Photo-reduction facilitated stachydrine oxidative N-demethylation reaction: A case study of Rieske non-heme iron oxygenase Stc2 from Sinorhizobium meliloti

Abstract

Rieske-type non-heme iron oxygenases (ROs) are an important family of non-heme iron enzymes. They catalyze a diverse range of transformations in secondary metabolite biosynthesis and xenobiotic bioremediation. ROs typically shuttle electrons from NAD(P)H to the oxygenase component via reductase component(s). This chapter describes our recent biochemical characterization of stachydrine demethylase Stc2 from Sinorhizobium meliloti. In this work, the eosin Y/sodium sulfite pair serves as the photoreduction system to replace the NAD(P)H-reductase system. We describe Stc2 protein purification and quality control details as well as a flow-chemistry to separate the photo-reduction half-reaction and the oxidation half-reaction. Our study demonstrates that the eosin Y/sodium sulfite photo-reduction pair is a NAD(P)H-reductase surrogate for Stc2-catalysis in a flow-chemistry setting. Experimental protocols used in this light-driven Stc2 catalysis are likely to be applicable as a photoreduction system for other redox enzymes.

1. Introduction

Rieske-type non-heme iron oxygenases (ROs) are an important family of non-heme iron enzymes. ROs catalyze a diverse range of transformations in the biosynthesis of secondary metabolites and in xenobiotics bioremediation (Barry & Challis, 2013; Perry, de Los Santos, Alkhalaf, & Challis, 2018). ROs showcase Nature’s strategy in site-selective C-H bond functionalization reactions (Liu, Knapp, Jo, Dill, & Bridwell-Rabb, 2022), including cis-dihydroxylation (Jeffrey et al., 1975; Karlsson et al., 2003; Zhu et al., 2023), N-dealkylation, N-demethylation (Daughtry et al., 2012; Hu et al., 2022; Summers et al., 2012), C-hydroxylation (Fei, Yin, Zhang, & Zabriskie, 2007; Li et al., 2011), C–S bond cleavage (Higgins, Davey, Trickett, Kelly, & Murrell, 1996; Higgins, De Marco, & Murrell, 1997), C–C bond desaturation (Julien, Tian, Reid, & Reeves, 2006), oxidative carbocyclization and heterocyclization (Sydor et al., 2011; Withall, Haynes, & Challis, 2015). In RO-catalyzed oxidation reactions, molecular oxygen (O₂) reduction needs four electrons and two of the electrons come in the form of a reduced nicotinamide adenine dinucleotide cofactor (NAD(P)H). A reductase alone or a reductase/ferredoxin pair is responsible for delivering electrons from NAD(P)H to the catalytic oxygenase component of the enzyme (Runda, de Kok, & Schmidt, 2023). In ROs, a tight coupling between O₂ consumption with substrate oxidation is important. Substrate binding induces changes in the non-heme iron electronic structure, and such alterations play key roles in controlling the iron-center activity (Liu, Tian, et al., 2022; Tian, Garcia, Donnan, & Bridwell-Rabb, 2023).

Stachydrine (1, N,N-dimethylproline) can serve as the sole nitrogen and carbon source for the soil bacterium Sinorhizobium meliloti 1021 (Phillips et al., 1998). Stachydrine catabolism depends on the stachydrine operon (stc), in which stc2 encodes a Rieske oxygenase and the encoded Stc3/Stc4 proteins serve as the Stc2 reductase pair (Scheme 1) (Burnet et al., 2000; Phillips et al., 1998). In many other Rieske-type oxygenases, the reductase and ferredoxin are fused into one protein (Kweon et al., 2008). Stachydrine demethylase (Stc2) catalyzes the first N-demethylation step of stachydrine to produce monomethylproline as the product. Stc2 is one of the structurally characterized ROs (Daughtry et al., 2012; Friemann et al., 2009; Hu et al., 2022), and is a homotrimer, which possesses a conserved mononuclear iron site and a Rieske [2Fe-2S] cluster (Daughtry et al., 2012). This Rieske [2Fe-2S] cluster is part of the electron-transfer chain, which shuttles electrons from NAD(P)H/reductase to the mononuclear iron site for O₂ activation and oxidative N-demethylation reaction.

Scheme 1.

Stc2-catalyzed N-demethylation reaction using a photo-reduction system.

The application of oxygenases in bioremediation or for generating value-added products is attracting increased interest (Dunham & Arnold, 2020; Gibson & Parales, 2000; Hu et al., 2022; Perry et al., 2018; Pyser, Chakrabarty, Romero, & Narayan, 2021; Sun et al., 2023; Wu, Snajdrova, Moore, Baldenius, & Bornscheuer, 2021; Xu et al., 2024; Zwick & Renata, 2020). Most studies on ROs employ an NAD(P)H-dependent reductase pair (Kweon et al., 2008) To apply ROs in biocatalysis, three issues need to be addressed: (1) enzymes must be obtained with a sufficient level of activity, (2) appropriate reductase or reductase pairs must be acquired to achieve an optimal enzymatic performance, and (3) the dependence on the expensive co-substrate NAD(P)H must be bypassed (Hu et al., 2022; Immanuel, Sivasubramanian, Gul, & Dar, 2020). For cytochrome P450 enzymes or flavin-dependent oxygenases, a few methods have been explored, including regenerating NAD(P)H by an enzymatic system (Uppada, Bhaduri, & Noronha, 2014), reducing NADP⁺ electrochemically or photochemically (Immanuel et al., 2020; Ozgen, Runda, & Schmidt, 2021), using a photo-reduction approach with a photo-sensitizer (PS) to regenerate NAD(P)H (Park et al., 2015), and delivering electrons to reduce iron-sulfur clusters with photo-activated NAD(P)H (Shanmugam, Quareshy, Cameron, Bugg, & Chen, 2021).

In this chapter, we describe the eosin Y/sodium sulfite photo-reduction system as a replacement for the NAD(P)H-reductase pair in the Stc2-catalyzed stachydrine N-demethylation reaction (Scheme 1) (Hu et al., 2022). In addition, using flow-chemistry to separate the reduction half-reaction and oxidation half-reaction, we achieve a performance comparable to that of the Stc2-NAD(P)H-reductase enzymatic system with the Stc2-eosin Y/sodium sulfite photo-biocatalytic system (Hu et al., 2022). The procedures developed in this study are likely be applicable to other oxidoreductase systems.

2. Heterologous expression and purification of Stc2

Key resources table.

