| The activation of molecular oxygen plays an important role in biological oxidation reactions. Various enzymes employ transition metals to facilitate this chemical process. The ferritin-like dimetal-carboxylate oxidases or oxygenases, which include soluble methane monooxygenase (sMMO), toluene/o-xylene monooxygenase, phenol hydroxylase (PhOH), alkene monooxygenase (AMO), plant soluble fatty acyl--acyl carrier protein desaturase, and the beta2 (R2) subunit of class Ia ribonucleotide reductase (RNR), as well as N-oxygenase AurF and cyanobacterial aldehyde decarbonylase (cAD), activate O2 at a carboxylate-bridged non-heme dimetal center. Such enzymes have been extensively investigated due to their extremely interesting and complicated chemistry, as well as their potential commercial usage. These enzymes usually function in multicomponent complexes including a reductase, a scaffold subunit, and a ferritin-like oxygenase/oxidase. Diiron is most commonly employed by these oxygenases/oxidases as the metal cofactor. A general reaction cycle of the diiron cofactor involves: 1) the reduction of the diferric form to diferrous by the reductase component, in which electrons are transferred from NADH or NADPH; 2) activation of molecular oxygen by the diferrous cofactor, forming some type of peroxo-diferric intermediate; 3) oxidation of the substrate as the peroxo species is converted to diferric form, ready to be reduced to start another catalytic cycle. Manganese has also been reported to replace one or both of the iron atoms of the metal cofactors, most notably in the beta 2 subunits of class Ib and Ic ribonucleotide reductases. The class Ic beta 2 subunit utilizes a heterodinuclear Mn/Fe cofactor while the beta 2 subunit of class Ib ribonucleotide reductase employs a dimanganese cofactor. Due to the presence of the transition metals, these enzymes usually possess rich spectroscopic features, allowing multiple spectroscopic methods such as Mossbauer spectroscopy, electron paramagnetic resonance (EPR) spectroscopy, UV/visible absorption spectrophotometry, electron-nuclear double resonance (ENDOR) spectroscopy, Raman spectroscopy, etc. to be used to study the reactions. Due to the speed at which most of the reactions catalyzed by these dimetal enzymes occur, the millisecond to second time scale is required, thereby necessitating fast quench or real time measurement; therefore, rapid-mix techniques are usually adjoined to spectroscopic methods to meet this requirement.;In this thesis, the reaction mechanism and intermediates of dinuclear ferritin-like oxygenase/oxidase are summarized and the progress I have made during my Ph.D. study on AurF and cADs is narrated in detail. |
