Flavocytochrome c sulfide dehydrogenase (FCC) is an important enzyme of sulfur metabolism in sulfur-oxidizing bacteria, and its catalytic properties have been extensively studied. However, the ultrafast dynamics of FCC is not well understood. We present ultrafast transient absorption and fluorescence spectroscopy measurements to unravel the early events upon excitation of the heme and flavin chromophores embedded in the flavocytochrome c (FccAB) from the bacterium Thiocapsa roseopersicina. The fluorescence kinetics of FccAB suggests that the majority of the photoexcited species decay nonradiatively within the first few picoseconds. Transient absorption spectroscopy supports these findings by suggesting two major dynamic processes in FccAB, internal conversion occurring in about 400 fs and the vibrational cooling occurring in about 4 ps, mostly affecting the heme moiety.

Department of Biotechnology and Microbiology University of Szeged Szeged H 6726 Hungary

Department of Medicinal Chemistry University of Szeged Dóm tér 8 Szeged H 6720 Hungary

Department of Theoretical Physics University of Szeged Tisza Lajos krt 84 86 Szeged H 6720 Hungary

Extreme Light Infrastructure ERIC Dolni Brezany CZ 25241 Czech Republic

Faculty of Science Faculty of Science Pavol Jozef Šafárik University in Košice Park Angelinum 19 Košice 040 01 Slovakia

Institute of Biophysics HUN REN Biological Research Centre Szeged H 6726 Hungary

Institute of Biotechnology Czech Academy of Sciences Vestec CZ 25250 Czech Republic

Institute of Physics University of Pécs Ifjúság útja 6 Pécs H 7624 Hungary

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Shimizu T.; Ida T.; Antelo G. T.; Ihara Y.; Fakhoury J. N.; Masuda S.; Giedroc D. P.; Akaike T.; Capdevila D. A.; Masuda T. Polysulfide metabolizing enzymes influence SqrR-mediated sulfide-induced transcription by impacting intracellular polysulfide dynamics. PNAS Nexus 2023, 2 (3), pgad04810.1093/pnasnexus/pgad048. PubMed DOI PMC

Imhoff J. F.; Bias-Imhoff U.. Lipids, quinones and fatty acids of anoxygenic phototrophic bacteria. In Anoxygenic Photosynthetic Bacteria; Blankenship R. E.; Madigan M. T.; Bauer C. E., Eds.; Kluwer Academic Publishers: The Netherland, 1995; pp 179–205.

Griesbeck C.; Schütz M.; Schödl T.; Bathe S.; Nausch L.; Mederer N.; Vielreicher M.; Hauska G. Mechanism of sulfide-quinone reductase investigated using site-directed mutagenesis and sulfur analysis. Biochemistry 2002, 41 (39), 11552–11565. 10.1021/bi026032b. PubMed DOI

Wang R. Hydrogen sulfide: The third gasotransmitter in biology and medicine. Antioxid. Redox Signaling 2010, 12, 1061–1064. 10.1089/ars.2009.2938. PubMed DOI

Li L.; Rose P.; Moore P. K. Hydrogen Sulfide and Cell Signaling. Annu. Rev. Pharmacol. Toxicol 2011, 51, 169–187. 10.1146/annurev-pharmtox-010510-100505. PubMed DOI

Osipov E. M.; Lilina A. V.; Tsallagov S. I.; Safonova T. N.; Sorokin D. Y.; Tikhonova T. V.; Popov V. O. Structure of the flavocytochrome c sulfide dehydrogenase associated with the copper-binding protein CopC from the haloalkaliphilic sulfur-oxidizing bacterium Thioalkalivibrio paradoxus ARh 1. Acta Crystallogr., Sect. D 2018, 74, 632–642. 10.1107/S2059798318005648. PubMed DOI

Dahl C.Inorganic sulfur compounds as electron donors in purple sulfur bacteria. In Sulfur Metabolism in Phototrophic Organisms; Hell R.; Dahl C.; Knaff D.; Leustek T., Eds.; Springer: Dordrecht, The Netherlands, 2008; pp 289–317.

Frigaard N.-U.; Bryant D. A.. Genomic insights into the sulfur metabolism of phototrophic green sulfur bacteria. In Sulfur Metabolism in Phototrophic Organisms; Hell R.; Dahl C.; Knaff D.; Leustek T., Eds.; Springer: Dordrecht, The Netherlands, 2008; pp 337–355.

