Name: Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry (Video) | JoVE Journal
Uploaded: 2012-03-18T14:00:00.000+00:00
Duration: 12 min 8 s
Description: Learn stopped-flow spectrophotometry to analyze reductive and oxidative half-reactions of flavin-dependent monooxygenase SidA from Aspergillus fumigatus. This method enables kinetic analysis of enzyme intermediates.

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5 minutes

Research supported by NSF award MCB-1021384.

1. Preparation of Anaerobic Buffer
<ol>
 <li>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.</li>
 <li>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.</li>
 <li>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.</li>
 <li>Flush the flask with argon for 10 seconds and disconnect it from the vacuum manifold. Place the flask inside the glove box.</li>
 <li>Leave the flask open in the glove box overnight with vigorous agitation.</li>
</ol>
2. Removing Oxygen from the Stopped-flow System
<ol>
 <li>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.</li>
 <li>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.</li>
 <li>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).</li>
 <li>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.</li>
 <li>Replace the solution of the reservoir syringes with the same buffer containing glucose oxidase and glucose. Repeat step 2.4.</li>
 <li>Repeat step 2.5 and allow the solution to stand in the flow system overnight.</li>
 <li>After overnight incubation, repeat step 2.5.</li>
</ol>
3. Preparation of Oxygen Saturated Buffer
<ol>
 <li>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.</li>
 <li>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.</li>
 <li>Bubble the solution with 100% oxygen for 1 hour with agitation.</li>
 <li>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.</li>
</ol>
4. Preparation of NADPH Solution
<ol>
 <li>Weigh out 1 mg of NADPH in a 1.5 mL Eppendorf tube and place it into the glove box.</li>
 <li>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 (ε340 nm= 6,270 M-1cm-1).</li>
</ol>
5. Removal of Oxygen from the Enzyme Solution
<ol>
 <li>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.</li>
 <li>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.</li>
 <li>Place the vial on ice and connect it to a Schlenk line by inserting a needle through the stopper of the vial.</li>
 <li>Degas the enzyme solution by performing 5 consecutive rounds of vacuum degassing and flushing with argon every 20 minutes for 1 hour.</li>
 <li>Flush the vial with argon for 10 seconds and disconnect it from the vacuum manifold. Place the vial on ice inside the glove box.</li>
</ol>
6. Reductive Half-reaction: Monitoring Flavin Reduction
<ol>
 <li>Prepare the stopped-flow instrument and the Pro-Data control software for single mixing mode following the manufacturer's protocol.</li>
 <li>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.</li>
 <li>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.</li>
 <li>In the Control Panel window of the Pro-Data control software select Photodiode Array, External trigger (to activate stopped-flow operation), and Logarithmic scale.</li>
 <li>Launch the Pro-Data viewer software and define the directory where the data files will be stored.</li>
 <li>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).</li>
 <li>In order to ensure that glucose oxidase has been removed completely from the flow circuit, perform step 6.6 four times in total.</li>
 <li>Click on Baseline in the Pro-Data control software.</li>
 <li>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).</li>
 <li>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.</li>
 <li>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.</li>
 <li>Using the A452 nm value obtained from the drives, determine the enzyme concentration before mixing (ε452 nm= 13,700 M-1cm-1) and prepare 4 mL of a NADPH solution 1.5-fold more concentrated.</li>
 <li>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.</li>
 <li>Repeat step 6.11. This yields spectra during the reduction of the enzyme.</li>
</ol>
7. Oxidative Half-reaction: Monitoring Flavin Oxidation
<ol>
 <li>Prepare the stopped-flow instrument and the Pro-Data control software for double mixing mode following the manufacturer's protocol.</li>
 <li>Repeat steps 6.2-6.5.</li>
 <li>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.</li>
 <li>Perform step 7.3 four times in total to completely remove the contents from the flow circuit.</li>
 <li>Click Baseline in the Pro-Data control software.</li>
 <li>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).</li>
 <li>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.</li>
 <li>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.</li>
 <li>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.</li>
 <li>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.</li>
 <li>Repeat step 7.8. This yields spectra during the reoxidation of the fully reduced enzyme.</li>
</ol>
8. Data Analysis
<ol>
 <li>Launch the Pro-Data converter software. Click on the Options icon and select Save in ProDataCSV.</li>
 <li>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.</li>
 <li>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.</li>
 <li>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.</li>
</ol>
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 kcat 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 (λmax 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.
<img alt="Enzymatic catalysis showing reductive, oxidative half-reactions, chemical structure diagram." src="/files/ftp_upload/3803/3803scheme1.jpg" data-altupdatedat="1787630408000" data-alt="Scheme 1"> 
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).
<img alt="Spectroscopy setup with PC workstation for transient absorption measurement; diagram and equipment labels." src="/files/ftp_upload/3803/3803fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
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.
<img alt="Absorbance spectra and kinetic data; transient absorption analysis; photonic measurements." src="/files/ftp_upload/3803/3803fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
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.
<img alt="Absorbance vs. wavelength graph; UV-Vis spectroscopy; indicates multiple concentrations; spectral peaks." src="/files/ftp_upload/3803/3803fig3.jpg" data-altupdatedat="1787630408000" data-alt="Figure 3"> 
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 (λmax of 380 nm), and the oxidized enzyme (λmax of 450 nm), respectively. B) Absorbance trace at 382 and 452 nm recorded as a function of time.

