Dioxygenases without Requirement for Cofactors and Their Chemical Model Reaction:  Compulsory Order Ternary Complex Mechanism of 1H-3-Hydroxy-4-oxoquinaldine 2,4-Dioxygenase Involving General Base Catalysis by Histidine 251 and Single-Electron Oxidation of the Substrate Dianion

• Published in: Biochemistry (Easton) 2004-11, Vol.43 (45), p.14485-14499
• Main Authors: Frerichs-Deeken, Ursula, Ranguelova, Kalina, Kappl, Reinhard, Hüttermann, Jürgen, Fetzner, Susanne
• Format: Article
• Language: English
• Subjects: Alanine - genetics; Anaerobiosis - genetics; Arthrobacter - enzymology; Arthrobacter - genetics; Bacterial Proteins - antagonists & inhibitors; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Binding Sites; Catalysis; Dioxygenases - antagonists & inhibitors; Dioxygenases - chemistry; Dioxygenases - genetics; Electron Spin Resonance Spectroscopy; Electrons; Enzyme Activation - genetics; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Histidine - chemistry; Histidine - genetics; Hydrogen Peroxide - chemistry; Hydrogen-Ion Concentration; Kinetics; Models, Chemical; Mutagenesis, Site-Directed; Oxidation-Reduction; Oxygen - chemistry; Quinaldines - chemistry; Spectrophotometry, Ultraviolet; Substrate Specificity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi048735u

1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) is a cofactor-less dioxygenase belonging to the α/β hydrolase fold family, catalyzing the cleavage of 1H-3-hydroxy-4-oxoquinaldine (I) and 1H-3-hydroxy-4-oxoquinoline (II) to N-acetyl- and N-formylanthranilate, respectively, and carbon monoxide. Bisubstrate steady-state kinetics and product inhibition patterns of HodC, the C69A protein variant of Hod, suggested a compulsory-order ternary-complex mechanism, in which binding of the organic substrate precedes dioxygen binding, and carbon monoxide is released first. The specificity constants, k cat/K m,A and k cat/K m,O 2 , were 1.4 × 108 and 3.0 × 105 M-1 s-1 with I and 1.2 × 105 and 0.41 × 105 M-1 s-1 with II, respectively. Whereas HodC catalyzes formation of the dianion of its organic substrate prior to dioxygen binding, HodC-H251A does not, suggesting that H251, which aligns with the histidine of the catalytic triad of the α/β hydrolases, acts as general base in catalysis. Investigation of base-catalyzed dioxygenolysis of I by electron paramagnetic resonance (EPR) spectroscopy revealed formation of a resonance-stabilized radical upon exposure to dioxygen. Since in D2O spectral properties are not affected, exchangeable protons are not involved, confirming that the dianion is the reactive intermediate that undergoes single-electron oxidation. We suggest that in the ternary complex of the enzyme, direct single-electron transfer from the substrate dianion to dioxygen may occur, resulting in a radical pair. Based on the estimated spin distribution within the radical anion (observed in the model reaction of I), radical recombination may produce a C4- or C2-hydroperoxy(di)anion. Subsequent intramolecular attack would result in the 2,4-endoperoxy (di)anion that may collapse to the reaction products.