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.
Dioxygenases catalyze a diverse range of chemical reactions that involve the incorporation of oxygen into a substrate and typically use a transition metal or organic cofactor for reaction. Bacterial (1H)-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) belongs to a class of oxygenases able to catalyze this energetically unfavorable reaction without any cofactor. In the quinaldine metabolic pathway, HOD breaks down its natural N-heteroaromatic substrate using a mechanism that is still incompletely understood. Experimental and computational approaches were combined to study the initial step of the catalytic cycle. We have investigated the role of the active site His-251/Asp-126 dyad, proposed to be involved in substrate hydroxyl group deprotonation, a critical requirement for subsequent oxygen reaction. The pH profiles obtained under steady-state conditions for the H251A and D126A variants show a strong pH effect on their kcat and kcat/Km constants, with a decrease in kcat/Km of 5500- and 9-fold at pH 10.5, respectively. Substrate deprotonation studies under transient-state conditions show that this step is not rate-limiting and yield a pKa value of approximately 7.2 for WT HOD. A large solvent isotope effect was found, and the pKa value was shifted to approximately 8.3 in D2O. Crystallographic and computational studies reveal that the mutations have a minor effect on substrate positioning. Computational work shows that both His-251 and Asp-126 are essential for the proton transfer driving force of the initial reaction. This multidisciplinary study offers unambiguous support to the view that substrate deprotonation, driven by the His/Asp dyad, is an essential requirement for its activation.
