Abstract
Dioxygenases catalyze a diverse range of biological reactions by incorporating molecular oxygen into organic substrates. Typically, they use transition metals or organic cofactors for catalysis. Bacterial 1-H-3-hydroxy-4-oxoquinaldine-2,4-dioxygenase (HOD) catalyzes the spin-forbidden transfer of dioxygen to its N-heteroaromatic substrate in the absence of any cofactor. We combined kinetics, spectroscopic and computational approaches to establish a novel reaction mechanism. The present work gives insight into the rate limiting steps in the reaction mechanism, the effect of first-coordination sphere amino acids as well as electron-donating/electron-withdrawing substituents on the substrate. We highlight the role of active site residues Ser101/Trp160/His251 and their involvement in the reaction mechanism. The work shows, for the first time, that the reaction is initiated by triplet dioxygen and its binding to deprotonated substrate and only thereafter a spin state crossing to the singlet spin state occurs. As revealed by steady- and transient-state kinetics the oxygen-dependent steps are rate-limiting, whereas Trp160 and His251 are essential residues for catalysis and contribute to substrate positioning and activation, respectively. Computational modeling further confirms the experimental observations and rationalizes the electron transfer pathways, and the effect of substrate and substrate binding pocket residues. Finally, we make a direct comparison with iron-based dioxygenases and explain the mechanistic and electronic differences with cofactor-free dioxygenases. Our multidisciplinary study confirms that the oxygenation reaction can take place in absence of any cofactor by a unique mechanism in which the specially designed fit-for-purpose active-site architecture modulates substrate reactivity toward oxygen.
Highlights
Dioxygenases are mechanistically intriguing enzymes since they are able to perform a spin-forbidden reaction in which the triplet spin ground-state of molecular oxygen reacts with either a cofactor or an organic molecule
Details of the reaction mechanism were established from density functional theory modeling to determine the rate-determining step in the reaction and the quantum mechanical features that affect the height of the barrier, and the rate constant
To gain further insight into the reaction mechanism, we studied the role of several active site residues, including the conserved residues His[251] and Ser[101,12] which correspond to the histidine/nucleophile residues[27] of the catalytic triad in the α/β fold superfamily, and the active site residue Trp[160]
Summary
Dioxygenases are mechanistically intriguing enzymes since they are able to perform a spin-forbidden reaction in which the triplet spin ground-state of molecular oxygen reacts with either a cofactor or an organic molecule. These enzymes catalyze the incorporation of molecular oxygen into their organic substrates as a means to initiate their metabolism. These enzymes show different cofactor requirements, ranging from transition metals (typically iron with either a heme and nonheme ligand environment) to flavins; they show diverse structural frameworks to drive oxygenation chemistry
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