The hydroxylase component (S5HH) of salicylate-5-hydroxylase catalyzes C5 ring hydroxylation of salicylate but switches to methyl hydroxylation when a C5 methyl substituent is present. The use of 18 O 2 reveals that both aromatic and aryl-methyl hydroxylations result from monooxygenase chemistry. The functional unit of S5HH comprises a nonheme Fe(II) site located 12 Å across a subunit boundary from a one-electron reduced Riesketype iron−sulfur cluster. Past studies determined that substrates bind near the Fe(II), followed by O 2 binding to the iron to initiate catalysis. Stopped-flow-single-turnover reactions (STOs) demonstrated that the Rieske cluster transfers an electron to the iron site during catalysis. It is shown here that fluorine ring substituents decrease the rate constant for Rieske electron transfer, implying a prior reaction of an Fe(III)-superoxo intermediate with a substrate. We propose that the iron becomes fully oxidized in the resulting Fe(III)-peroxo-substrate-radical intermediate, allowing Rieske electron transfer to occur. STO using 5-CD 3 -salicylate-d 8 occurs with an inverse kinetic isotope effect (KIE). In contrast, STO of a 1:1 mixture of unlabeled and 5-CD 3 -salicylate-d 8 yields a normal product isotope effect. It is proposed that aromatic and aryl-methyl hydroxylation reactions both begin with the Fe(III)-superoxo reaction with a ring carbon, yielding the inverse KIE due to sp 2 → sp 3 carbon hybridization. After Rieske electron transfer, the resulting Fe(III)-peroxo-salicylate intermediate can continue to aromatic hydroxylation, whereas the equivalent aryl-methyl intermediate formation must be reversible to allow the substrate exchange necessary to yield a normal product isotope effect. The resulting Fe(III)-(hydro)peroxo intermediate may be reactive or evolve through a high-valent iron intermediate to complete the arylmethyl hydroxylation.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.