Reagent or resource	Source	Identifier
Bacterial strains
Escherichia coli BL21-pDB1281	Carrying pDB1281 plasmid for iron-sulfur cluster maturation (Daughtry et al., 2012)
Chemicals
D-desthiobiotin	Sigma-Aldrich	D1411
Strep-Tactin® Sepharose® resin	IBA life sciences	2-1201-025
Lysozyme	Sigma-Aldrich	L4919-5G
Bradford reagent	Sigma-Aldrich	B6916-500 mL
LB media	Thermo Fisher Scientific	BP1426-2
Tris(hydroxymethyl)methylamine	Thermo Fisher Scientific	10103203
Ammonium bicarbonate (NH4HCO3)	Sigma-Aldrich	A6141-500G
Hydrochloric acid (HCl)	Thermo Fisher Scientific	A144-212
Ethylenediaminetetraacetic acid (EDTA)	Thermo Fisher Scientific	A15161.0B
Sodium chloride (NaCl)	Thermo Fisher Scientific	S271-3
Ampicillin sodium	GoldBio	A-301-5
Glycerol	Thermo Fisher Scientific	A 16205.0F
Dimethyl sulfoxide (DMSO)	Thermo Fisher Scientific	J66650. AK
30% (w/v) Acrylamide/bis-acrylamide solution (37.5:1)	Merck	A3699-100 mL
Ammonium persulfate	Thermo Fisher Scientific	17874
Coomassie protein assay reagent	Thermo Fisher Scientific	PI23238
Coomassie brilliant blue R-250	Thermo Fisher Scientific	11876744
Ethanoic acid	Merck	8187552500
2-Mercaptoethanol	Thermo Fisher Scientific	J66742.30
Methanol	Merck	20864.320
3-(N-Morpholino) propanesulfonic acid	Merck	M3183-100G
Tetramethylethylenediamine (TEMED)	Thermo Fisher Scientific	17919
Precision Plus Protein™ all blue prestained protein standards (10–250 kDa)	Bio-Rad	161-0373
2-[4′-Hydroxy-benzeneazo] benzoic acid (HABA)	Sigma-Aldrich	H5126-5G
Anhydrotetracycline hydrochloride (AHT)	Sigma-Aldrich	PHR3175-50MG
Ammonium peroxydisulfate (APS)	Sigma-Aldrich	A3678
Sodium dodecyl sulfate (SDS)	Sigma-Aldrich	L3771
Recombinant DNA
IBA-pASK5plus plasmid encoding Stc2	This study	None

3. Materials and equipment

3.1. Equipment

• VWR 1525 Digital Incubator.

• C25KC Incubator shaker.

• Beckman Coulter Avanti JXN-26.

• Coy Lab’s Vinyl Anaerobic Chamber.

• Fisher Scientific accuSpin Micro 17.

• Fisherbrand^™ Model 705 Sonic Dismembrator.

• Agilent Cary 60 UV-Vis Spectrophotometer.

• Electrophoresis Cells, Power supplies for PAGE applications.

• Chemglass Life Sciences Airfree Vacuum System, Welch 8907 Vacuum Pump.

• Mettler-Toledo FiveEasy pH meter F20-Std-Kit.

• Milli-Q^® 7000 Ultrapure Water Systems.

• P10, P200, and P1000 Eppendorf adjustable volume pipettes, and corresponding tips.

• Digital thermometer.

• 200 and 50 mL Amicon^® Stirred Cells.

3.2. Solutions and consumables

• Miller Luria-Bertani (LB) broth (autoclaved).

• Miller Luria-Bertani (LB) media containing 2% (w/v) agar (autoclaved).

• 100 mg/mL ampicillin (filter sterilized) (1000× stock).

• Anhydrotetracycline hydrochloride in DMSO (filter sterilized) (25 mg/mL).

• Tris-HCl buffer pH 8.0, 1 M (100×).

• Sodium chloride 5 M (100×).

• Lysis buffer 100 mM Tris-HCl (pH 8.0), 50 mM NaCl, 10% glycerol.

• d-Desthiobiotin in lysis buffer (50 mM)

• Ammonium bicarbonate 1 M (100×)

• IBA-pASK5plus plasmid containing Stc2 coding sequence (10 μg/mL) in sterile TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0).

• Nalgene^™ Oak Ridge High-Speed PPCO Centrifuge Tubes.

• 50 mL Falcon tubes, 15 mL tubes (bought sterile).

• 10 and 25 mL serological pipettes (bought sterile).

• P10, P200 and P1000 pipette tips, 1.5 mL Eppendorf tubes (autoclaved).

• pH meter calibration solutions at pH 4.0, 7.0, and 10.0.

4. Step-by-step procedure details

All solutions were prepared using 18.2 MΩ/cm Milli-Q water (Millipore). All consumables were sterilized by autoclaving at 121 °C for 20 min and 0.1 MPa (15 pounds per square inch, PSI) or in a commercial sterilized package. Working stock solutions were prepared freshly each day before use.

4.1. Escherichia coli starter culture

Timing: Day 1 to morning of day 2.

1. Using aseptic technique, the E. coli BL21-pDB1281 strain carrying IBA-pASK5plus_Stc2 construct was streaked from a frozen glycerol stock (−80 °C) and inoculated on a LB plate supplemented with 100 μg/mL ampicillin and 50 μg/mL kanamycin (10 mL). The plate was incubated in a 37 °C incubator overnight.

2. In the morning of day 2, colonies were picked using a sterile P200 tip to inoculate 100 mL of LB media supplemented with the 100 μg/mL ampicillin and 50 μg/mL kanamycin in a 250 mL Erlenmeyer flask. The culture was incubated at 37 °C and 250 rpm for ~8 h, serving as a seed culture.

4.2. Stc2 protein overexpression

Timing: Day 2 to 3.

• 3.The seed culture was diluted in a 1:100 ratio into 4 × 2 L of LB media, supplemented with 100 μg/mL ampicillin, 50 μg/mL kanamycin, 0.1 mM ferrous ammonium sulfate, and 1 mM cysteine. This step required the use of 4 L Erlenmeyer flasks. The cultures were incubated at 37 °C and 180 rpm. Cell growth was monitored by measuring the optical density at 600 nm (OD600).
• 4.After ~2 h, when the OD600 reached ~0.2–0.4, the temperature of the culture was lowered to room temperature and a few mL of the culture were collected as the pre-induction sample and labeled as [PRE] sample. The Fe-S cluster maturation operon carried in pDB1281 was induced by L-arabinose at a 0.2 g/L final concentration.
• 5.After L-arabinose induction, cell growth was continued at 25 °C and 180 rpm for an additional ~2 h to reach OD600 of 0.6~0.8. Stc2 protein overexpression was induced by the addition of AHT to a final concentration of 500 μg/L. After AHT-mediated induction, the culture was left at 25 °C and 180 rpm for an additional 12~14 h. A few mL of cell culture was collected and labeled as [POST] sample.
• 6.Cells were harvested by centrifugation (Beckman Coulter Avanti JXN-26, JLA 8.1 rotor, 5000 rpm, 20 min, 4 °C) and stored at −80 °C before use. The typical yield is ~3.5 g of wet cells per liter of culture.
• 7.On this day, all buffers to be used for protein purification were prepared anaerobically and stored in the Coy-chamber. All other needed materials, including columns, tubes, and other consumables were moved to the Coy chamber.

4.3. Anaerobic Stc2 protein purification (Daughtry et al., 2012)

Timing: Day 4.

