The use of nickel by enzymes is unusual as evidenced by the fact that there are only eight nickel-based enzyme systems currently known, one of which is methyl-coenzyme M reductase (MCR). The site of catalysis in MCR is a highly reduced nickelcontaining prosthetic group named coenzyme F<sub>430</sub>. In the catalytic reaction of MCR, the substrates methyl-coenzyme M and coenzyme B are converted to methane and a heterodisulfide. Although MCR has been studied for decades, it is not clear how it carries out the difficult chemistry of methane formation. The length of the alkanoyl chain in coenzyme B is apparently important for enzymatic turnover. This thesis describes the X-ray crystallographic characterization of the MCR enzyme in complex with four different coenzyme B analogues. Structure analysis suggests why the coenzyme B analogues range from poor substrates to inhibitors. This thesis also describes a crystal structure of a putative catalytic intermediate of MCR with a methyl group bound to the nickel atom. This is the first crystal structure of MCR that resembles a proposed catalytic intermediate. In contrast to nickel, iron is much more widely used in enzymatic systems. A common iron-containing prosthetic group is known as heme. The enzyme hydroxylamine oxidoreductase (HAO) contains 24 hemes and is therefore one of the most complex heme proteins known. The site of catalysis in HAO is a heme with unusual properties proposed to have a single covalent crosslink to a tyrosine residue of the protein. This thesis describes a HAO crystal structure that suggests a different model for this heme: an unprecedented double crosslink between the heme porphyrin and tyrosine. The crystal structure also revealed that HAO is complexed to a protein identified as a hypothetical protein of unknown function in the <italic>Nitrosomonas europaea</talic> genome. As the HAO sample was purified from <italic>N. europaea</italic> this interaction is likely of biological relevance. A crystal structure of HAO in complex with hydroxylamine substrate is also reported. The closing chapter of the thesis describes the cloning and transformation of the <italic>N. europaea hao</italic> gene into <italic>Shewanella oneidensis</italic> that potentially could support recombinant expression of HAO.
University of Minnesota Ph.D. dissertation. July 2012. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Dr. Carrie M. Wilmot. 1 computer file (PDF); ix, 124 pages.
Cedervall, Peder E..
Structural studies of two metallo-enzymes: methyl-coenzyme M reductase and hydroxylamine oxidoreductase.
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