posted on 2022-12-30, 13:05authored byMelanie
S. Rogers, Adrian M. Gordon, Todd M. Rappe, Jason D. Goodpaster, John D. Lipscomb
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.