Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry

1. Preparation of Anaerobic Buffer

1. Prepare 1 L of 100 mM potassium phosphate buffer, pH 7.5. Pour 250 mL of the buffer into a 500-mL Büchner flask with a stir bar.
2. Seal the flask with a rubber stopper and place it on a stir plate. Connect the short tube of the flask to a Schlenk line.
3. Degas the buffer under vacuum for 5 hours at room temperature with agitation. During this time period, perform 5 consecutive rounds of vacuum degassing and flushing with argon every hour.
4. Flush the flask with argon for 10 seconds and disconnect it from the vacuum manifold. Place the flask inside the glove box.
5. Leave the flask open in the glove box overnight with vigorous agitation.

2. Removing Oxygen from the Stopped-flow System

1. Prepare 1 L of 0.1 M sodium acetate, pH 5.0. Pour 125 mL of this buffer into a 250-mL Büchner flask and perform steps 1.2-1.4.
2. Weigh out 5 mg glucose oxidase from Aspergillus niger (181300 U/g) and 0.9 g glucose using a 1.5 mL Eppendorf tube and 50 mL Falcon conical tube, respectively. Place both containers into the glove box.
3. Dissolve glucose oxidase and glucose in 50 mL of anaerobic 0.1 M sodium acetate, pH 5.0 (final concentrations of 18.13 U/mL and 100 mM, respectively). Fill the reservoir syringes with this solution (Figure 1).
4. Ensure that the drive valves are in the “load” position and fill the drive syringes (Figure 1). Turn the F valve to the “drive” position. Empty the stop syringe by clicking on Empty in the Pro-Data control software. Push the buffer from drive syringe F through the flow circuit by manually raising the corresponding drive ram. Repeat this step ten times in total. Perform the same procedure with the other three drive syringes. Wait one hour.
5. Replace the solution of the reservoir syringes with the same buffer containing glucose oxidase and glucose. Repeat step 2.4.
6. Repeat step 2.5 and allow the solution to stand in the flow system overnight.
7. After overnight incubation, repeat step 2.5.

3. Preparation of Oxygen Saturated Buffer

1. Prepare 100 mM potassium phosphate buffer (pH 7.5) and pour 50 mL into a 50-mL vial with a stir bar. Cap the vial with a Wheaton stopper and a Wheaton aluminum seal.
2. Place the vial on ice. Immerse a long needle connected to a tank containing 100% oxygen into the solution. Insert another short needle through the stopper of the vial as a vent.
3. Bubble the solution with 100% oxygen for 1 hour with agitation.
4. First remove the short needle from the vial and wait 10 seconds before removing the long needle. Place the closed vial in the glove box.

4. Preparation of NADPH Solution

1. Weigh out 1 mg of NADPH in a 1.5 mL Eppendorf tube and place it into the glove box.
2. Dissolve the NADPH in 300 μL of anaerobic 100 mM potassium phosphate buffer, pH 7.5. Remove 30 μL of this solution from the glove box to determine the NADPH concentration with a spectrophotometer (ε_{340 nm}= 6,270 M^-1cm^-1).

5. Removal of Oxygen from the Enzyme Solution

1. Take 400 μL of enzyme stock solution from the freezer and thaw in the glove box. The enzyme stock solution (360 μM) was previously prepared in 100 mM potassium phosphate buffer (pH 7.5) containing 100 mM NaCl, and frozen in liquid nitrogen.
2. Transfer the enzyme solution into a 25-mL vial with a stir bar, cap the vial with a Wheaton stopper and a Wheaton aluminum seal and remove from the glove box.
3. Place the vial on ice and connect it to a Schlenk line by inserting a needle through the stopper of the vial.
4. Degas the enzyme solution by performing 5 consecutive rounds of vacuum degassing and flushing with argon every 20 minutes for 1 hour.
5. Flush the vial with argon for 10 seconds and disconnect it from the vacuum manifold. Place the vial on ice inside the glove box.