Sousa F. M.; Pereira J. G.; Marreiros B. C.; Pereira M. M. Taxonomic distribution, structure/function relationship and metabolic context of the two families of sulfide dehydrogenases: SQR and FCSD. Biochim. Biophys. Acta, Bioenerg. 2018, 1859, 742–753. 10.1016/j.bbabio.2018.04.004. PubMed DOI

Visser J. M.; de Jong G. A. H.; Robertson L. A.; Kuenen J. G. A novel membrane-bound flavocytochrome c sulfide dehydro-genase from the colourless sulfur bacterium Thiobacillus sp. W5. Arch. Microbiol. 1997, 167, 295–301. 10.1007/s002030050447. PubMed DOI

Kostanjevecki V.; Brigé A.; Meyer T. E.; Cusanovich M. A.; Guisez Y.; van Beeumen J. A membrane-bound flavocytochrome c-sulfide dehydrogenase from the purple phototrophic sulfur bacterium Ectothiorhodospira vacuolata. J. Bacteriol. 2000, 182 (11), 3097–3103. 10.1128/JB.182.11.3097-3103.2000. PubMed DOI PMC

Chen Z. W.; Koh M.; Van Driessche G.; Van Beeumen J. J.; Bartsch R. G.; Meyer T. E.; Cusanovich M. A.; Mathews F. S. The structure of flavocytochrome c sulfide dehydrogenase from a purple phototrophic bacterium. Science 1994, 266 (1979), 430–432. 10.1126/science.7939681. PubMed DOI

Hirano Y.; Kimura Y.; Suzuki H.; Miki K.; Wang Z. Y. Structure analysis and comparative characterization of the cytochrome c’ and flavocytochrome c from thermophilic purple photosynthetic bacterium Thermochromatium tepidum. Biochemistry 2012, 51, 6556–6567. 10.1021/bi3005522. PubMed DOI

Cunane L. M.; Chen Z. W.; Durley R. C.; Barton J. D.; Mathews F. S. Flavocytochromes: structures and implications for electron transfer. Biochem. Soc. Trans. 1999, 27 (2), 179–184. 10.1042/bst0270179. PubMed DOI

Barends T. R. M.; Gorel A.; Bhattacharyya S.; Schirò G.; Bacellar C.; Cirelli C.; Colletier J. P.; Foucar L.; Grünbein M. L.; Hartmann E.; Hilpert M.; et al. Influence of pump laser fluence on ultrafast myoglobin structural dynamics. Nature 2024, 626 (8000), 905–911. 10.1038/s41586-024-07032-9. PubMed DOI PMC

Shank C. V.; Ippen E. P.; Bersohn R. Time-Resolved Spectroscopy of Hemoglobin and Its Complexes with Subpicosecond Optical Pulsess. Science 1976, 193 (4247), 50–51. 10.1126/science.935853. PubMed DOI

Ferrante C.; Batignani G.; Pontecorvo E.; Montemiglio L. C.; Vos M. H.; Scopigno T. Ultrafast dynamics and vibrational relaxation in six-coordinate heme proteins revealed by femtosecond stimulated Raman spectroscopy. J. Am. Chem. Soc. 2020, 142 (5), 2285–2292. 10.1021/jacs.9b10560. PubMed DOI PMC

Van Den Berg P. A.; Mulrooney S. B.; Gobets B.; Van Stokkum I. H.; Van Hoek A.; Williams J. R. C. H.; Visser A. J. Exploring the conformational equilibrium of E. coli thioredoxin reductase: characterization of two catalytically important states by ultrafast flavin fluorescence spectroscopy. Protein Sci. 2001, 10 (10), 2037–2049. 10.1110/ps.06701. PubMed DOI PMC

Andrikopoulos P. C.; Chaudhari A. S.; Liu Y.; Konold P. E.; Kennis J. T.; Schneider B.; Fuertes G. QM calculations predict the energetics and infrared spectra of transient glutamine isomers in LOV photoreceptors. Phys. Chem. Chem. Phys. 2021, 23 (25), 13934–13950. 10.1039/D1CP00447F. PubMed DOI PMC