• 8.Frozen cell pellets (~10 g) were transferred into the Coy-chamber and left at room temperature for 20 min to be thawed. They were then resuspended using the anaerobic lysis buffer (~40 mL). Lysozyme (40 mg) was then added to a final concentration of 1 mg/mL to lyse the cells. The cell suspension was incubated on ice with gentle shaking for an additional 30 min.

  Items transferred to the Coy-chamber include: E. coli cells, lysozyme (50 mg), 50 and 15 mL falcon tubes, Nalgene centrifuge tubes, sonication steel beaker (100 mL), digital thermometer, one bucket of ice. Eppendorf pipette and pipette tips.

• 9.After incubation, NH₄Fe(SO₄)₂ (0.2 mM final concentration), sodium ascorbate (0.4 mM final concentration), and DTT (1 mM final concentration) were added to reconstitute Stc2 with Fe²⁺ under a reducing environment. Cells were then disrupted by sonication (10 cycles: 8 s on/2 min off, amplitude: 80%). The temperature of the cell lysate mixture was carefully monitored to ensure that it is below 10 °C to avoid protein denaturation. The cell disruption process was monitored by measuring the absorbance at 260 nm after the cell lysate supernatant was diluted by 1000-fold. After all cells were disrupted, the resulting cell lysate was clarified by centrifugation (Beckman JA-20 rotor, 19,500 rpm, 45 min, 4 °C) and the supernatant was collected. Approximately 20 μL of the cell lysate were collected and labeled as the [CL] sample for subsequent SDS-PAGE analysis.

  Note: To protect the Step-Tactin resin, the supernatant after the first centrifugation step was collected and centrifuged again (Beckman JA-20 rotor, 19,500 rpm, 45 min, 4 °C) to obtain a clear cell lysate solution.

• 10.To ensure that the O₂ level in the Coy chamber reamins low, when material is transferred into the Coy-chamber, the chamber is re-equilibrated for at least 20 min before conducting any subsequent experiments.

  Transferred items include: tubes after centrifugation, Bradford solution (~2 mL), d-desthiobiotin and NH₄HCO₃ powder, Strep-Tactin^® Sepharose^® resin (~15 mL), columns, ice, and ice buckets.

• 11.After the Strep-Tactin^® Sepharose^® resin was transferred into the Coy chamber, it was pre-conditioned by washing with at least 100 mL of ice-cold anaerobic lysis buffer (~6-fold resin volume). The Strep-Tactin^® Sepharose^® resin in the anaerobic lysis buffer was kept in the Coy-chamber for at least one more hour before using it in the next step. The supernatant (~20 μL) was collected and labeled as [SP] and for subsequent SDS-PAGE analysis. Cell pellets were resuspended in 50 mL lysis buffer and ~20 μL of the resuspension was collected and labeled as [CP] and for subsequent SDS-PAGE analysis. The cell lysate supernatant was mixed with 15 mL Strep-tag resin and incubated on ice for 40 min. Every 5 min, the resin was resuspended by gentle agitation to ensure well mixing between the resin and the cell lysate.
• 12.The supernatant/resin suspension was loaded onto a column. Flow through (~20 μL) was collected and labeled as [FT] for subsequent SDS-PAGE analysis. After draining the supernatant, the resin was washed by anaerobic lysis buffer (105 mL, ~7-fold resin volume). The washing process was carefully monitored by collecting the UV–visible spectra of the flow through. The washing step was stopped when the OD₂₈₀ was lower than 0.1. At the end of the washing steps, the first 3 resin volumes of the washes and the last 4 resin volumes of the washing solutions were combined. ~20 μL of each of the combined washing solution was collected and labeled as [W1] and [W2] separately, for subsequent SDS-PAGE analysis.

  Note: The washing process was stopped when the absorbance at 280 nm reached 0.1, or the ratio between the absorbance at 280 nm (A_{280 nm}) and 260 nm (A_{260 nm}) was higher than 1.5 (Fig. 1).

• 13.The target Stc2 protein was eluted from the resin using two rounds of elution buffer I (0.5 resin volume of lysis buffer supplemented with 5 mM d-desthiobiotin each time), then switched to 2–3 rounds of elution buffer II (0.5 resin volume of lysis buffer supplemented with 2.5 mM d-desthiobiotin each time). After elution, all elution fractions were combined. Approximately 20 μL was collected from the combined elution solutions and labeled as [E] for SDS-PAGE analysis (Fig. 2). All samples were stored on ice when purification was analyzed by SDS-PAGE analysis.

  Note:

  1. Desthiobiotin solution preparation: the solubility of the acidic form of biotin from Sigma is low. To prepare a 50 mM d-desthiobiotin stock solution, d-desthiobiotin (54 mg) from Sigma was dissolved using 0.5 mL of 1 M NH₄HCO₃ solution, and the final volume was adjusted to 5 mL using lysis buffer.

  2. The first two rounds of elution were carried out using 5 mM d-desthiobiotin-containing elution buffer, and the subsequent elution steps were conducted using the 2.5 mM d-desthiobiotin-containing elution buffer.

  3. For each round of elution, 0.5 resin volume of ice-cold elution buffer was used to resuspend the resin and the mixture was incubated on ice for 10 min before elution. At the end of each elution, a small aliquot (~10 μL) was collected for the Bradford Assay to estimate the protein concentration. This assay provides information on whether the elution process is done. These samples were also analyzed by SDS-PAGE to further assess the degree of purity of the eluted protein.

  4. Strep-Tactin^® Sepharose^® resin regeneration: Wash the Strep-Tactin^® Sepharose^® resin from step 13 using the wash buffer (100 mM Tris-HCl buffer, 50 mM sodium chloride, pH 8.0) for 10× resin volume. The Strep-Tactin^® Sepharose^® resin was washed with 3 resin volumes of the regeneration buffer (100 mM Tris-HCl buffer, pH 8.0, 150 mM sodium chloride, 1 mM HABA, 1 mM EDTA, pH 8.0) first. Then the resin was incubated with 1 resin volume of regeneration buffer (100 mM Tris-HCl buffer, pH 8.0, 150 mM sodium chloride, 1 mM HABA, 1 mM EDTA, pH 8.0) for 15 min. After drainage, the regeneration process was repeated three more times. After resin regeneration by the HABA solution, the excess HABA was removed by washing the resin with washing buffer (100 mM Tris-HCl buffer, 50 mM sodium chloride, pH 8.0). The regenerated resin was stored at 4 °C.

Fig. 1.

UV–visible spectra of some of the washing step samples in Stc2 purification. Wash 4: sample from washing the resin with the 4th resin volume buffer. Wash 5: sample from washing the resin with the 5th resin volume buffer. Wash 6: sample from washing the resin with the 6th resin volume buffer.

Fig. 2.