6. Reductive Half-reaction: Monitoring Flavin Reduction

1. Prepare the stopped-flow instrument and the Pro-Data control software for single mixing mode following the manufacturer’s protocol.
2. Turn on a circulating water bath (15 °C). Remove oxygen from the water by bubbling nitrogen through it for 20 minutes. This prevents the oxygen contamination through the flow circuit.
3. Maintain the temperature of the drive syringes and the observation cell at 15 °C by connecting the water bath housing of the stopped-flow to the circulating bath.
4. In the Control Panel window of the Pro-Data control software select Photodiode Array, External trigger (to activate stopped-flow operation), and Logarithmic scale.
5. Launch the Pro-Data viewer software and define the directory where the data files will be stored.
6. Replace the solution of reservoir syringes C and F with anaerobic 100 mM potassium phosphate buffer (pH 7.5). Turn the C and F valves to the “drive” position. Empty the stop syringe by clicking Empty in the Pro-Data control software. Push the buffer from both drive syringes through the flow circuit by manually raising the corresponding drive ram five times in total (click Empty before each movement of the ram).
7. In order to ensure that glucose oxidase has been removed completely from the flow circuit, perform step 6.6 four times in total.
8. Click on Baseline in the Pro-Data control software.
9. Mix 100 μL of enzyme stock solution with 1100 μL of anaerobic 100 mM potassium phosphate buffer (final concentration after mixing in the stopped flow of 15 μM).
10. Replace the solution of reservoir syringe F with the enzyme solution. Turn the F valve to the “drive” position. Empty the stop syringe by clicking Empty in the Pro-Data control software. Push the enzyme solution from drive syringe F through the flow circuit by manually raising the corresponding drive ram. Perform this three times in total. In the Pro-Data control software, set the acquisition time to 60 s.
11. Ensure that both drive syringes are filled with their corresponding solutions and the drive rams are in contact with the drive syringe pistons. Turn both valves to the “drive” position and click Acquire in the Pro-Data control software to perform the drive. Repeat this step two times more to obtain the data in triplicate. This yields spectra of the oxidized form of the enzyme.
12. Using the A_{452 nm} value obtained from the drives, determine the enzyme concentration before mixing (ε_{452 nm}= 13,700 M^-1cm^-1) and prepare 4 mL of a NADPH solution 1.5-fold more concentrated.
13. Replace the solution of reservoir syringe C with the NADPH solution and empty and refill the stop syringe three times as described in 6.10.
14. Repeat step 6.11. This yields spectra during the reduction of the enzyme.

7. Oxidative Half-reaction: Monitoring Flavin Oxidation

1. Prepare the stopped-flow instrument and the Pro-Data control software for double mixing mode following the manufacturer’s protocol.
2. Repeat steps 6.2-6.5.
3. Replace the solution in the four reservoir syringes with anaerobic 100 mM potassium phosphate buffer (pH 7.5). Turn the F valve to the “drive” position. Empty the stop syringe by clicking Empty in the Pro-Data control software. Push the buffer from the drive syringe F through the flow circuit by manually raising the corresponding drive ram. Perform this step five times in total with each drive syringe.
4. Perform step 7.3 four times in total to completely remove the contents from the flow circuit.
5. Click Baseline in the Pro-Data control software.
6. Mix 200 μL of enzyme stock solution with 1000 μL of anaerobic 100 mM potassium phosphate buffer (final concentration after mixing in the stopped flow of 15 μM).
7. Replace the solution of reservoir syringe A with the enzyme solution. Turn the A valve to the “drive” position. Empty the stop syringe by clicking Empty in the Pro-Data control software. Push the enzyme solution from drive syringe A through the flow circuit by manually raising the corresponding drive ram. Perform this step three times in total. In the Pro-Data control software, set the delay time to 0.5 s and acquisition time to 60 s.
8. Ensure that all drive syringes are filled with their corresponding solutions and the drive rams are in contact with the drive syringe pistons. Turn all valves to the “drive” position and click Acquire in the Pro-Data control software to perform the drive. Repeat this step two more times to obtain the data in triplicate. This yields spectra of the oxidized form of the enzyme.
9. Replace the solution of reservoir syringe B with an NADPH solution 1.5-fold more concentrated than the enzyme solution in syringe A. Turn the B valve to the “drive” position and empty and refill the stop syringe three times as described in 7.7.
10. Replace the solution of reservoir syringe C with oxygenated buffer. Turn the C valve to the “drive” position and empty and refill the stop syringe three times as described in 7.7. In the Pro-Data control software, set the delay and acquisition time at 15 and 900 s, respectively.
11. Repeat step 7.8. This yields spectra during the reoxidation of the fully reduced enzyme.

