Laser Flash Photolysis of Deazariboflavin

Laser flash photolysis experiments were conducted at 21 °C on an Edinburgh LP920 laser flash photolysis spectrometer, as previously described.^{40 (link)} Briefly, sample contained ~ 20 μM deazariboflavin (dRF) and 5 mM semicarbazide in a pH 7.6 buffer (40 mM bis-Tris propane, 400 mM NaCl, 1 mM Ca²⁺, 2 mM l-Arg, 20 μM H₄Band 10% glycerol). The crowder (Ficoll 70 or Dextran 70) was added to the solution when necessary. The dRF solution without the protein in a cuvette was vigorously deaerated by a CO/Ar (v/v 1:3) mixed gas for ~ 90 minutes. The surface of introduced protein aliquot was then purged by the gas to remove traces of O₂ before it was mixed into the dRF solution. The sample was illuminated for 3 – 4 minutes by white light to obtain the [Fe(II)−CO][FMNH^•] form. The protein sample was subsequently flashed with 446 nm laser excitation to initiate the FMN ‒ heme IET, which can be monitored by the decrease of absorption of FMNH· and Fe(II) at 580 nm and 465 nm, respectively.^{41 (link)} The CO photolysis experiments were conducted at least twice for one sample. The averaged kinetic traces were analyzed by OriginPro 2019 (OriginLab, MA).