Iuliano J. N.; French J. B.; Tonge P. J.. Vibrational spectroscopy of flavoproteins. In Methods in Enzymology; Elsevier, 2019; Vol. 620, pp 189–214. PubMed

Yang J.; Zhang Y.; He T. F.; Lu Y.; Wang L.; Ding B.; Zhong D. Ultrafast nonequilibrium dynamics of short-range protein electron transfer in flavodoxin. Phys. Chem. Chem. Phys. 2021, 24 (1), 382–391. 10.1039/D1CP04445A. PubMed DOI

He T. F.; Guo L.; Guo X.; Chang C. W.; Wang L.; Zhong D. Femtosecond dynamics of short-range protein electron transfer in flavodoxin. Biochemistry 2013, 52 (51), 9120–9128. 10.1021/bi401137u. PubMed DOI PMC

Tumbic G. W.; Li J.; Jiang T.; Hossan M. Y.; Feng C.; Thielges M. C. Interdomain Interactions Modulate the Active Site Dynamics of Human Inducible Nitric Oxide Synthase. J. Phys. Chem. B 2022, 126 (36), 6811–6819. 10.1021/acs.jpcb.2c04091. PubMed DOI PMC

Pfennig N. Eine vollsynthetische Nährlösung zur selektiven Anreicherung einiger Schwefelpurpurbakter. Naturwissenschaften 1961, 48, 13610.1007/BF00631938. DOI

Maestro S.–2 Schrödinger LLCNew York NY. 2022.

Li H.; Robertson A. D.; Jensen J. H. ″Very Fast Empirical Prediction and Interpretation of Protein pKa Values″. Proteins:Struct., Funct., Bioinf. 2005, 61, 704–721. 10.1002/prot.20660. PubMed DOI

Andrikopoulos P. C.; Liu Y.; Picchiotti A.; Lenngren N.; Kloz M.; Chaudhari A. S.; Precek M.; Rebarz M.; Andreasson J.; Hajdu J.; Schneider B.; Fuertes G. ″Femtosecond-to-nanosecond dynamics of flavin mononucleotide monitored by stimulated Raman spectroscopy and simulations.″. Phys. Chem. Chem. Phys. 2020, 22, 6538–6552. 10.1039/C9CP04918E. PubMed DOI

Lórenz-Fonfría V. A.; Kandori H. Bayesian Maximum Entropy (Two-Dimensional) Lifetime Distribution Reconstruction from Time-Resolved Spectroscopic Data. Appl. Spectrosc. 2007, 61, 428–443. 10.1366/000370207780466172. PubMed DOI

Lórenz-Fonfría V. A.; Kandori H. Transformation of Time-Resolved Spectra to Lifetime-Resolved Spectra by Maximum Entropy Inversion of the Laplace Transform. Appl. Spectrosc. 2006, 60, 407–417. 10.1366/000370206776593654. PubMed DOI

Lórenz-Fonfría V. A.; Kandori H. Practical Aspects of the Maximum Entropy Inversion of the Laplace Transform for the Quantitative Analysis of Multi-Exponential Data. Appl. Spectrosc. 2007, 61, 74–84. 10.1366/000370207779701460. PubMed DOI

Chaudhari A. S.; Chatterjee A.; Domingos C. A.; Andrikopoulos P. C.; Liu Y.; Andersson I.; Schneider B.; Lórenz-Fonfría V. A.; Fuertes G. Genetically encoded non-canonical amino acids reveal asynchronous dark reversion of chromophore, backbone, and side-chains in EL222. Protein Sci. 2023, 32, e459010.1002/pro.4590. PubMed DOI PMC

Liu Y.; Chaudhari A. S.; Chatterjee A.; Andrikopoulos P. C.; Picchiotti A.; Rebarz M.; Kloz M.; Lorenz-Fonfria V. A.; Schneider B.; Fuertes G. Sub-Millisecond Photoinduced Dynamics of Free and EL222-Bound FMN by Stimulated Raman and Visible Absorption Spectroscopies. Biomolecules 2023, 13, 16110.3390/biom13010161. PubMed DOI PMC

Stock G.; Hamm P. A non-equilibrium approach to allosteric communication. Philos. Transactions R. Soc., B 2018, 373, 2017018710.1098/rstb.2017.0187. PubMed DOI PMC

