Light-driven Oxidative Demethylation Reaction Catalyzed by a Rieske-type Non-heme Iron Enzyme Stc2

Abstract

Rieske-type non-heme iron oxygenases/oxidases catalyze a wide range of transformations. Their applications in bioremediation or biocatalysis face two key barriers: the need of expensive NAD(P)H as a reductant and a proper reductase to mediate the electron transfer from NAD(P)H to the oxygenases. To bypass the need of both the reductase and NAD(P)H, using Rieske-type oxygenase (Stc2) catalyzed oxidative demethylation as the model system, we report Stc2 photocatalysis using eosin Y/sulfite as the photosensitizer/sacrificial reagent pair. In a flow-chemistry setting to separate the photo-reduction half-reaction and oxidation half-reaction, Stc2 photo-biocatalysis outperforms the Stc2-NAD(P)H-reductase (GbcB) system. In addition, in a few other selected Rieske enzymes (NdmA, CntA, and GbcA), and a flavin-dependent enzyme (iodotyrosine deiodinase, IYD), the eosin Y/sodium sulfite photo-reduction pair could also serve as the NAD(P)H-reductase surrogate to support catalysis, which implies the potential applicability of this photo-reduction system to other redox enzymes.

Keywords: N-demethylation; O2 delivery; Rieske-type oxygenase; eosin Y and sulfite; flow-chemistry; photo-biocatalysis.

Figure 1.. Stc2-catalysis using a [Ru(bpy)₃]²⁺-ascorbate pair as a photo-reduction system.

a), Scheme of Stc2-catalyzed N-demethylation reaction with [Ru(bpy)₃]²⁺ as the photo-sensitizer (PS). b), Stc2 reduction time-course monitored by UV-visible spectroscopy. The control used is the [Ru(bpy)₃]²⁺ solution. c), EPR spectrum of reduced Stc2, which contains two species, A and B (red traces). Experimental data (in blue) are overlaid with simulation (black trace). The insert is the full EPR spectrum, showing no other species. d), EPR spectrum of NO treated reduced Stc2. The features near g = 4 correspond to {S = 3/2} species, with E/D = 0.015. The signal at g = 2.04 is DNIC, and the g = 1.92 and 1.75 signals are from [2Fe-2S]⁺. e), ¹H-NMR spectra of Stc2-catalyzed stachydrine N-demethylation reaction [N-methyl group signals of the substrate (1) and product (2) in ¹H-NMR spectrum], under single turnover condition (trace i); under dark (trace ii); in the absence of Stc2 (trace iii); and under multiple turnover conditions (trace iv) using [Ru(bpy)₃]²⁺ as the photo-sensitizer and ascorbate as the sacrficial reagent.

Figure 2.. ¹H-NMR Screening of the Stc2 photo-reduction system components under multiple turnover conditions.

Photo sensitizer, sacrificial reagent, and electron mediators were screened in a sequential manner. First, photo-sensitizers were screened using sodium ascorbate as a sacrificial reagent in Tris-buffer (e.g., zinc porphyrin in trace i, eosin Y in trace ii, [Ru(bpy)₃]²⁺ in trace iii). Second, with eosin Y as the photosensitizer, sacrificial reagents were screened (e.g., Na₂SO₃ in trace iv; Na₂S₂O₄ in v; and formic acid in vi). Third, using eosin Y/Na₂SO₃ pair, additional electron mediators were screened (e.g., titanium dioxide in trace vii; CdSe quantum dots in viii; and methyl viologen in ix). More complete list is in Table S1.

Figure 3.. Eosin Y/sodium sulfite photo-reduction system for Stc2 reaction.

a), Scheme of Stc2-catalyzed N- demethylation reaction with eosin Y as the photo-sensitizer and sulfite as the sacrificial reagent. b), Time course of Stc2 reduction under various eosin concentrations. The reaction mixture contained 2 mM stachydrine, 100 μM Stc2, 0.15 – 0.6 μM of eosin Y, and 20 mM sodium sulfite in 20 mM Tris, pH 8.0 buffer. c), Stc2 reduction time-course monitored by UV-visible spectroscopy. The UV spectra were collected at 0 min (red trace) and 40 min (dark blue trace). The same mixture without Stc2 is set up as control (black trace, with 0.3 μM of eosin Y) to show the change of UV-visible spectra during the photoreduction process.

Figure 4.. Further optimization of Stc2 photo-biocatalysis and a comparative characterization between the Stc2-photo-biocatalysis and the Stc2-NAD(P)H-reductase system.

a), Flow-chemistry setting in Stc2-catalysis by separating the photo-reduction half-reaction and the oxidation half-reaction. To address the O₂ delivery challenge in flow-chemistry, chlorite dismutase (CLD) mediated in situ O₂ production was further introduced into Stc2-catalysis. b), a representative ¹H-NMR spectrum [the methyl group signals for substrate (1) and product (2) in ¹H-NMR] of Stc2 reaction under the multiple turnover reaction condition using eosin Y/Na₂SO₃ photo-reduction system (trace i) in batch reaction; under an optimized eosin Y concentration flow-chemistry setting, which further improved the Stc2 turnover number to ~65-70 (trace ii); and Stc2 reaction using GbcB as the reductase and NADH as the reductant (trace iii).

Figure 5.. Additional examples of eosin Y/sodium sulfite pair supported photo-biocatalytic transformations.

a), Reaction scheme and HPLC profile of NdmA reaction, under multiple turnover condition (trace i), a control without protein (trace ii) and the 2^nd control without substrate (trace iii). b), Reaction scheme and ¹H-NMR analysis of CntA reaction under single turnover condition (trace i) and a control without protein (trace ii) [methyl signals for the substrate (5) and the product (6) in ¹H-NMR spectrum]. c), Reaction scheme and ¹H-NMR analysis of GbcA reaction [methyl signals for the substrate (7) and the product (8) in ¹H-NMR spectrum]. d), Reaction scheme and ¹H-NMR analysis of IYD reaction (aromatic hydrogen signals for the substrate (9) and product (10)].

Scheme 1.. The Stc2-catalyzed stachydrine N-demethylation reaction.

a), The Stc operon from S. meliloti 1021 and the proposed Stc2-catalyzed N-demethylation reaction using its biological reductases Stc3 and Stc4. b), Stc2-catalyzed N-demethylation reaction using a photo-reduction system with the help of photo-sensitizers (PS). c), The overall structure of Stc2 trimer. d), Stc2 active site, including its mononuclear non-heme iron site, the Rieske-type [2Fe-2S]²⁺ cluster on an adjacent subunit, and Glu201 used to link them.