SDS-PAGE analysis of Stc2 purification samples. CL, cell lysate; CP, cell pellet suspension; SP, supernatant; FT, flow through after resin treatment; W1, combined wash I solution; W2, combined wash II solution; E, eluted Stc2 protein by D-desthiobiotin solution; M, protein molecular weight marker.

4.4. Concentrating the eluted Stc2 in the COY chamber

Timing: Day 4.

• 14.Samples collected in steps 4–13 were analyzed on a 12% SDS-PAGE gel (Table 1).

  SDS-PAGE samples were prepared in a proper dilution with 5× loading dye (10% SDS, 500 mM DTT, 50% Glycerol, 500 mM Tris-HCl and 0.05% bromophenol blue dye) in a 1.5 mL Eppendorf tube. (CL, CP and SP samples were diluted 10 times; FT and all wash (W) samples were diluted 3-fold; Elution samples (E) samples were diluted 4-fold). 4 μL of each sample (~1–5 μg of Stc2 protein) were used for SDS-PAGE analysis using Tris/glycine/SDS running buffer (Gibbins, 2004). Gels were stained using Coomassie Brilliant Blue R-250 stain (Fig. 2) (Neuhoff, Arold, Taube, & Ehrhardt, 1988).

• 15.Based on the SDS-PAGE results, proper elution fractions were combined and concentrated using 200 mL Amicon^® Stirred Cell with EMD Millipore ultrafiltration disc membranes (10 kDa, 76 mm diameter). When the protein solution volume was ~10 mL, it was transferred to a 50 mL Amicon^® Stirred Cell with ultrafiltration disc membranes (10 kDa, 43 mm diameter) and proteins were concentrated to ~100 mg/mL (the final volume is ~2 mL). The concentrated Stc2 was aliquoted and stored in −80 °C.

  Note:

  In general, if the washing process is carefully monitored, there should be only minor contaminating bands in the elution factions. The elution fractions can be combined directly for biochemical and spectroscopic characterizations.

Table 1.

Components for casting SDS-PAGE gels.

Reagent	Volume for 12% resolving gel	Volume for 4% stacking gel
40% (w/v) acrylamide stock solution (37.5:1)	1.5 mL	0.24 mL
Deionized water	1.5 mL	1.43 mL
1 M Tris-HCl (pH8.8)	1.88 mL	0
1 M Tris-HCl (pH6.8)	0	0.25 mL
10% (w/v) SDS	50 μL	20 μL
10% (w/v) APS (100 mg/mL)	50 μL	20 μL
TEMED	5 μL	2 μL

5. Expected outcomes, advantages, and disadvantages

The host cell E. coli BL21-pDB1281-pASK-IBA5plus system was used for Stc2 protein production for a few reasons. First of all, pDB1281 has the iron-sulfur maturation system, which will greatly improve the iron-sulfur cluster content in Stc2 protein (Daughtry et al., 2012; Xiao, Zhao, & Liu, 2008) Secondly, pASK-IBA3plus protein expression system can be induced by anhydrotetracycline (AHT, order no.: PHR3175) at a final concentration of 500 μg/L and it is compatible with the induction system used for iron-sulfur cluster maturation system. Lastly, proteins can be eluted with 2.5 mM of d-desthiobiotin solution from the Strep-Tactin resin because d-desthiobiotin does not interfere with downstream enzymatic reactions. This statement means that the eluted protein can be used for biochemical assays directly, which gives a great chance of obtaining high quality proteins.

The two cofactors in ROs, a Rieske [2Fe-2S] cluster and the mononuclear iron center, are susceptible to oxidative damage (Daughtry et al., 2012; Hu et al., 2022). Due to this reason, all of our proteins were purified under anaerobic conditions in a Coy chamber. Besides preparing anaerobic buffers, all materials were transferred into the Coy chamber ahead of the time and were given enough equilibrating time to ensure that O₂ levels were low. The Coy chamber was maintained in the N₂/H₂ atmosphere with O₂ concentration lower than 5 ppm. In our studies, Stc2 protein was also concentrated in the Coy chamber after purification.

It is also important to monitor every step of the purification process because different proteins may have different expression levels and their binding affinity to the resin might be slightly different.

6. Optimization and troubleshooting

The above methods are robust and work well for several of our nonheme iron enzymes (Cheng et al., 2022; Wang et al., 2023; Zhu et al., 2022). In some cases, in order to produce the target protein in a high yield, fresh transformation of the protein expression plasmid into the E. coli BL21-pDB1281 competent cell was essential. Stc2 protein has two cofactors: the mononuclear iron site and the [2Fe-2S] cluster. To increase the holoenzyme yield, besides the incorporation of the Fe-S cluster maturation system, the culture medium was also supplemented with iron salt and cysteine. Because both the [2Fe-2S] cluster and the mononuclear iron site in Stc2 protein can be oxidatively damaged, the purification was conducted under an anaerobic environment. In addition, during the cell lysate process, extra reductants (e.g., DTT and ascorbate) were included to maintain a reducing environment. In our hands, when the purification was conducted under anaerobic conditions, high quality holo-Stc2 was obtained without the need of an additional reconstitution step.

7. Safety considerations and standards

Anything encountering cells, cell lysate, or protein solutions were decontaminated using the appropriate procedures for biohazards. Eye protection and appropriate Personal Protective Equipment (PPE) should be worn (earmuffs, safety glasses, gloves and laboratory coat when handling hazardous chemicals). Chemical waste should be disposed of through approved routes. Acrylamide and bis(acrylamide) are toxic if swallowed, inhaled, or absorbed through the skin and should be handled properly. The Coy-chamber is maintained in 95:5 mixture of N₂/H₂ and the chamber should be placed in a room with a well-functioning ventilation system.

8. Stc2 characterization

Key resources table.

Reagent or resource	Source	Identifier
Chemicals
Iron standard for AAS	Thermo Fisher Scientific	SI124-100
Nitric acid	Sigma-Aldrich	438073-500 mL
Zinc acetate	Sigma-Aldrich	1724703
Sodium hydroxide (NaOH)	Sigma-Aldrich	S5881-1KG
N, N-dimethyl-p-phenylenediamine	Thermo Fisher Scientific	AAA1417514
Iron (III) chloride, anhydrous	Thermo Fisher Scientific	012357. A1
Hydrochloric acid (12 M)	Thermo Fisher Scientific	A144-212
Tris(hydroxymethyl)methylamine	Thermo Fisher Scientific	10103203
Bovine serum albumin standard (BSA, 2.0 mg/mL)	Thermo Fisher Scientific	23209
Bradford Reagent	Sigma-Aldrich	B6916-500 mL
Software and Algorithms
Prism	GraphPad	v.8.4.3

9. Materials and equipment

9.1. Equipment

• Coy Lab’s Vinyl Anaerobic Chamber.

• Beckman Coulter Microfuge^® 22R Centrifuge.

• Agilent 5800 ICP-OES spectrometer.

• Agilent Cary 60 UV-Vis Spectrophotometer.

• Milli-Q^® 7000 Ultrapure Water Systems.