Bozovic O.; Zanobini C.; Gulzar A.; Jankovic B.; Buhrke D.; Post M.; Wolf S.; Stock G.; Hamm P. Real-time observation of ligand-induced allosteric transitions in a PDZ domain. Proc. Natl. Acad. Sci. U.S.A. 2020, 117, 26031–26039. 10.1073/pnas.2012999117. PubMed DOI PMC

Berera R.; van Grondelle R.; Kennis J. T. Ultrafast transient absorption spectroscopy: principles and application to photosynthetic systems. Photosynth. Res. 2009, 101, 105–118. 10.1007/s11120-009-9454-y. PubMed DOI PMC

Lórenz-Fonfría V. A.; Schultz B. J.; Resler T.; Schlesinger R.; Bamann C.; Bamberg E.; Heberle J. Pre-gating conformational changes in the ChETA variant of channelrhodopsin-2 monitored by nanosecond IR spectroscopy. J. Am. Chem. Soc. 2015, 137 (5), 1850–1861. 10.1021/ja5108595. PubMed DOI

Groma G. I.; Heiner Z.; Makai A.; Sarlós F. Estimation of kinetic parameters from time-resolved fluorescence data: A compressed sensing approach.. RSC Adv. 2012, 2 (30), 11481–11490. 10.1039/c2ra21773b. DOI

Macheroux P.UV-Visible Spectroscopy as a Tool to Study Flavoproteins. In Flavoprotein Protocols. Methods in Molecular Biology; Chapman S. K.; Reid G. A., Eds.; Humana Press, 1999; Vol. 131. PubMed

Ponka P. Cell biology of heme. Am. J. Med. Sci. 1999, 318 (4), 241–256. 10.1016/S0002-9629(15)40628-7. PubMed DOI

Bergmann A.; Dou Z. Fluorescence-based Heme Quantitation in Toxoplasma Gondii.. Bio Protoc. 2021, 11 (12), e406310.21769/BioProtoc.4063. PubMed DOI PMC

Champion P. M.; Perreault G. J. ″Observation and quantitation of light emission from cytochrome c using Soret band laser excitation.″. J. Chem. Phys. 1981, 75 (1), 490–491. 10.1063/1.441846. DOI

Steer R. P. Concerning correct and incorrect assignments of Soret (S 2–S 0) fluorescence in porphyrinoids: a short critical review. Photochem. Photobiol. Sci. 2014, 13, 1117–1122. 10.1039/c4pp00122b. PubMed DOI

Zheng W.; Dong L.; Yan Z.; Yi L.; Jianan Y. Q. Two-photon excited hemoglobin fluorescence. Biomed. opt. express 2011, 2 (1), 71–79. 10.1364/BOE.2.000071. PubMed DOI PMC

Islam S. D. M.; Susdorf T.; Penzkofer A.; Hegemann P. Fluorescence quenching of flavin adenine dinucleotide in aqueous solution by pH dependent isomerisation and photo-induced electron transfer. Chem. Phys. 2003, 295 (2), 137–149. 10.1016/j.chemphys.2003.08.013. DOI

Sengupta A.; Khade R. V.; Hazra P. pH dependent dynamic behavior of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) in femtosecond to nanosecond time scale. J. Photochem. Photobiol., A 2011, 221 (1), 105–112. 10.1016/j.jphotochem.2011.04.033. DOI

Vasyutinski̊ O. S.; Gorbunova L. A.; Danilova M. K.; et al. Determination of fluorescence quantum yields and decay times of NADH and FAD in water–alcohol mixtures: the analysis of radiative and nonradiative relaxation pathways. J. Photochem. Photobiol., A 2003, 436, 11438810.2139/ssrn.4214523. DOI

Kurabayashi Y.; Koichi H. K.; Youkoh H.; Kaizu K.; Hiroshi K. S2. fwdarw. S0 fluorescence of some metallotetraphenylporphyrins. J. Phys. Chem. A 1984, 88 (7), 1308–1310. 10.1021/j150651a013. DOI

Kolenc O. I.; Kyle P. Q. Evaluating cell metabolism through autofluorescence imaging of NAD (P) H and FAD.. Antioxid. Redox Signaling 2019, 30 (6), 875–889. 10.1089/ars.2017.7451. PubMed DOI PMC

