Contrasting Mechanisms of Aromatic and Aryl-Methyl Substituent Hydroxylation by the Rieske Monooxygenase Salicylate 5-Hydroxylase

Abstract

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.

Custom tuning of Rieske oxygenase reactivity

“…Recent studies have indicated that an Fe(III)-superoxo species is likely the key oxidant that is used to facilitate the monooxygenation and dioxygenation reactions performed by salicylate 5-hydroxylase and benzoate 1,2-dioxygenase, respectively (Supplementary Fig. 1 ) 32 – 34 . However, to date, despite several investigations that indicate formation of the reactive Fe-based intermediate for catalysis is the rate limiting step of a Rieske oxygenase catalyzed reaction, the nature of the oxidizing species for most members of this enzyme class remains to be defined 35 – 37 .…”

Rieske Oxygenases and Other Ferredoxin‐Dependent Enzymes: Electron Transfer Principles and Catalytic Capabilities

“…Another recent example reported by Rogers et al shows that the Rieske monooxygenase salicylate 5-hydroxylase (S5HH) catalyzes C5 ring hydroxylation of salicylate, but switches to methyl hydroxylation when a C5 methyl substituent is present. [172] Interestingly, it was shown that S5HH exhibits high sensitivity to the electronic structure of the substrate in the rate constant of electron transfer from the Rieske cluster to the mononuclear Fe site. When S5HH switches from aromatic to methyl hydroxylation, the Rieske cluster oxidation kinetics and product formation exhibit opposite deuterium isotope effects during the hydroxylation of the methyl substituent, giving new insight into the mechanisms of Rieske monooxygenation.…”

“…Another recent example reported by Rogers et al. shows that the Rieske monooxygenase salicylate 5‐hydroxylase (S5HH) catalyzes C5 ring hydroxylation of salicylate, but switches to methyl hydroxylation when a C5 methyl substituent is present [172] . Interestingly, it was shown that S5HH exhibits high sensitivity to the electronic structure of the substrate in the rate constant of electron transfer from the Rieske cluster to the mononuclear Fe site.…”