• Scientific Industries SI-T236 Vortex-Genie 2T.

9.2. Solutions and consumables

• P10, P200 and P1000 pipette tips, 1.5 mL Eppendorf tubes (autoclaved).

• Iron Reference Standard solution (1000 ppm).

• 2% (w/w) nitric acid.

• 12% (w/v) NaOH solution.

• 0.1% (w/v) N, N-dimethyl-p-phenylenediamine solution (DMPD) in 5 M HCl.

• Deionized water.

• 1% (w/w) zinc acetate solution.

• 23 mM FeCl₃ solution in 1.2 M HCl.

• 6 M HCl solution.

• 1.2 M HCl solution.

• 10 mM Tris-HCl buffer, pH 8.0.

• 50 mL, and 15 mL Falcon tubes, 1.5 mL Eppendorf tubes (bought sterile).

10. Step-by-step details

All solutions were prepared using 18.2 MΩ/cm Milli-Q water (Millipore). Unless stated, all Stc2 related operations were conducted in the anaerobic Coy-chamber.

10.1. Stc2 protein analysis

• 16.SDS-PAGE analysis of Stc2 samples is shown in Fig. 3.
• 17.Protein standard solutions (0.05–0.5 mg/mL, Table 2) were prepared by serial dilutions of bovine serum albumin (BSA) using a lysis buffer.
• 18.Stc2 proteins in various dilutions (10×, 50×, and 100×) were prepared from the concentrated Stc2 solution by diluting it using the lysis buffer.
• 19.Bradford reagents (960 μL) was mixed with 40 μL protein solution (BSA standards or Stc2 samples). After they were thoroughly mixed, the samples were incubated at room temperature for three minutes.
• 20.The mixture samples were transferred to disposable cuvettes. The sample to be used as a blank sample was prepared by mixing 960 μL Bradford reagents with 40 μL of lysis buffer.
• 21.A standard curve was prepared by correlating the absorbance at 595 nm (A_{595 nm}) with protein concentration (μg/mL) (Fig. 4).
• 22.Stc2 protein concentration in mg/mL was calculated with the standard curve, a dilution factor, and its A595. The concentration (with the unit μM) was calculated using Stc2 molecular weight of 46 kDa.

  e.g., in one of the samples shown (Fig. 4): Stc2 concentration=C′ / 46,000×1000×1000 = [(0.64–0.1167)/1.487×100]/46,000×1000×1000 = 765 μM. (C′, concentration of protein in mg/mL).

Fig. 3.

SDS-PAGE analysis of anaerobically purified Rieske oxygenases Stc2 (final sample).

Table 2.

Standard curve of protein quantification.

[BSA] (mg/mL)	A595 nm
0.05	0.1648
0.1	0.2879
0.2	0.4183
0.3	0.5663
0.4	0.7201
0.5	0.8476

Fig. 4.

Bovine serum albumin-based standard curve using Bradford assay for Stc2 protein concentration determination. Stc2 concentration=C′/46,000 × 1000 × 1000 = [(0.64–0.1167)/1.487 × 100]/46,000 × 1000 × 1000 = 765 μM. (C′, concentration of protein in mg/mL).

10.2. Stc2 iron content quantification (Abbasi, Abbina, Gill, Bhagat, & Kizhakkedathu, 2021)

• 23.Iron standards at various concentrations (0.125, 0.25, 0.5, 0.75, 1, 1.5 and 2 ppm) were prepared by serial dilution from an iron stock solution (1000 ppm) using 2% (w/w) HNO₃ solution.
• 24.Holo-Stc2 protein should have three iron atoms per Stc2 subunit. Based on the concentration of the protein Stc2 stock solution (590 μM), the theoretical iron content should be 98.8 ppm. Therefore, the Stc2 stock solution was diluted by 50-fold using 2% (w/w) nitric acid solution to prepare 1 mL of the Stc2 solution with iron content in the 0.125–2 ppm iron standard curve range. After mixing the protein with nitric acid solution, the mixture was vortexed for 3 min
• 25.The Stc2 protein sample was then centrifuged at 15,500 rpm for 10 min to remove the protein precipitates and the supernatant was transferred to a new 1.5 mL Eppendorf tube.
• 26.The iron content in iron standards and the Stc2 samples were then analyzed using Agilent 5800 ICP-OES spectrometer by measuring the emission spectrum at 371.993 nm.

  Note: The sample uptake time was 15 s and stabilization time was 10 s. The emission spectrum at 371.993 nm was recorded for iron concentration calculation. The Agilent January 2021 Edition Microwave Plasma Atomic Emission Spectroscopy (MP-AES) Application eHandbook was used as the guide, especially the procedures of Inductively coupled plasma-atomic emission spectrometry (ICP-AES) outlined in the manual. Data analysis samples are shown in Fig. 5 and Table 3.

  Standard curve: y = 8105x + 522.7, R² = 0.9980.

  Iron concentration [Fe] = (10,020.13–522.7)/8105 = 1.17 ppm = (1.17/55.845) × 1000 μM = 20.1 μM.

  Sample dilution factor D = (18 + 982)/18 = 55.56.

  Sample protein concentration [Stc2] = 590 μM.

  Iron content of Stc2 protein [Fe]% = [Fe] × D/[Stc2] = (20.1 μM) × 55.56/590 μM = 1.89.

  Note: One molecule of Stc2 should have two irons from the Fe-S cluster and one mononuclear iron. We have tried to adjust the iron and sulfur in the culture medium. In the absence of reconstitution of cofactors in the protein purification step, this iron content is a typical number for Stc2 system Fig. 5.

Fig. 5.

Iron quantification of purified Stc2 by atomic emission spectroscopy. Iron concentration from Stc2 [Fe] = (10,020.13–522.7)/8105 = 1.17 ppm = (1.17/55.845) × 1000 μM = 20.1 μM. Sample dilution factor D = (18 + 982)/18 = 55.56. Sample protein concentration [Stc2] = 590 μM. Iron content of Stc2 protein [Fe]% = [Fe] × D/[Stc2] = (20.1 μM) × 55.56/590 μM = 1.89.

Table 3.

Iron content measurement from AES.

Iron concentration (ppm)	Average intensity	Intensity read 1	Intensity read 2	Intensity read 3	Intensity SD	Intensity %RSD
0	0	11.8	−15.02	3.22	13.69	–
0.125	1645.1	1645.51	1644.99	1644.8	0.37	0.02
0.25	2668.65	2652.23	2676.03	2677.68	14.24	0.53
0.5	4775.29	4747.14	4782.93	4795.81	25.21	0.53
0.75	6776.07	6782.26	6773.09	6772.84	5.37	0.08
1	8716.35	8627.39	8876.61	8645.06	139.07	1.6
1.5	12,725.51	12,661.05	12,778.85	12,736.62	59.68	0.47
2	16,515.91	16,456.06	16,438.24	16,653.42	119.42	0.72
Stc2 sample	10,020.13	10,096.13	10,044.24	9920.01	90.5	0.9

10.3. Quantification of labile sulfur in Stc2 based on the reaction shown in Fig. 6 (Beinert, 1983)

Fig. 6.

