Photoirradiation Generates an Ultrastable 8-Formyl FAD Semiquinone Radical with Unusual Properties in Formate Oxidase

Formate oxidase (FOX) was previously shown to contain a noncovalently bound 8-formyl FAD (8-fFAD) cofactor. However, both the absorption spectra and the kinetic parameters previously reported for FOX are inconsistent with more recent reports. The ultraviolet–visible (UV–vis) absorption spectrum reported in early studies closely resembles the spectra observed for protein-bound 8-formyl flavin semiquinone species, thus suggesting FOX may be photosensitive. Therefore, the properties of dark and light-exposed FOX were investigated using steady-state kinetics and site-directed mutagenesis analysis along with inductively coupled plasma optical emission spectroscopy, UV–vis absorption spectroscopy, circular dichroism spectroscopy, liquid chromatography and mass spectrometry, and electron paramagnetic resonance (EPR) spectroscopy. Surprisingly, these experimental results demonstrate that FOX is deactivated in the presence of light through generation of an oxygen stable, anionic (red) 8-fFAD semiquinone radical ca...