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 18O2 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 Rieske-type iron-sulfur cluster. Past studies determined that substrates bind near the Fe(II), followed by O2 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-CD3-salicylate-d8 occurs with an inverse kinetic isotope effect (KIE). In contrast, STO of a 1:1 mixture of unlabeled and 5-CD3-salicylate-d8 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 sp2 → sp3 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 aryl-methyl hydroxylation.
Bibliographical noteFunding Information:
The authors acknowledge financial support of this work from National Institutes of Health (NIH) Grant GM118030 (to J.D.L.) and the National Science Foundation Grant No. CHE-1945525 (to J.D.G.). Mass spectrometry analysis was performed at the University of Minnesota Department of Chemistry Mass Spectrometry Laboratory, supported by the Office of the Vice President of Research, College of Science and Engineering, and the Department of Chemistry at the University of Minnesota, as well as the National Science Foundation Award CHE-1336940. H NMR spectroscopy at the UMN Chemistry NMR Laboratory was supported by the Office of the Vice President of Research, College of Science and Engineering, and the Department of Chemistry at the University of Minnesota. Structures of fluorinated S5HH reaction products were determined using the University of Minnesota NMR Center supported by NIH Grant 1S10OD021536. The authors acknowledge the Minnesota Supercomputing Institute (MSI) at the University of Minnesota and the National Energy Research Scientific Computing Center (NERSC), a DOE Office of Science User Facility supported by the Office of Science of the U.S. Department of Energy under Contract DE-AC02-05CH11231. The authors thank Sean Murray and Joseph Dalluge for assistance with GCMS data collection and analysis. The authors thank Letitia Yao and Phillipe Buhlmann for insightful discussions regarding the 5-hydroxylmethyl-salicylate and 3-hydroxylmethyl-benzoate S5HH reaction product determination via H NMR. The authors thank De-Feng Li for generously providing the salicylate-bound structural model of salicylate 5-hydroxylase (derived from PDB ID: 7C8Z). 1 1
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