Reaction scheme for the reaction used for quantification of labile sulfur in purified Stc2.

• 27.20 μL of Stc2 protein stock solution was mixed with 180 μL of Tris-HCl buffer (20 mM, pH = 8.0) to make a 200 μL solution containing 90 μM of Stc2.
• 28.To the above Stc2 protein solution, 600 μL freshly prepared (or freshly diluted) 1% (w/w) zinc acetate solution was added and mixed gently.
• 29.Immediately to the above mixture, 30 μL 12% (w/w) NaOH solution was added, and the mixture was stirred gently until the schlieren of NaOH disappear. The mixture was left at room temperature in the Coy chamber for about 1 h.
• 30.Next, 150 μL of 0.1% DMPD solution was added to the mixture and it was stirred gently until only the top 2-mm layer of liquid had undissolved zinc hydroxide. Then, 30 μL of 23 mM FeCl₃ solution was added into the mixture under the surface, followed by an immediate acceleration of the stirring bar to make a homogeneous solution quickly.
• 31.After 30 min of color development at room temperature, the mixture was centrifuged at 15,500 rpm for 20 min to precipitate the protein.
• 32.The supernatant was transferred to a cuvette and the A_{670 nm}, A_{710 nm}, and A_{750 nm} were recorded. The A_{670 nm} value was used to calculate the sulfide concentration (Fig. 7) (Beinert, 1983; Hu et al., 2022).
• 33.Based on the A_{670 nm} = 0.7087 and ε_{670 nm} = 34.5 cm⁻¹•mM⁻¹ (Beinert, 1983), the value of sulfide concentration can be calculated and a sample of calculation is shown here:

  the sulfur concentration [S] = A_{670 nm}/(ε_{670 nm} × l) = 0.7087/(34.5 cm⁻¹•mM⁻¹ × 1 cm) = 20.5 μM.

  Final Stc2 concentration: [Stc2]_start/dilution factor = 90 μM/5.05 = 17.8 μM.

  Labile sulfur content = [S]/[Stc2] = (20.5 μM)/(17.8 μM) = 1.15.

  Because each Rieske iron-sulfur cluster has 2 equivalents of sulfide, the above calculation suggests that our Rieske iron-sulfur content is ~58%.

  Note:

  1. Zinc acetate solution (~10%, w/w) is stable for only a few days. It is better to prepare fresh solution.

  2. For the labile iron-sulfur containing proteins, the zinc hydroxide precipitation step takes no longer than 5 min whereas for other stable Fe-S proteins, l ~ 2 h might be required.

  3. Standardization may be performed using Na₂S•9H₂O.

Fig. 7.

UV–visible spectrum of one of the Stc2 samples used for labile sulfur quantification. The sulfur concentration [S] = A_{670 nm}/(ε_{670 nm} × l) = 0.7087/(34.5 cm⁻¹•mM⁻¹ × 1 cm) = 20.5 μM. Final Stc2 concentration: [Stc2]_start/dilution factor = 90 μM/5.05 = 17.8 μM. Labile sulfur content = [S] / [Stc2] = (20.5 μM)/(17.8 μM) = 1.15.

10.4. Optimization and troubleshooting

The above protein characterization methods are well-established methods and highly reproducible. For spectroscopic studies, protein concentration determined from amino acid analysis instead of the Bradford Assay might be needed. In addition, the Coy-chamber needs to be maintained well to ensure low O₂ levels. The O₂ detector needs to be calibrated on a regular basis.

11. Stc2 photo-reduction using eosin Y and Na₂SO₃

Reagent table.

Reagent or resource	Source	Identifier
Chemicals
Tris(hydroxymethyl)methylamine	Thermo Fisher Scientific	10103203
Sodium sulfite (Na2SO3)	Sigma-Aldrich	08981
Stachydrine	Biosyn	FS65548
Eosin Y (EY)	Sigma-Aldrich	E4009-5G
Deuterium oxide	Cambridge isotope Laboratories, Inc.	DLM-4-100
Ethyl viologen dibromide	Sigma-Aldrich	384097-1G
Chloroform	Sigma-Aldrich	650498
Hemin	Sigma-Aldrich	51280
Kanamycin	Merck	420311
Isopropyl-β-d-1-thiogalactopyrano-side (IPTG)	Merck	I5502
Ammonium sulfate	Sigma-Aldrich	7783-20-2
Glycerol	Thermo Fisher Scientific	A 16205.0F
L-ascorbate sodium	Sigma-Aldrich	A7631-100G
Software and algorithms
MestReNova	Mestrelab Research	v.14.1.0
ChemOffice	PerkinElmer	v. 20.0
Prism	GraphPad	v.8.4.3
SpinCount	The Hendrich Metalloprotein Group

12. Materials and equipment

12.1. Equipment

• Coy Lab’s Vinyl Anaerobic Chamber.

• Fisher Scientific accuSpin Micro 17.

• Agilent Cary 60 UV-Vis Spectrophotometer.

• Agilent 500 MHz VNMRS spectrometer.

• Scientific Industries SI-T236 Vortex-Genie 2T.

• Labconco FreeZone 10 L −84 °C Freeze Dryer System.

• Fisher Scientific Isotemp^™ digital hotplate stirrer.

• Light Bulb, full spectrum, 24 W (BRIM, #SOL24W)

• Screw-tight quartz cuvette (MES Supplies LLC, LS0703).

12.2. Solutions and consumables

• P10, P200, and P1000 pipette tips, 1.5 mL Eppendorf tubes (autoclaved).

• 10 mM NaOH solution.

• 20 mM Tris-HCl buffer, pH 8.0 (anaerobic preparation).

• 6 M HCl solution (5 mL HCl stock solution in 5 mL deionized water).

• 50 mM Fe(NH₄)(SO₄) solution.

• 100 mM EDTA solution, pH 8.0.

• 50 mM HEPES solution, pH 8.0.

• Deionized water.

• Glass vial, clear.

13. Step-by-step method details

All solutions were prepared using 18.2 MΩ/cm Milli-Q water (Millipore). All of the experimental operations were conducted under anaerobic conditions in a Coy chamber unless stated otherwise.

13.1. Light-driving Stc2 Fe-S cluster reduction using eosin Y and Na₂SO₃ as photosensitizer/sacrificial reagent pair

• 34.At least 20 mL of anaerobic buffer was prepared (20 mM Tris-HCl, pH = 8.0) and transferred into the Coy chamber one-day before the planned experiments.
• 35.The eosin Y solution was prepared using the anaerobic buffer. In general, the stock solution was prepared at a concentration of ~500 μM. To accurately measure the eosin Y concentration, the stock solution was diluted by 100× and the UV–visible spectrum of the diluted solution was collected (300–800 nm spectrum in Fig. 8 as an example). The eosin Y concentration could be calculated based on its extinction coefficient: ε_{525 nm} = 95 cm⁻¹∙mM⁻¹.