<ol><li>Hissen, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Aspergillus+fumigatus+siderophore+biosynthetic+gene+sidA%2C+encoding+L-ornithine+N5-oxygenase%2C+is+required+for+virulence%5BTitle%5D)+AND+%22Infect.+Immun%22%5BJournal%5D)">The Aspergillus fumigatus siderophore biosynthetic gene sidA, encoding L-ornithine N5-oxygenase, is required for virulence.</a> Infect. Immun. 73 (9), 5493-5503 (2005).</li>
<li>Chocklett, S. W., Sobrado, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aspergillus+fumigatus+SidA+is+a+highly+specific+ornithine+hydroxylase+with+bound+flavin+cofactor%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Aspergillus fumigatus SidA is a highly specific ornithine hydroxylase with bound flavin cofactor.</a> Biochemistry. 49 (31), 6777-6783 (2010).</li>
<li>Mayfield, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive+spectroscopic%2C+steady+state%2C+and+transient+kinetic+studies+of+a+representative+siderophore-associated+flavin+monooxygenase%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">Comprehensive spectroscopic, steady state, and transient kinetic studies of a representative siderophore-associated flavin monooxygenase.</a> J. Biol. Chem. 285 (40), 30375-30388 (2010).</li>
<li>Fierke, C. A., Hammes, G. G. Transient kinetic approaches to enzyme mechanisms. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aFierke%2C+CA+%22Contemporary+enzyme+kinetics+and+mechanism%22">Contemporary enzyme kinetics and mechanism</a>. , Elsevier. USA. (2009).</li>
<li>Palfey, B. A., McDonald, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Control+of+catalysis+in+flavin-dependent+monooxygenases%5BTitle%5D)+AND+%22Arch.+Biochem.+Biophys%22%5BJournal%5D)">Control of catalysis in flavin-dependent monooxygenases.</a> Arch. Biochem. Biophys. 493 (1), 26-26 (2010).</li>
<li>van Berkel, W. J. H., Kamerbeek, N. M., Fraaije, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flavoprotein+monooxygenases%2C+a+diverse+class+of+oxidative+biocatalysts%5BTitle%5D)+AND+%22J.+Biotechnol%22%5BJournal%5D)">Flavoprotein monooxygenases, a diverse class of oxidative biocatalysts.</a> J. Biotechnol. 124 (4), 670-689 (2006).</li>
<li>Chapman, S. K., Reid, G. A. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aChapman%2C+SK+%22Flavoprotein+Protocols%22">Flavoprotein Protocols</a>. , Humana Press. Totowa, USA. (1999).</li>
<li>Sobrado, P., Fitzpatrick, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Solvent+and+primary+deuterium+isotope+effects+show+that+lactate+CH+and+OH+bond+cleavage+are+concerted+in+Y254F+flavocytochrome+b2%2C+consistent+with+a+hydride+transfer+mechanism%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Solvent and primary deuterium isotope effects show that lactate CH and OH bond cleavage are concerted in Y254F flavocytochrome b2, consistent with a hydride transfer mechanism.</a> Biochemistry. 42, 15208-15214 (2003).</li>
<li>Kumar, S., Gawandi, V. B., Capito, N., Phillips, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Substituent+effects+on+the+reaction+of+%C3%8E%C2%B2-benzoylalanines+with+Pseudomonas+fluorescens+kynureninase%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Substituent effects on the reaction of Î²-benzoylalanines with Pseudomonas fluorescens kynureninase.</a> Biochemistry. 49 (36), 7909-7913 (2010).</li>
<li>Yun, D., García-Serres, R., Chicalese, B. M., An, Y. H., Huynh, B. H., Bollinger, J. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%28%CE%BC-1%2C2-Peroxo%29diiron%28III%2FIII%29+complex+as+a+precursor+to+the+diiron%28III%2FIV%29+intermediate+X+in+the+assembly+of+the+iron-radical+cofactor+of+ribonucleotide+reductase+from+mouse%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">(μ-1,2-Peroxo)diiron(III/III) complex as a precursor to the diiron(III/IV) intermediate X in the assembly of the iron-radical cofactor of ribonucleotide reductase from mouse.</a> Biochemistry. 46 (7), 1925-1932 (2007).</li>
<li>Pennati, A., Zanetti, G., Aliverti, A., Gadda, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+salt+and+pH+on+the+reductive+half-reaction+of+Mycobacterium+tuberculosis+FprA+with+NADPH+Biochemistry%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Effect of salt and pH on the reductive half-reaction of Mycobacterium tuberculosis FprA with NADPH Biochemistry.</a> Biochemistry. 47 (11), 3418-3425 (2008).</li>
<li>Patil, P. V., Allou, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+use+of+protocatechuate+dioxygenase+for+maintaining+anaerobic+conditions+in+biochemical+experiments%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">The use of protocatechuate dioxygenase for maintaining anaerobic conditions in biochemical experiments.</a> Analytical Biochemistry. 286 (2), 187-192 (2000).</li>
</ol>

