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The work outlined herein presents an investigation of synthetic model complexes of relevance to the active sites of Ni(II)-containing enzymes, particularly urease, glyoxalase I, and acireductone dioxygenase. The research focuses on studying the structural and reactivity features of nickel complexes with biologically relevant substrates. The anion of acetohydroxamic acid is a well-known inhibitor of urease enzymes, including those isolated from Klebsiella aerogenes and Bacillus pasteurii. A precursor to the acetohydroxamate coordination in ureases is proposed to be an interaction between Ni(II) and acetohydroxamic acid. By using a novel supporting chelate ligand capable of secondary hydrogen bonding interactions a novel pseudo-octahedral, Ni(II) acetohydroxamic acid complex has been isolated and characterized. Detailed analysis of the structural features and acetohydroxamic displacement reactivity of this complex has provided fundamental chemical insight toward understanding of the inhibition mechanism in urease enzymes. Glyoxalase I (Glx I) catalyzes one step of the cellular detoxification pathway for α-ketoaldehydes (e.g. methylglyoxal) in humans and bacteria. The GlxI enzyme from E. coli is a Ni(II)-containing enzyme that catalyzes the isomerization of a hemithioacetal to produce a thioester. Of relevance to this enzyme, the first example of a Ni(II) complex that promotes a hemithioacetal isomerization is reported herein. In order to monitor this type of reaction a new approach involving a a deuterium-labeled hemithioacetal (PhC(O)CH(OH)SCD3) and 2H NMR was employed. Acireductone dioxygenases (ARDs) catalyze aliphatic oxidative C-C bond cleavage of an acireductone (1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene) intermediate in the methionine salvage pathway. A unique aspect of these enzymes is that the regioselectivity of the dioxygenase reaction depends on the metal ion bound in the active site. Outlined herein are descriptions of the synthesis, characterization, and O2 reactivity of a novel trinuclear Ni(II) enediolate complex of relevance to the proposed enzyme/substrate adduct in Ni(II)-ARD. Efforts have also been made toward the preparation of C(1)-H acireductone compounds using a combined synthetic/enzymatic approach. A phenyl appended-C(1)-H acireductone was isolated and introduced to a Ni(II) precursor complex. This reaction produced spectroscopic changes consistent with the formation of a new Ni(II) acireductone complex. Preliminary studies of the O2 reactivity of this complex are reported.

Strikt anaerobe Bakterien wie Clostridium difficile und C. scatologenes verwenden GRE, um die chemisch ungünstige Decarboxylierung von 4-Hydroxyphenylacetat zu p-Cresol zu katalysieren. Das Enzymsystem besteht aus einer Decarboxylase und dem zugehörigen Aktivierungsenzym. Die 4-Hydroxyphenylacetat-Decarboxylase (4Hpad) besitzt zusätzlich zum Protein-basierten Glycinradikal eine weitere Untereinheit mit bis zu zwei [4Fe-4S] Clustern und repräsentiert hierdurch eine neue Klasse von Fe/S-Cluster-haltigen GREs, die aromatische Verbindungen umsetzen. Das Aktivierungsenzym (4Hpad-AE) weicht vom Standardtypus ab, indem es zusätzlich zum S-Adenosylmethionin(SAM)-bindenden [4Fe-4S]-Cluster (RS-Cluster) mindestens einen weiteren [4Fe-4S]-Cluster bindet. In dieser Studie wurden heterologe Expressions- und Reinigungsprotokolle für 4Hpad und 4Hpad-AE entwickelt. Kristallstrukturen von 4Hpad cokristallisiert mit den Substraten (4-Hydroxyphenylacetat, 3,4-Dihydroxyphenylacetat) und dem Inhibitor (4-Hydroxyphenylacetamid) zeigten geringe strukturelle Änderungen im aktiven Zentrum des Proteins. Die Radikalbildung am 4Hpad-AE wurde durch die Überprüfung einer klassischen reduktiven Spaltung von SAM zu den Reaktionsprodukten 5’-Deoxyadenosin und Methionin bestätigt. EPR- und Mössbauer-Spektroskopische Analysen zeigten, dass 4Hpad-AE mindestens einen zusätzlichen [4Fe-4S] Cluster neben dem einzelnen RS-Cluster enthält. Die katalytische Notwendigkeit eines zusätzlichen Clusters wurde durch eine Mutationsanalyse untersucht, wobei eine verkürzte Version des Enzyms ohne die zusätzliche Cystein-reiche Insertion konstruiert wurde. Das verkürzte Mutante ohne die Bindungsmotive für die zusätzlichen Cluster gekennzeichnet, die Konfiguration, Stöchiometrie und die Funktion der zusätzlichen Cluster diagnostizieren. / 4-hydroxyphenylacetate decarboxylase (4Hpad) is a two [4Fe-4S] cluster containing glycyl radical enzyme proposed to use a glycyl/thiyl radical dyad to catalyze the last step of tyrosine fermentation in Clostridium difficile and C. scatologenes by a Kolbe-type decarboxylation. The decarboxylation product p-cresol is a virulence factor of the human pathogen C. difficile. The small subunit of 4Hpad may have a regulatory function with the Fe/S clusters involved in complex formation and radical dissipation in the absence of substrate. The respective activating enzyme (4Hpad-AE) has one or two [4Fe-4S] cluster(s) in addition to the SAM-binding [4Fe-4S] cluster (RS cluster). The role of these auxiliary clusters is still under debate with proposed functions including structural integrity and conduit for electron transfer to the RS cluster. This study shows the optimized expression and purification protocols for the decarboxylase and the co-crystallization experiments and binding studies with 4-hydroxy-phenylacetate and 3,4-dihydroxyphenylacetate and with the inhibitor 4-hydroxy-phenylacetamide. The purification and characterization of active site mutants of decarboxylase are also done. Concerning 4-HPAD-AE, we report on the purification of code-optimized variants, and on spectroscopic and kinetic studies to characterize the respective i) SAM binding enthalpies, ii) rates for reductive cleavage of SAM and iii) putative functions of the additional Fe/S clusters. The truncated mutant lacking the binding motifs for the auxiliary clusters is characterized to diagnose the configuration, stoichiometry and function of the auxiliary clusters.