  Example. The eosin Y stock solution’s concentration [EY] = A_{525 nm} × D/(ε × l) = 0.7931 × 100/(95 cm⁻¹•mM⁻¹ × 1 cm) = 835 μM ([EY]: EY concentration; D: dilution factor; ε: EY extinction coefficient).

• 36.The eosin Y stock solution can be aliquoted into small fractions, frozen by liquid nitrogen, and be stored in −20 °C refrigerator in dark (covered by aluminum foil).
• 37.1 M of sodium sulfite stock solution was prepared using the anaerobic buffer (20 mM Tris-HCl, pH = 8.0).
• 38.To monitor Stc2 reduction, the photoreduction reaction was conducted in a screw-tight quartz cuvette directly in the Coy-chamber. A typical 1 mL reaction mixture contained 100 μM Stc2 enzyme, 20 mM sacrificial reagent Na₂SO₃, and 1.5 μM photosensitizer eosin Y in the anaerobic buffer (20 mM Tris-HCl, pH = 8.0).
• 39.The sealed quartz cuvette was placed on ice and photoreduction was conducted under either white light using mercury lamp or a specific wavelength with LED lamp. When white light was used, to avoid heating up the protein samples, our photo-reduction reaction was conducted on ice.
• 40.At various time points (0 and 20 min), the UV–visible spectra of the sample were collected to monitor the reducing rate (350–600 nm UV–visible spectra in Fig. 9 as samples).
• 41.The absorbance at 460 nm, which is related to Stc2 [2Fe-2S]²⁺ cluster, could be used for monitoring the extent of Stc2-reduction reaction.

Fig. 8.

UV–visible spectrum of an eosin Y for its concentration calculation. The eosin Y stock solution’s concentration [EY] = A_525nm × D / (ε × l) = 0.7931 × 100/(95 cm⁻¹•mM⁻¹ × 1 cm) = 835 μM ([EY], EY concentration; D, dilution factor; ε, EY extinction coefficient).

Fig. 9.

Time-course of Stc2 reduction monitored by UV–visible spectroscopy. The UV spectra were collected at 0 min (red trace) and 20 min (blue trace). The same mixture without Stc2 is set up as control (black trace, with 1.5 μM of eosin Y) to show the change of UV–visible spectra during the photoreduction process.

13.2. Light-driving demethylation of stachydrine using eosin Y and Na₂SO₃ under multiple turnover condition (Hu et al., 2022; Xu et al., 2024)

• 42.A stachydrine stock solution was prepared by dissolving stachydrine power using the 20 mM Tris-HCl anaerobic buffer. In general, we have an ethyl viologen solution with a known concentration. The stachydrine concentration was determined by using a known concentration of an ethyl viologen with as an internal standard. ¹H-NMR was then used to determine the stachydrine concentration. Fig. 10 shows a sample of stachydrine concentration determination used in this work.
• 43.Eosin Y concentration calculation has been discussed in prior section (Fig. 8).
• 44.Sodium sulfite stock solution (1 M) was prepared by dissolving Na₂SO₃ power in the anaerobic buffer (20 mM Tris-HCl, pH = 8.0).
• 45.To prepare O₂ saturated buffer, the anaerobic buffer (20 mL, 20 mM Tris-HCl, pH = 8.0) in a 100 mL three-neck flask was degassed under vacuum first. An O₂ balloon was then connected to the flask and the mixture was stirred at room temperature for at least 30 min. Buffer de-gassing and the subsequent O₂ purge were repeated for at least two more times to prepare O₂ saturated buffer.
• 46.A 250 μL reaction mixture in a 5 mL glass vial (Uline, WI, USA) contained 20 μM Stc2 enzyme, 2 mM stachydrine, 10 μM eosin Y, and 20 mM Na₂SO₃ in the anaerobic buffer (20 mM Tris-HCl, pH = 8.0). Then the mixture was stirred for 30 s. This process should be conducted in the Coy chamber anaerobically.
• 47.The anaerobic mixture in step 46 was then transferred out of the anaerobic coy chamber and 250 μL oxygenated buffer was immediately mixed with it to initiate the reaction.
• 48.After reaction initiation, the mixture was stirred in an open vial under white light and aerobic environment for 1 h. The reaction was conducted on ice to avoid protein denaturing due to heating by lamp.
• 49.The reaction mixture was quenched by 300 μL of chloroform and thoroughly mixed by vortexing for 2 min to inactivate the Stc2 protein.
• 50.The aqueous and chloroform layers were separated by centrifugation (15,500 rpm, 10 min).
• 51.The aqueous layer was transferred to a falcon tube, frozen by liquid nitrogen, and lyophilized.
• 52.The lyophilized sample was re-dissolved by 350 μL deuterated water. After centrifugation (15,500 rpm, 10 min), the supernatant was analyzed by ¹H NMR.
• 53.The turnover numbers were calculated based on the relative intensities of the signals for the N-methyl group in the substrate (stachydrine) vs. the product (mono-methyl proline) in a ¹H NMR spectrum (see Fig. 11 as an example).

Fig. 10.

¹H NMR spectrum of stachydrine with a known concentration of ethyl viologen for stachydrine concentration determination. The NMR samples contain 10 μL of stock stachydrine stock solution, 20 μL of 50 mM ethyl viologen and 300 μL D₂O. Each of the two the N-methyl group signals (~3.17 and ~2.97 ppm) from stachydrine contain 3 protons, and the methyl group signal (~1.54 ppm) from the ethyl viologen contains 6 protons. The concentration of the stachydrine stock solution [stachydrine] = (0.86 + 0.84)/2×(6/3)×2 × 50 mM = 170 mM.

Fig. 11.

¹H NMR assay of Stc2 reaction using the N-methyl group signals from stachydrine and mono-methylproline for quantification.

