Departamento de Bioquímica e Biologia Molecular, Setor de Ciências Biológicas, Universidade Federal do Paraná, Curitiba, PR, Brazil.

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1 Supplementary information - Supplementary Figures 1-8 Excited singlet molecular O 2 ( 1 Δ g ) is generated enzymatically from excited carbonyls in the dark Camila M. Mano 1, Fernanda M. Prado 1, Júlio Massari 1, Graziella E. Ronsein 1, Glaucia R. Martinez 2, Sayuri Miyamoto 1, Jean Cadet 3, Helmut Sies 4, Marisa H. G. Medeiros 1, Etelvino J.H. Bechara 1, 5 & Paolo Di Mascio 1 1 Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CEP , CP 26077, São Paulo, SP, Brazil. 2 Departamento de Bioquímica e Biologia Molecular, Setor de Ciências Biológicas, Universidade Federal do Paraná, Curitiba, PR, Brazil. 3 Institut Nanosciences et Cryogénie, CEA/Grenoble, F Grenoble Cedex 9, France. 4 Institute of Biochemistry and Molecular Biology I, and Leibniz Research Institute for Environmental Medicine, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany. 5 Departamento de Ciências Exatas e da Terra, Instituto de Ciências Ambientais, químicas e Farmacêuticas, Universidade Federal de São Paulo, SP, Brazil. Correspondence to: Paolo Di Mascio (pdmascio@iq.usp.br) & Etelvino H. Bechara (ebechara@iq.usp.br)

2 Supplementary Figure 1: Quenching studies of TMD chemiluminescence with the sorbate ion. (A) UV visible light emission of triplet excited acetone and (B) Monomol light emission time course of O 2 ( 1 Δ g ) monitored during incubation of 2 mm TMD in CCl 4 at 70ºC (line a), in the presence of 0.5 mm (line b) or 1.0 mm sorbate (line c).

3 O O O 2 ( 1 Δ g ) N H TEMP N O TEMPO Supplementary Figure 2: EPR spin trapping studies of TMD thermolysis in the presence of TEMP. (A) EPR spectra obtained upon incubation of 30 mm TEMP with 4 mm TMD in D 2 O at 60ºC recorded for 20 min (line a) and 40 min (line b). The spectrum c was obtained after adding 0.4 µm TEMPO. Line d is the computer simulation of spectrum b traced in order to calculate the hyperfine coupling constant (a N = 1.60 mt). (B) EPR spectra obtained after incubation of 30 mm TEMP with 20 µm HPR and 50 mm IBAL in D 2 O at 37ºC. EPR spectra obtained in the absence of IBAL (line a) or HRP (line b); in the complete TEMP/IBAL/HRP system (line c); upon the addition of 0.4 µm TEMPO (line d) or 8mM sorbate (line e) and simulation spectrum traced in order to calculate the hyperfine coupling constant of the signal (a N = 1.60 mt).

4 Supplementary Figure 3: EAS chemical quenching studies of O 2 ( 1 Δ g ) produced during thermal cleavage of TMD in the presence of molecular oxygen. HPLC-ESI- MS/MS analysis of 8 mm EAS incubated for 24 h with 8 mm TMD at 70ºC in deuterated buffer (pd 7.4). (A) UV chromatogram recorded at 210 nm. Endoperoxide EAS 16 O 16 O eluted at 7.2 min. (B) SRM chromatogram of EAS 18 O 18 O (m/z ). (C) SRM chromatogram of EAS 16 O 16 O (m/z ). (D) Full mass spectrum obtained from peak at 7.2 min within mass range ( m/z). (E) Product ion spectrum of precursor ion with m/z 228.

5 Supplementary Figure 4: EAS chemical quenching studies of O 2 ( 1 Δ g ) generated by HRP-catalyzed oxidation IBAL in the presence of [ 18 O]-labeled molecular oxygen. HPLC-ESI-MS/MS analysis of 8 mm EAS incubated with 8 mm TMD for 2 h at 70ºC in deuterated phosphate buffer (pd 7.4). (A) UV chromatogram recorded at 210 nm. Endoperoxide EASO 2 containing 16 O or 18 O eluted at 7.8 min. (B) SRM chromatogram of EAS 18 O 18 O (m/z ). (C) SRM chromatogram of EAS 18 O 16 O (m/z ). (D) SRM chromatogram of EAS 16 O 16 O (m/z ). (E) Full mass spectrum obtained from peak at 7.8 min within a mass range id m/z. (E) Product ion spectrum of precursor ion at m/z 230. (F) Product ion spectrum of precursor ion at m/z 228.

6 Supplementary Figure 5: EAS chemical trapping of O 2 ( 1 Δ g ) generated by the HRP-catalyzed oxidation of IBAL in the presence of molecular oxygen. HPLC-ESI- MS/MS analysis of 8 mm EAS incubated for 24 h with 5 µm HRP and 50 mm IBAL at 37ºC in deuterated phosphate buffer (pd 7.4). (A) UV chromatogram recorded at 210 nm. Endoperoxide EAS 16 O 2 eluted at 7.9 min. (B) SRM chromatogram of EAS 18 O 18 O (m/z ). (C) SRM chromatogram of EAS 16 O 16 O (m/z ). (D) Full mass spectrum obtained from peak at 7.9 min within a mass range of m/z.

7 Supplementary Figure 6: EAS chemical trapping of O 2 ( 1 Δ g ) generated by the HRP-catalyzed oxidation of IBAL in the presence of molecular oxygen. UHR-ESI- Q-TOF analysis of 8 mm EAS upon incubation for 24 h with 5 µm HRP and 50 mm IBAL at 37ºC in deuterated phosphate buffer (pd 7.4). (A) Extracted chromatogram of EASO 16 O 2 and EAS 18 O 2 showing 9,10-endoperoxides at 6.6 min. (B) High resolution spectrum obtained from EASO 16 O 2 peak at 6.6 min. (C) Product ion spectrum of EASO 16 O 2 at m/z 228.

8 Figure 7: EAS chemical trapping of O 2 ( 1 Δ g ) generated by the HRP-catalyzed oxidation of IBAL in the presence of [ 18 O]-labeled molecular oxygen. UHR-ESI-Q- TOF analysis of 8 mm EAS upon incubation for 24 h with 5 µm HRP and 50 mm IBAL at 37ºC in deuterated phosphate buffer (pd 7.4). (A) Extracted chromatogram of EASO 18 O 2 and EAS 16 O 2 showing endoperoxides at 6.6 min. (B) High resolution spectrum obtained from EASO 18 O 2 peak at 6.6 min. (C) Product ion spectrum from EASO 18 O 2 at m/z 230.

9 Supplementary Figure 8: Quenching efficiency of sorbate anion upon the generation of O 2 ( 1 Δ g ) by HRP-catalyzed oxidation of IBAL in the presence of EAS and 16 O 2. (A) UV detection of EAS 16 O 2 at 210 nm in the absence (line a) and presence (line b) of 5 mm sorbate. (B) HPLC-ESI-MS/MS detection of EAS 16 O 2 monitoring the mass transition m/z in the absence (line a, peak intensity: 5,437 A.U) and presence (line b, peak intensity: 1,807 A.U) of sorbate.