Salicylate 5-Hydroxylase: Intermediates in Aromatic Hydroxylation by a Rieske Monooxygenase

摘要: Rieske oxygenases (ROs) catalyze a large range of oxidative chemistry. We have shown that cis-dihydrodiol-forming dioxygenases first react with their aromatic substrates via an active site nonheme Fe(III)-superoxide; electron transfer from the cluster then completes product-forming reaction. Alternatively, two-electron-reduced Fe(III)-peroxo or hydroxo-Fe(V)-oxo activated oxygen intermediates are possible and may be utilized by other ROs to expand catalytic range. Here, reaction monooxygenase, salicylate 5-hydroxylase, does not form cis-dihydrodiol is examined. Single-turnover kinetic studies show fast binding O2. Transfer required gentisate product occurs through bonds over ∼12 A must also very fast. However, observed rate constant for this much slower than expected sensitive substrate type. This suggests initial using same Fe(III)-superoxo-level intermediate as limits transfer. transient (λmax = 700 nm) paramagnetic resonance (EPR) at g 4.3 after formed in site. The use 17O2 (I 5/2) results hyperfine broadening signal, showing binds mononuclear iron its C5-OH intermediate. chromophore EPR signal allow study release cycle. Comparison kinetics single- multiple-turnover reactions shows re-reduction metal centers accelerates ∼300-fold, providing insight into regulatory mechanism ROs.