13.3. Stc2-catalysis in a flow-setting

• 54.E. coli BL21 competent cells carrying a pET41a-(+)-CLD construct for chlorite dismutase (CLD) was kindly provided by Prof. Yisong Guo. A single colony was inoculated into LB broth supplemented with 50 μg/mL kanamycin (10 mL) and incubated at 37 °C overnight. A 10 mL of seed culture was inoculated into 1 L of LB media supplemented with 90 μM hemin and 50 μg/mL kanamycin. The overexpression of CLD was induced using IPTG at a final concentration of 0.2 mM when OD600 reached 0.6~0.8.
• 55.The CLD protein was overexpressed at 20 °C for 14 h and the cells were harvested by centrifugation according to the method described previously. E. coli BL21-pET41a-CLD cells (8 g) were resuspended in 40 mL of lysis buffer (100 nM Tris-HCl, 50 mM NaCl, pH 8.0) and 40 mg lysozyme was added. Cells were disrupted as outlined in Step 9 except that no iron salt or reductants were included.
• 56.The supernatant (40 mL) was transferred into a beaker on ice, and 12.04 g ammonium sulfate crystals were added slowly with stirring to reach a level of ~50% ammonium sulfate saturation. This process took about 1 h. After all ammonium sulfate crystals were added to the solution, the mixture was stirred on ice for an additional 30 min
• 57.The CLD sample was centrifuged at 15,000 rpm for 20 min. The pellet was collected and resuspended in 5 mL of lysis buffer (100 mM Tris-HCl, 50 mM NaCl, pH 8.0), and incubated on ice for an additional 30 min to fully dissolve CLD. The partially purified CLD protein solution was quickly frozen using liquid nitrogen for future use in Stc2 reactions.
• 58.A 5 mL reaction mixture that contained 20 μM Stc2, 2 mM stachydrine, 20 mM Na₂SO₃ and 20 μM eosin Y in 20 mM Tris-HCl, pH 8.0 buffer was placed in a reservoir serving as the starting solution. The flow system was set up as shown in Fig. 12.
• 59.The reaction mixture was circulated between the two halves (reduction half by photocatalysis and oxidation half with O₂ generated from chlorite by CLD) driven by a peristaltic pump. The solution was pumped into a 3 mL PFA tube, which was illuminated by either the LED light or by the white light. The peristaltic pump used in this experiment was operated at a flow rate of 6 mL/min. Therefore, the photoreduction half-reaction time was ~30 s before exiting the PFA tube. The time of the reduction half was determined based on the reduction kinetics determined from the experiments outlined in steps 34–41 in the prior section.
• 60.O₂ was generated in situ by syringe pump delivery and mixing of two solutions: Solution A was 5 μM CLD in 20 mM Tris-HCl, pH 8.0 buffer and Solution B was 1.25 mM NaClO₂ in 20 mM Tris-HCl, pH 8.0 buffer. The CLD/chlorite ratio was 1:1200. The O₂ generating mixture was then mixed with the Stc2 photo-reduced mixture using a three-way valve as shown in the Fig. 12 setup.
• 61.The rate of O₂ delivery was achieved by controlling the rate of chlorite delivery and it varied from 7.2 to 2.0 μmol/h to match with the Stc2 circulation rate in the reaction mixture.
• 62.In some reactions, some additional eosin Y was supplemented to compensate for the loss of eosin Y due to photo-bleaching. At the end of the reaction, the mixture was lyophilized and dissolved in 1 mL D2O and analyzed by ¹H NMR (Fig. 13).

Fig. 12.

Schematic diagram of the Stc2-catalysis in flow setting with in situ O₂ generation using CLD-NaClO₂ system.

Fig. 13.

¹H NMR assay of Stc2 flow reaction using the N-methyl group signals from stachydrine and mono-methylproline for quantification.

13.4. Optimization and troubleshooting

1. When the protocol is applied to other systems, several parameters might be optimized to fit the particular system:

2. The biocatalyst is temperature dependent, and the lamp generates heat that might denature the enzyme. In our studies, we run the reaction on a water or ice bath to avoid enzyme denaturing by heat.

3. Light sources used in the photo-reduction might influence the reduction efficiency. According to the absorbance spectrum analysis conducted by EY, it has a pronounced absorbance feature at 525 nm. In our hands (Fig. 14), the Stc2 batch reaction utilizing green light (525 nm) have an observed turnover that is lower than those of white, blue (467 nm), or purple light (370 nm). When applied in other enzymatic reactions, the light source will likely need to be examined.

4. In Stc2-catalysis under a flow setting, the rate of the pumping cycle is a crucial factor. Upon doubling the circulation speed of the pump, transitioning from 3 to 6 mL/min, we observed a corresponding doubling in the reaction conversion rate, escalating from approximately 30 to about 65. This phenomenon underscores the significance of pump circulation speed in optimizing reaction efficiency. Given the inherent instability of the protein and the susceptibility of EY to photobleaching under light, modulating the pump’s circulation will be needed to achieve an optimal performance.

5. Several O₂ delivery methods have been examined in this Stc2 photo-catalytic process. Employing an O₂-saturated buffer as the O₂ source proved to be effective in initiating the reaction. However, this method also posed challenges due to the inherent limitations of O₂ concentration within the buffer. The necessity to deliver a substantial volume of O₂-containing buffer to compensate for this limitation resulted in considerable dilution of the reaction solution. Direct delivery of O₂ gas as slugs used in organic flow-chemistry was also examined (Shimizu, Kato, & Kobayashi, 2023). Unfortunately, this method did not seem to work for Stc2 reaction due to protein denaturation. To resolve the O₂ delivery issue in flow-chemistry setting, we introduced CLD-catalyzed NaClO₂ decomposition to O₂ as an in situ O₂ production method. CLD activity is a few orders of magnitude greater than that of Stc2 (Mehboob et al., 2009). To match the O₂ delivery rate with that of Stc2-catalysis, co-delivering CLD and NaClO₂ at a proper ratio led to the best Stc2-catalysis performance. When this method is applied in other systems, an optimization of the O₂ delivery rate is needed to fit the particular system of interest.

Fig. 14.

¹H NMR analysis of Stc2-catalyzed reaction under representative light source when eosin Y/sulfite serve as the photosensitizer/sacrificial reagent pair. (A) White light lamp, positive control; (B) Greem light, 525 nm wavelength; (C) Blue light, 480 nm wavelength; (D) Purple light, 370 nm wavelength. A typical 500 μL reaction in 20 mM Tirs-HCl buffer (pH 8.0) includes 20 μM Stc2 protein, 2 mM stachydrine, 10 μM eosin Y, 20 mM Na₂SO₃. The reactions were then conducted under aerobic conditions in an open vial under different light source for 1 h before quenching for analysis.

Abbreviations

ROs

Rieske-type non-heme iron oxygenases

Stc2

Rieske oxygenase subunit of stachydrine demethylase

GbcB

glycine-betaine demethylase reductase subunit

NAD(P)H

nicotinamide adenine dinucleotide (phosphate) reduced form

FAD

flavin adenine dinucleotide

NMR

nuclear magnetic resonance

EDTA

ethylenediaminetetraacetic acid

rpm

revolutions per minute

Tris

tris(hydroxymethyl)methylamine

E. coli

Escherichia coli

IPTG

isopropyl-β-D-galactopyranoside

LB media

Luria-Bertani media

APS

ammonium persulfate

SDS

sodium dodecyl sulfate

SDS-PAGE

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TEMED

tetramethylethylenediamine

UV-Vis

ultraviolet-visible spectroscopy

OD

optical density

ppm

parts per million

PS

photo-sensitizer

His

histidine

Cys

cysteine

HABA

2-[4′-hydroxy-benzeneazo] benzoic acid

AHT

anhydrotetracycline hydrochloride

DMPD

N,N-dimethyl-p-phenylenediamine solution

BSA

bovine serum albumin

Ru(bpy)₃

tris (2,2′-bipyridyl) dichlororuthenium (II) hexahydrate

DTT

dithiothreitol