Spectroscopic Studies and Molecular Dynamics Simulations of Mononuclear Dioxygen Activation Enzymes

The dioxygen activation enzymes are ubiquitous in plants, animals, and microorganisms. These enzymes perform the reactions that propel the circulation of oxygen atoms on the planet. The dioxygen molecule supports many critical biochemical processes strictly regulated by enzymes, which in many cases are metalloenzymes. The iron-containing enzymes are very common in performing dioxygen activation. Among them, the mononuclear non-heme iron (MNHI) enzymes, constituting a large subcategory, are capable of performing various types of reactions using the mono-iron center. The mechanistic investigation of the MNHI enzymes is the main subject of this thesis.The iron (II) and 2-oxoglutarate (Fe/2OG) dependent enzyme is a subfamily of MNHI enzymes that use 2OG as co-substrate for dioxygen activation. The Fe/2OG enzymes are proposed to have conserved mechanisms for the initial dioxygen activation reaction with 2OG. We investigate different Fe/2OG enzymes to reconstruct the potential reaction pathways after the initial reaction with dioxygen. We also investigate the effect of different 2OG binding configurations to the iron center on the dioxygen activation. Besides the Fe/2OG enzymes, we investigate a co-substrate independent MNHI enzyme to explore the mechanism that leads to novel reactivity.The investigation of iron-containing enzymes shows a historical trend, where the research focus is initially on the heme enzymes, followed by Fe/2OG MNHI enzymes, and then co-substrate independent MNHI enzymes. In this sequence, the versatility of the mononuclear iron center is gradually untamed. The heme enzymes install a rigid porphyrin frame to fix the iron, in which the iron has very limited flexibility. The Fe/2OG enzymes coordinate the Fe through protein residues and 2OG, such that variation of residues and different configurations of 2OG introduces more flexibility. The co-substrate independent MNHI enzymes delegate the initial dioxygen activation from 2OG to the substrate, therefore the substrate can use the initial dioxygen activation step together with the subsequent oxidation steps to perform complex reactions. The research in this thesis will advance the understanding of dioxygen activation in iron-containing enzymes and the mechanism that couples dioxygen activation with various enzymatic reactivity.