We have nothing to disclose.

Enzymes that catalyze redox reactions usually contain cofactors such as hemes and flavins that undergo significant absorbance changes during the catalytic cycle. The oxidized form of the flavin presents absorbance maxima at ~ 360 and 450 nm, and its reduction is typically monitored by following the absorbance decrease at 450 nm7. In general, some transient intermediates are present but form and decay too fast to be measured in regular spectrophotometers. Using the Applied Photophysics SX20 stopped-flow spectrophotometer (or similar instruments), it is possible to measure absorbance changes in the millisecond time scale (dead-time, 2 ms). Here we studied the reductive and oxidative half-reactions of the flavin-dependent monooxygenase Af SidA, serving as a model. The rate of hydride transfer was determined by measuring the change of absorbance at 452 nm after mixing the enzyme with NADPH under anaerobic conditions. Subsequently, taking advantage of the double mixing mode of the stopped-flow instrument, the enzyme was first reacted with NADPH, until full reduction was achieved, then the reduced enzyme-NADP+ complex was mixed with oxygen. Following this procedure, it is possible to detect transient oxygenated flavin intermediates and to measure rates of formation and decay. The identification of these intermediates provides experimental data about the nature of the reacting species in catalysis. In the case of Af SidA, the formation of the C4a-hydroperoxyflavin (typically monitored at 370-380 nm), which is the hydroxylating species. In addition, measuring the rate constant of each step allows one to obtain information about the rate-determining step of the reaction and help to elucidate the kinetic and chemical mechanisms of the enzyme. 
In general, similar approaches can be used for other flavoenzymes, or proteins for which absorbance changes occurred, such as proteins that contain heme, pyridoxal-phosphate, or non-heme iron8-10. A limitation to this method is that large amounts of purified enzyme are required, but this can be overcome by using an expression system with high yields. One determines the optimal protein concentration for recording spectra by using enough protein so that a strong enough signal can be observed, but not too much so that enzyme is not wasted. Typically, the lowest enzyme concentration for flavin-containing enzymes used in stopped-flow experiments is 6-10 &mu;M (after mixing) and is determined using the corresponding molar extinction coefficient of the enzyme. In the case of Af SidA, the percentage of the enzyme bound FAD is 50-65%2. Apo-protein is regarded as inactive in these experiments because a bound FAD cofactor is necessary for catalysis. Another possible limitation to this method is if processes in an enzyme occur faster than 2 ms (dead time of the stopped-flow) they will not be observed, but there are reported strategies where rates can be decreased to overcome this issue. One example for this includes using a high NaCl concentration in the reaction of a ferredoxin-NADP+ reductase11. The scrubbing of oxygen from the flow circuit of the stopped-flow is often a tricky step in this experiment and requires special attention. The glucose oxidase-glucose system described here is used successfully in most laboratories as it is an effective and inexpensive method. However, there are some drawbacks which include the production of H2O2 and for some applications other alternatives as the protocatechuate dioxygenase-protocatechuate system should be considered12. The utilization of an anaerobic glove box makes it easier to ensure anaerobic conditions, but is not essential. Oxygen must be removed from the flow circuit of the stopped-flow as we want the enzyme to be reduced in the absence of oxygen or react with oxygen at concentrations that we specify. Although the stopped-flow is in the glove box, there is oxygen in the flow circuit if we used aerobic buffers in previous experiments. In addition to absorbance measurements, fluorescence and circular dichroism assays can be performed in the Applied Photophysics SX20 stopped-flow spectrophotometer with the corresponding accessories.