8. Data Analysis

1. Launch the Pro-Data converter software. Click on the Options icon and select Save in ProDataCSV.
2. Open the directory where the data files were stored. Drag the files to the APL Pro-Data converter window. The data will be saved automatically as CSV format files in the same directory.
3. Open the CSV format files using Microsoft Excel (Microsoft, Redmond, WA, USA). Normalize the baseline of the stopped-flow traces by subtracting the corresponding absorption at 700 nm.
4. Analyze the corrected traces using KaleidaGraph (Synergy Software, Reading, PA) by fitting the plot of the absorbance at the corresponding maximum versus time to the appropriate exponential function. As shown in Figure 2B, the absorbance at 452 nm was plotted as a function of time in order to determine the rate of flavin reduction after mixing the enzyme and NADPH in the stopped-flow instrument. In this case, the best fit of the data was obtained using a double exponential equation and rates of 0.65 and 0.23 s^-1 were calculated for the first and second phase of the reduction, respectively. To determine the rate of formation of the C4a-hydroperoxyflavin intermediate and the reoxidation after reacting the reduced enzyme with oxygen, we analyzed the stopped-flow traces at 380 and 452 nm, respectively (Figure 3B). Using a single exponential equation, we calculated rates of 1.4 and 0.006 s^-1 for the first and second step of the oxidative half-reaction, respectively.

9. Representative Results

The results from the experiments described in the previous sections show how the reductive half-reaction of Af SidA can be monitored by measuring the changes in the absorbance at 452 nm, which correspond to the changes in the redox state of the flavin. The rate of this step can be determined by fitting the data to the appropriate equation (Figure 2; Step 8.4). The reduction rate obtained (0.65 s^-1) is similar to the k_cat value calculated with steady-state experiments², suggesting that the reduction is the rate-determining step of the reaction. Taking advantage of the double mixing mode of the stopped-flow, the rate of oxidation and the intermediates in this half-reaction can be determined (Figure 3; Step 8.4). In the reaction catalyzed by Af SidA, the C4a-hydroperoxyflavin is clearly detected (λ_max of 380 nm), and the rate of formation and decay can be calculated. The slow rate of the reoxidation obtained (0.006 s^-1) indicates that the C4a-hydroperoxyflavin is very stable in absence the ornithine.

Scheme 1. Mechanism of Af SidA. The isoallozaxine ring of the FAD cofactor is shown. The oxidized flavin (A) binds to NADPH (B) and reacts to form reduced flavin and NADP⁺ (C). After reaction with molecular oxygen and binding of ornithine, the C4a-hydroperoxyflavin is formed (D). This is the hydroxylating species. After hydroxylation of ornithine, the hydroxyflavin (E) must be dehydrated to form the oxidized enzyme. NADP⁺ remains bound throughout the catalytic cycle and is the last product to be released (F).

Figure 1. The stopped-flow instrument. A) Picture of the components of the Applied Photophysics SX20 stopped-flow spectrophotometer. B) Picture of the sample handling unit. C) Scheme of the flow circuit in double mixing mode.

Figure 2. Anaerobic reduction of SidA with NADPH in the stopped-flow instrument. A) Spectral changes recorded after mixing equal volumes of 30 μM SidA and 45 μM NADPH. The first spectrum (oxidized SidA) and last spectrum (fully reduced SidA) is highlighted in blue and red, respectively. B) Absorbance trace at 452 nm recorded as a function of time.

Figure 3. Oxidation of SidA in the stopped-flow instrument. A) Spectral changes recorded after mixing equal volumes of the fully reduced SidA and oxygenated buffer. The final concentrations were 15 μM SidA, 22.5 μM NADPH, and 0.95 mM oxygen. The spectrum recorded at 0.034, 4.268, and 727.494 s corresponds to the fully reduced enzyme, the C4a-hydroperoxyflavin intermediate (λ_max of 380 nm), and the oxidized enzyme (λ_max of 450 nm), respectively. B) Absorbance trace at 382 and 452 nm recorded as a function of time.