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Cell Biology JoVE (Open Access) Citable · DOI

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

DOI: 10.3791/3803-v
What you'll learn
  • ✓ Set up and operate a stopped-flow spectrophotometer under anaerobic conditions
  • ✓ Monitor flavin reduction and oxidation half-reactions in real-time
  • ✓ Identify and quantify kinetic parameters for flavin-dependent monooxygenase reactions
  • ✓ Interpret UV-Vis spectral data to distinguish enzyme intermediates
Protocol

Biopharma Insights 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.

Difficulty
advanced
Total time
~4–6 hours per experiment (including setup, equilibration, and multiple kinetic runs)
Biosafety
BSL-1

Steps

1
Prepare anaerobic buffer solutions

Prepare degassed buffer solutions required for anaerobic stopped-flow experiments. This ensures the reaction environment is free of dissolved oxygen to accurately monitor enzyme kinetics.

▶ 01:37
2
Remove oxygen from stopped-flow system

Flush the stopped-flow instrument with anaerobic buffer and remove residual oxygen from all tubing and mixing chambers. This step is critical for preventing unwanted oxidation during measurements.

▶ 02:24
3
Prepare key reagent solutions

Prepare concentrated stock solutions of SidA enzyme, substrates, and cofactors (e.g., NADPH) in anaerobic buffer. Maintain solutions under anaerobic conditions prior to injection.

▶ 03:22
4
Monitor flavin reduction half-reaction kinetics

Mix SidA with NADPH using stopped-flow and record real-time absorbance changes to track flavin reduction. Calculate rate constants from the resulting time-resolved spectra.

▶ 04:45
5
Monitor flavin oxidation half-reaction kinetics

Mix reduced SidA with oxidizing substrate using stopped-flow and record absorbance changes to track flavin oxidation. Determine rate constants for the oxidative phase.

▶ 07:31
6
Acquire and analyze spectral intermediates

Collect full UV-Vis spectra at multiple time points during both half-reactions to identify and characterize enzyme-bound flavin species (oxidized, reduced, charge-transfer complexes).

▶ 10:10
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