<table><tbody><tr><td>Vacuum pump</td><td>Welch Allyn</td><td>-</td><td></td></tr><tr><td>B&amp;#252;chner flasks</td><td>Fisher Scientific</td><td>70340-500</td><td></td></tr><tr><td>Stir bars</td><td>Fisher Scientific</td><td>14-512-129</td><td></td></tr><tr><td>Stir plates</td><td>Fisher Scientific</td><td>11-100-49S</td><td></td></tr><tr><td>Schlenk lines</td><td>Kontes Corp</td><td>-</td><td></td></tr><tr><td>Argon tank </td><td>Airgas</td><td>AR UPC300</td><td></td></tr><tr><td>Nitrogen tank</td><td>Airgas</td><td>NI200</td><td></td></tr><tr><td>Nitrogen tank, ultra high purity grade</td><td>Airgas</td><td>NI UHP200</td><td></td></tr><tr><td>Oxygen tank</td><td>Airgas</td><td>OX 40</td><td></td></tr><tr><td>5% Hydrogen balance nitrogen tank</td><td>Airgas</td><td>X02NI95B200H998</td><td></td></tr><tr><td>SX20 Stopped-flow spectrophotometer</td><td>AppliedPhotophysics</td><td>-</td><td></td></tr><tr><td>Glove box</td><td>Coy Laboratories, Inc.</td><td>-</td><td></td></tr><tr><td>Water bath</td><td>Lauda Brinkmann</td><td>-</td><td></td></tr><tr><td>50 mL BD Falcon tubes</td><td>Fisher Scientific</td><td>14-432-23</td><td></td></tr><tr><td>15 mL BD Falcon conical tubes</td><td>Fisher Scientific</td><td>05-527-90</td><td></td></tr><tr><td>1.5 mL Eppendorf microcentrifuge tubes</td><td>Fisher Scientific</td><td>05-402-18</td><td></td></tr><tr><td>50 and 25 mL glass vials</td><td>Fisher Scientific</td><td>06-402</td><td></td></tr><tr><td>Rubber stoppers</td><td>Fisher Scientific</td><td>06-447H</td><td></td></tr><tr><td>Aluminum seals</td><td>Fisher Scientific</td><td>06-406-15</td><td></td></tr><tr><td>Potassium phosphate, monobasic</td><td>Fisher Scientific</td><td>AC2714080025</td><td></td></tr><tr><td>Potassium phosphate, dibasic</td><td>Fisher Scientific</td><td>P288-500</td><td></td></tr><tr><td>Sodium acetate</td><td>Sigma-Aldrich</td><td>S-2889</td><td></td></tr><tr><td>Glucose oxidase from &lt;em&gt;A. niger&lt;/em&gt;</td><td>Sigma-Aldrich</td><td>G7141-250KU</td><td></td></tr><tr><td>D-Glucose</td><td>Fisher Scientific</td><td>D16-500</td><td></td></tr><tr><td>&lt;em&gt;&amp;#946;&lt;/em&gt;-NADPH</td><td>Fisher Scientific</td><td>ICN10116783</td><td></td></tr><tr><td>L(+)-Ornithine hydrochloride</td><td>Fisher Scientific</td><td>ICN10116783</td><td></td></tr></tbody></table>

monitoring the reductive and oxidative half-reactions of a flavin-dependent monooxygenase using stopped-flow spectrophotometry

