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 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
1. Preparation of Anaerobic Buffer
2. Removing Oxygen from the Stopped-flow System
3. Preparation of Oxygen Saturated Buffer
4. Preparation of NADPH Solution
5. Removal of Oxygen from the Enzyme Solution
6. Reductive Half-reaction: Monitoring Flavin Reduction
7. Oxidative Half-reaction: Monitoring Flavin Oxidation
8. Data Analysis
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.
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 (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.
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 μM (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.
The authors have nothing to disclose.
Research supported by NSF award MCB-1021384.
General Laboratory Equipment | Company | Catalogue Number |
Vacuum pump | Welch | – |
Büchner flasks | Fisher | 70340-500 |
Stir bars | Fisher | 14-512-129 |
Stir plates | Fisher | 11-100-49S |
Schlenk lines | Kontes Glass | – |
Argon tank | Airgas | AR UPC300 |
Nitrogen tank | Airgas | NI200 |
Nitrogen tank, ultra high purity grade | Airgas | NI UHP200 |
Oxygen tank | Airgas | OX 40 |
5% Hydrogen balance nitrogen tank | Airgas | X02NI95B200H998 |
SX20 Stopped-flow spectrophotometer | AppliedPhotophysics | – |
Glove box | Coy | – |
Water bath | Brinkmann Lauda | – |
Supplies | ||
50 mL BD Falcon tubes | Fisher | 14-432-23 |
15 mL BD Falcon conical tubes | Fisher | 05-527-90 |
1.5 mL Eppendorf microcentrifuge tubes | Fisher | 05-402-18 |
50 and 25 mL glass vials | Fisher | 06-402 |
Rubber stoppers | Fisher | 06-447H |
Aluminum seals | Fisher | 06-406-15 |
Reagents | ||
Potassium phosphate, monobasic | Fisher | AC2714080025 |
Potassium phosphate, dibasic | Fisher | P288-500 |
Sodium acetate | Sigma | S-2889 |
Glucose oxidase from A. niger | Sigma | G7141-250KU |
D-Glucose | Fisher | D16-500 |
β-NADPH | Fisher | ICN10116783 |
L(+)-Ornithine hydrochloride | Fisher | ICN10116783 |