liquid chromatography (HPLC) analysis. Kinetic analysis showed that the enzyme has a K [subscript m] of 0.43 mM for its substrate, phosphoserine, and a K [subscript m] of 0.05 mM for its required nucleophile, sulfite. Four compounds were found to be inhibitors and IC₅₀ values were determined. The results show that CS carries out a new reaction and narrows the gap in our knowledge of Class II Methanogen CoM biosynthesis. In the second part of this dissertation, five enzymes in a newly discovered but poorly characterized pathway for the degradation of halogenated aromatic compounds in Leptothrix cholodnii SP-6 were examined. The pathway reportedly culminates in the production of 2-chloroacetaldehyde, a well-known alkylating agent. In order to determine if 2-chloroacetaldehyde is produced and how the organism survives in its presence, the pathway intermediates are being identified. To this end, 4-oxalocrotonate tautomerase (4-OT), 4-oxalocrotonate decarboxylase (4-OD), vinylpyruvate hydratase (VPH), pyruvate aldolase (PA) and acetaldehyde dehydrogenase (AAD) were cloned, expressed and characterized. 4-OT was found to process the 5-(chloro)-2-hydroxymuconate, but only when the equilibrium was shifted by the addition of 4-OD and VPH. Steady state kinetic analysis showed that while there is a slight decrease in K [subscript m] for the halogenated substrate when compared to the non-halogenated substrate, indicating a difference in binding. There is also a 30-fold decrease in the turnover number, indicating a preference for the non-halogenated substrate. The identity of the product, 5-(chloro)-2-oxo-4-hydroxypentanoate, was verified by ¹H NMR spectroscopy. A stereochemical analysis was also carried out. / text