Deniz E.; Valiño-Borau L.; Löffler J. G.; Eberl K. B.; Gulzar A.; Wolf S.; Durkin P. M.; Kaml R.; Budisa N.; Stock G.; Bredenbeck J. Through bonds or contacts? Mapping protein vibrational energy transfer using non-canonical amino acids. Nat. Commun. 2021, 12 (1), 328410.1038/s41467-021-23591-1. PubMed DOI PMC

Cao J.; Cogdell R. J.; Coker D. F.; Duan H. G.; Hauer J.; Kleinekathöfer U.; Jansen T. L.; Mančal T.; Miller R. D.; Ogilvie J. P.; Prokhorenko V. I.; et al. Quantum biology revisited. Sci. Adv. 2020, 6 (14), eaaz488810.1126/sciadv.aaz4888. PubMed DOI PMC

Bigwood R.; Gruebele M.; Leitner D. M.; Wolynes P. G. The vibrational energy flow transition in organic molecules: Theory meets experiment. Proc. Natl. Acad. Sci. U.S.A. 1998, 95 (11), 5960–5964. 10.1073/pnas.95.11.5960. PubMed DOI PMC

Förster T. Zwischenmolekulare energiewanderung und fluoreszenz. Ann. Phys. 1948, 437 (1–2), 55–75. 10.1002/andp.19484370105. DOI

Löwenich D.; Kleinermanns K.; Karunakaran V.; Kovalenko S. A. Transient and stationary spectroscopy of cytochrome c: ultrafast internal conversion controls photoreduction. Photochem. Photobiol. 2008, 84, 193–201. 10.1111/j.1751-1097.2007.00219.x. PubMed DOI

Negrerie M.; Cianetti S.; Vos M. H.; Martin J.-L.; Kruglik S. G. Ultrafast Heme Dynamics in Ferrous versus Ferric CytochromecStudied by Time-Resolved Resonance Raman and Transient Absorption Spectroscopy. J. Phys. Chem. B 2006, 110, 12766–12781. 10.1021/jp0559377. PubMed DOI

Kovalenko S. A.; Schanz R.; Hennig H.; Ernsting N. P. Cooling dynamics of an optically excited molecular probe in solution from femtosecond broadband transient absorption spectroscopy. J. Chem. Phys. 2001, 115, 3256–3273. 10.1063/1.1380696. DOI

Weigel A.; Dobryakov A.; Klaumunzer B.; Sajadi M.; Saalfrank P.; Ernsting N. P. Femtosecond Stimulated Raman Spectroscopy of Flavin after Optical Excitation. J. Phys. Chem. B 2011, 115, 3656–3680. 10.1021/jp1117129. PubMed DOI

Kottke T.; Heberle J.; Hehn D.; Dick B.; Hegemann P. Phot-LOV1: Photocycle of a Blue-Light Receptor Domain from the Green Alga Chlamydomonas reinhardtii. Biophys. J. 2003, 84, 1192–1201. 10.1016/S0006-3495(03)74933-9. PubMed DOI PMC

Langenbacher T.; Immeln D.; Dick B.; Kottke T. Microsecond Light-Induced Proton Transfer to Flavin in the Blue Light Sensor Plant Cryptochrome. J. Am. Chem. Soc. 2009, 131, 14274–14280. 10.1021/ja901628y. PubMed DOI

Liu H.; Ruan M.; Mao P.; Wang Z.; Chen H.; Weng Y. Unraveling the excited-state vibrational cooling dynamics of chlorophyll-a using femtosecond broadband fluorescence spectroscopy. J. Chem. Phys. 2024, 160 (20), 20510110.1063/5.0203819. PubMed DOI

Cina J. A.; Kovac P. A.; Jumper C. C.; Dean J. C.; Scholes G. D. Ultrafast transient absorption revisited: Phase-flips, spectral fingers, and other dynamical features. J. Chem. Phys. 2016, 144 (17), 17510210.1063/1.4947568. PubMed DOI

Zhang Y.; Yang J.; Liu N.; Wang L.; Lu F.; Chen J.; Zhong D. Ultrafast Nonequilibrium Dynamics of Vibrationally Hot Electron Transfer in Flavodoxin. J. Phys. Chem. Lett. 2023, 14 (47), 10657–10663. 10.1021/acs.jpclett.3c02438. PubMed DOI