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

Aspergillus fumigatus siderophore A (SidA) is an FAD-containing monooxygenase that catalyzes the hydroxylation of ornithine in the biosynthesis of hydroxamate siderophores that are essential for virulence (e.g. ferricrocin or N',N",N'''-triacetylfusarinine C)1. The reaction catalyzed by SidA can be divided into reductive and oxidative half-reactions (Scheme 1). In the reductive half-reaction, the oxidized FAD bound to Af SidA, is reduced by NADPH2,3. In the oxidative half-reaction, the reduced cofactor reacts with molecular oxygen to form a C4a-hydroperoxyflavin intermediate, which transfers an oxygen atom to ornithine. Here, we describe a procedure to measure the rates and detect the different spectral forms of SidA using a stopped-flow instrument installed in an anaerobic glove box. In the stopped-flow instrument, small volumes of reactants are rapidly mixed, and after the flow is stopped by the stop syringe (Figure 1), the spectral changes of the solution placed in the observation cell are recorded over time. In the first part of the experiment, we show how we can use the stopped-flow instrument in single mode, where the anaerobic reduction of the flavin in Af SidA by NADPH is directly measured. We then use double mixing settings where Af SidA is first anaerobically reduced by NADPH for a designated period of time in an aging loop, and then reacted with molecular oxygen in the observation cell (Figure 1). In order to perform this experiment, anaerobic buffers are necessary because when only the reductive half-reaction is monitored, any oxygen in the solutions will react with the reduced flavin cofactor and form a C4a-hydroperoxyflavin intermediate that will ultimately decay back into the oxidized flavin. This would not allow the user to accurately measure rates of reduction since there would be complete turnover of the enzyme. When the oxidative half-reaction is being studied the enzyme must be reduced in the absence of oxygen so that just the steps between reduction and oxidation are observed. One of the buffers used in this experiment is oxygen saturated so that we can study the oxidative half-reaction at higher concentrations of oxygen. These are often the procedures carried out when studying either the reductive or oxidative half-reactions with flavin-containing monooxygenases. The time scale of the pre-steady-state experiments performed with the stopped-flow is milliseconds to seconds, which allow the determination of intrinsic rate constants and the detection and identification of intermediates in the reaction4. The procedures described here can be applied to other flavin-dependent monooxygenases.5,6

We describe the use of a stopped-flow instrument to investigate both the reductive and oxidative half-reactions of Aspergillus fumigatus siderophore A (SidA), a flavin-dependent monooxygenase. We then show the spectra corresponding to the species in the reaction of SidA and we calculate the rate constants for their formation.

Aspergillus fumigatus siderophore A (SidA) is an FAD-containing monooxygenase that catalyzes the hydroxylation of ornithine in the biosynthesis of ...

