PMID- 30226367 OWN - NLM STAT- MEDLINE DCOM- 20190715 LR - 20190715 IS - 1520-4995 (Electronic) IS - 0006-2960 (Linking) VI - 57 IP - 40 DP - 2018 Oct 9 TI - Photoirradiation Generates an Ultrastable 8-Formyl FAD Semiquinone Radical with Unusual Properties in Formate Oxidase. PG - 5818-5826 LID - 10.1021/acs.biochem.8b00571 [doi] AB - 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 capable of persisting either in an aerobic environment for multiple weeks or in the presence of a strong reducing agent like sodium dithionite. Herein, we study the photoinduced formation of the 8-fFAD semiquinone radical in FOX and report the first EPR spectrum of this radical species. The stability of the 8-fFAD semiquinone radical suggests FOX to be a model enzyme for probing the structural and mechanistic features involved in stabilizing flavin semiquinone radicals. It is likely that the photoinduced formation of a stable 8-fFAD semiquinone radical is a defining characteristic of 8-formyl flavin-dependent enzymes. Additionally, a better understanding of the radical stabilization process may yield a FOX enzyme with more robust activity and broader industrial usefulness. FAU - Robbins, John M AU - Robbins JM AUID- ORCID: 0000-0002-5730-2531 AD - School of Chemical and Biomolecular Engineering , Georgia Institute of Technology , Atlanta , Georgia 30332-0100 , United States. AD - Engineered Biosystems Building (EBB) , Georgia Institute of Technology , Atlanta , Georgia 30332-2000 , United States. FAU - Geng, Jiafeng AU - Geng J AUID- ORCID: 0000-0001-9488-0974 AD - School of Chemistry and Biochemistry, Parker H. Petit Institute of Bioengineering and Bioscience , Georgia Institute of Technology , Atlanta , Georgia 30332-0363 , United States. FAU - Barry, Bridgette A AU - Barry BA AUID- ORCID: 0000-0003-3421-1407 AD - School of Chemistry and Biochemistry, Parker H. Petit Institute of Bioengineering and Bioscience , Georgia Institute of Technology , Atlanta , Georgia 30332-0363 , United States. FAU - Gadda, Giovanni AU - Gadda G AUID- ORCID: 0000-0002-7508-4195 AD - Department of Chemistry , Georgia State University , Atlanta , Georgia 30302-3965 , United States. AD - Center for Diagnostics and Therapeutics , Georgia State University , Atlanta , Georgia 30302-3965 , United States. AD - Center for Biotechnology and Drug Design , Georgia State University , Atlanta , Georgia 30302-3965 , United States. AD - Department of Biology , Georgia State University , Atlanta , Georgia 30302-3965 , United States. FAU - Bommarius, Andreas S AU - Bommarius AS AUID- ORCID: 0000-0003-4658-1675 AD - School of Chemical and Biomolecular Engineering , Georgia Institute of Technology , Atlanta , Georgia 30332-0100 , United States. AD - Engineered Biosystems Building (EBB) , Georgia Institute of Technology , Atlanta , Georgia 30332-2000 , United States. AD - School of Chemistry and Biochemistry, Parker H. Petit Institute of Bioengineering and Bioscience , Georgia Institute of Technology , Atlanta , Georgia 30332-0363 , United States. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't PT - Research Support, U.S. Gov't, Non-P.H.S. DEP - 20180927 PL - United States TA - Biochemistry JT - Biochemistry JID - 0370623 RN - 0 (Benzoquinones) RN - 0 (Fungal Proteins) RN - 146-14-5 (Flavin-Adenine Dinucleotide) RN - 3225-29-4 (semiquinone radicals) RN - 35919-91-6 (flavin semiquinone) RN - EC 1.- (Oxidoreductases) SB - IM MH - Aspergillus/*enzymology/genetics MH - Benzoquinones/*chemistry MH - Flavin-Adenine Dinucleotide/*analogs & derivatives/chemistry MH - Fungal Proteins/*chemistry/genetics MH - Mutagenesis, Site-Directed MH - Oxidoreductases/*chemistry MH - *Ultraviolet Rays EDAT- 2018/09/19 06:00 MHDA- 2019/07/16 06:00 CRDT- 2018/09/19 06:00 PHST- 2018/09/19 06:00 [pubmed] PHST- 2019/07/16 06:00 [medline] PHST- 2018/09/19 06:00 [entrez] AID - 10.1021/acs.biochem.8b00571 [doi] PST - ppublish SO - Biochemistry. 2018 Oct 9;57(40):5818-5826. doi: 10.1021/acs.biochem.8b00571. Epub 2018 Sep 27.