This procedure uses a stopped flow instrument to detect the different spectral forms of SID A and measure rates. The reaction catalyzed by SID A can be divided into reductive and oxidative half reactions of FAD. The oxidized flavin binds to N-A-D-P-H and reacts to form reduced flavin and NADP plus reaction of molecular oxygen and binding of ornithine results in formation of C four.A hydroxy flavin after hydroxylation of ornithine, the hydroxy flavin dehydrates to form the oxidized enzyme. NA DP plus remains bound throughout the catalytic cycle and is the last substrate to be released. The stopped flow instrument facilitates rapid mixing of small volumes of reactants and records.The spectral changes over time Stuff loss spectrophotometer allows one to measure the rate of formation and of a enzyme catalyzed reaction specifically of transient intermediates. Two members of my lab, Rita Robinson and Enviro Romero will demonstrate these experiments using set A at flavor mono oxygenase as a model system. Pour 250 milliliters of 100 millimolar potassium phosphate buffer into a 500 milliliter buchner flask containing a stir bar.Seal the flask with a rubber stopper, then place it on a stir plate and connect the short tube of the flask to a sch flank line. Degas the buffer under vacuum for an hour at room temperature with agitation, followed by flushing with argonne. Repeat five times.Finally flush the flask with Argonne for 10 seconds. Disconnect the flask from the vacuum manifold and place it inside the glove box. Continue to stir vigorously with the flask open.Fill the reservoir syringes with 18.13 units per milliliter glucose oxidase, and 100 millimolar glucose solutions. Now set the drive valves to the load position. Fill the drive syringes.Next, turn the F valve to the drive position. Using the pro data control software, click empty the stop syringe, then push the buffer from the drive syringe F through the flow circuit by manually raising the corresponding drive ram. Continue with the other three drive syringes equilibrate for an hour.Now refill the reservoir syringes and repeat flushing of the stopped flow system. To prepare oxygen saturated buffer. Bubble the solution with 100%oxygen for one hour with agitation.Then remove the short needle from the vial and wait 10 seconds. Proceed to remove the long needle and place the close vial in the glove box. For the N-A-D-P-H solution.Place a 1.5 milliliter einor tube containing one milligram of N-A-D-P-H into the glove box. Dissolve in 300 microliters of anaerobic, 100 millimolar potassium phosphate buffer. pH 7.5.Remove 30 microliters to determine the N-A-D-P-H concentration. Now to remove oxygen from the enzyme stock, transfer the solution into a 25 milliliter vial containing a stir bar cap with a wheat and stopper and aluminum seal. Place on ice.Connect to a schlink line, then degas by vacuum for 20 minutes, followed by flushing with argonne. Finally, flush the vial with Argonne for 10 seconds and disconnect it from the vacuum manifold. Place the vial on ice inside the glove box.In the control panel window of the pro data control software. Select photo diode array, external trigger and logarithmic scale. Also launch the pro data viewer software and define the directory for data file storage.Now replace the solution in the reservoir syringes C and F with anaerobic 100 millimolar potassium phosphate buffer pH 7.5. Turn the C and F valves to the drive position. Click empty the stop syringe in the pro data control software.Then push the buffer from both drive syringes through the flow circuit by manually raising the corresponding drive ram. Repeat flushing with reservoir syringes C and F three more times. To ensure complete removal of glucose oxidase from the flow circuit, proceed to click on baseline in the pro data control software.Replace the solution of the reservoir syringe F with the enzyme solution. Turn the F valve to the drive position. Click empty the stop syringe 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. Return to the pro data control software to set the acquisition time to 60 seconds. Verify that both drive syringes are filled with their corresponding solutions and that the drive rams are in contact with the drive syringe.Pistons.Turn both valves to the drive position and click on acquire in the pro data control software to perform the drive. Acquire spectral data of the oxidized form of the enzyme in triplicate using the A 4 52 nanometer value obtained from the drives. Determine the enzyme concentration before mixing and prepare four milliliters of N-A-D-P-H solution.1.5 fold more concentrated fill reservoir syringe C with the N-A-D-P-H solution and empty and refill the stop syringe three times as shown earlier. Now, acquire spectra during the reduction of the enzyme flush contents from the flow circuit with anaerobic 100 millimolar potassium phosphate buffer pH 7.5. Now click on baseline in the pro data control software.Then mix 200 microliters of enzyme stock solution with 1000 microliters of anaerobic, 100 millimolar potassium phosphate buffer. pH 7.5. Replace the solution of the reservoir syringe A with the enzyme solution.Turn the A valve to the drive position in the pro data control software. Click the stop syringe empty. Push the enzyme solution from drive syringe A through the flow circuit by manually raising the corresponding drive ram.Set the delay time to 0.5 seconds and acquisition time to 60 seconds. Verify that all drive syringes are filled with their corresponding solutions and the drive rams are in contact with the drive syringe pistons. Then turn all drive valves to the drive position and click on acquire in the pro data control software.Obtain spectral data of the oxidized form of the enzyme in triplicate. Next, replace the solution of the reservoir syringe B with an N-A-D-P-H 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 shown earlier in the control panel window.Set the delay time at 0.5 seconds. An acquisition at 20 seconds, replace the solution of the reservoir syringe sea with the oxygenated buffer. Turn the C valve to the drive position.Flush three times in the pro data control software. Set the delay time at 15 seconds and acquisition time at 900 seconds. Continue to acquire spectra during the re oxidation of the fully reduced enzyme in the stopped flow instrument.The anaerobic reduction of SID A with N-A-D-P-H can be monitored by measuring changes in the redox state of the flavin using absorbent at 452 here. Spectral changes are recorded over time from oxidized SID A to the last spectrum of fully reduced SID A.The rate of this enzymatic step can be determined by fitting the data to the appropriate equation. The rate of oxidation and the reaction intermediates can also be determined using the double mixing mode of the stopped flow in this reaction by AF Sid A.The C four A Hydroxi flavin intermediate is clearly detected with a lambda max of three 80 nanometers and the oxidized enzyme has a lambda max of 450 nanometers.Once mastered, this technique can be completed in four to six hours if it is performed properly. Remember to ensure the solutions are anaerobic. After watching this video, you should have a good understanding of how to investigate both the relative and the ulcerative ion of FLA independent mono further downstream experiment like rapid chemical.Quentin can help to elucidate the sad nature of the intermediates.

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Learn stopped-flow spectrophotometry to analyze reductive and oxidative half-reactions of flavin-dependent monooxygenase SidA from Aspergillus fumigatus. This method enables kinetic analysis of enzyme intermediates.

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

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