Skip to main content
SpringerLink
Log in

Investigation of the pH-dependence of the oxidation of FAD in VcCRY-1, a member of the cryptochrome-DASH family

  • Original Papers
  • Published:
Photochemical & Photobiological Sciences Aims and scope Submit manuscript

Abstract

Vibrio cholerae cryptochrome-1 (VcCRY-1) is a member of the cryptochrome DASH family. The flavoprotein appears to use blue light both for repair of cyclobutane pyrimidine dimers (CPDs) on DNA and signal transduction. Earlier, we found that it was almost impossible to oxidize the FADH· state upon binding to a CPD, and, in the absence of substrate, the rate of FADH· oxidation was much larger at high pH (Gindt et al. in Biochemistry 54:2802–2805, 2015). Here, we present the pH-dependence of the oxidation of FADH· by ferricyanide, which revealed a switch between slow and fast oxidation with a pKa ≈ 7.0. Stopped-flow mixing was used to measure the oxidation of FADH− to FADH· at pH 6.7 and 7.5. Substrate binding was required to slow down this oxidation such that it could be measured with stopped flow, but there was only a small effect of pH. In addition, resonance Raman measurements of FADH· in VcCRY-1 at pH 6.5 and 7.5 were performed to probe for structural changes near the FAD cofactor related to the observed changes in rate of FADH· oxidation. Only substrate binding seemed to induce a change near the FAD cofactor that may relate to the change in oxidation kinetics. The pH-effect on the FADH· oxidation rate, which is rate-limited by the proton acceptor, does not seem to be due to a protein structural change near the FAD cofactor. Instead, a conserved glutamate in CRY-DASH may control the deprotonation of FADH· and give rise to the pH-effect.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Kiontke, S., Goebel, T., Brych, A., & Batschauer, A. (2020). DASH-type cryptochromes—solved and open questions. Journal of Biological Chemistry, 401(12), 1487–1493. https://doi.org/10.1515/hsz-2020-0182

    Article  CAS  PubMed  Google Scholar 

  2. Fortunato, A. E., Annunziata, R., Jaubert, M., Bouly, J.-P., & Falciatore, A. (2015). Dealing with light: The widespread and multitasking cryptochrome/photolyase family in photosynthetic organisms. Journal of Plant Physiology, 172, 42–54.

    Article  CAS  Google Scholar 

  3. Mueller, M., & Carell, T. (2009). Structural biology of DNA photolyases and cryptochromes. Current Opinion in Structural Biology, 19(3), 277–285. https://doi.org/10.1016/j.sbi.2009.05.003

    Article  CAS  Google Scholar 

  4. Batschauer, A., Banerjee, R., & Pokorny, R. (2007). Cryptochromes. Annual Plant Review, 30, 17–48. (Light and Plant Development).

    CAS  Google Scholar 

  5. Brudler, R., Hitomi, K., Daiyasu, H., Toh, H., Kucho, K., Ishiura, M., et al. (2003). Identification of a new cryptochrome class: Structure, function, and evolution. Molecular Cell, 11, 59–67.

    Article  CAS  Google Scholar 

  6. Ahmad, M. (2003). Cryptochromes and flavoprotein blue-light photoreceptors. In Handbook of photochemistry and photobiology, 4 (pp. 159–182). American Scientific Publishers.

  7. Sancar, A. (2003). Structure and function of DNA photolyase and cryptochrome blue-light photoreceptors. Chemical Reviews, 103, 2203–2237.

    Article  CAS  Google Scholar 

  8. Tagua, V. G., Pausch, M., Eckel, M., Gutierrez, G., Miralles-Duran, A., Sanz, C., et al. (2015). Fungal cryptochrome with DNA repair activity reveals an early stage in cryptochrome evolution. Proceedings of the National Academy of Sciences of the United States of America, 112(49), 15130–15135. https://doi.org/10.1073/pnas.1514637112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Ahmad, M. (2016). Photocycle and signaling mechanisms of plant cryptochromes. Current Opinion in Plant Biology, 33, 108–115. https://doi.org/10.1016/j.pbi.2016.06.013

    Article  CAS  PubMed  Google Scholar 

  10. Wang, J., Du, X., Pan, W., Wang, X., & Wu, W. (2015). Photoactivation of the cryptochrome/photolyase superfamily. Journal of Photochemistry and Photobiology C, 22, 84–102. https://doi.org/10.1016/j.jphotochemrev.2014.12.001

    Article  CAS  Google Scholar 

  11. Mueller, P., Brettel, K., Grama, L., Nyitrai, M., & Lukacs, A. (2016). Photochemistry of wild-type and N378D mutant E. coli DNA photolyase with oxidized FAD cofactor studied by transient absorption spectroscopy. ChemPhysChem, 17(9), 1329–1340. https://doi.org/10.1002/cphc.201501077

    Article  CAS  Google Scholar 

  12. Pokorny, R., Klar, T., Hennecke, U., Carell, T., Batschauer, A., & Essen, L.-O. (2008). Recognition and repair of UV lesions in loop structures of duplex DNA by DASH-type cryptochrome. Proceedings of the National Academy of Sciences of the United States of America, 105(52), 21023–21027. https://doi.org/10.1073/pnas.0805830106

    Article  PubMed  PubMed Central  Google Scholar 

  13. Sokolowsky, K., Newton, M., Lucero, C., Wertheim, B., Freedman, J., Cortazar, F., et al. (2010). Spectroscopic and thermodynamic comparisons of Escherichia coli DNA photolyase and Vibrio cholerae cryptochrome 1. Journal of Physics and Chemistry B, 114(20), 7121–7130. https://doi.org/10.1021/jp102275r

    Article  CAS  Google Scholar 

  14. Selby, C. P., & Sancar, A. (2006). A cryptochrome/photolyase class of enzymes with single-stranded DNA-specific photolyase activity. Proceedings of the National Academy of Sciences of the United States of America, 103, 17696–17700.

    Article  CAS  Google Scholar 

  15. Garcia-Esquivel, M., Esquivel-Naranjo, E. U., Hernandez-Onate, M. A., Ibarra-Laclette, E., & Herrera-Estrella, A. (2016). The Trichoderma atroviride cryptochrome/photolyase genes regulate the expression of blr1-independent genes both in red and blue light. Fungal Biology, 120(4), 500–512. https://doi.org/10.1016/j.funbio.2016.01.007

    Article  CAS  PubMed  Google Scholar 

  16. Wang, F., Song, X., Dong, X., Zhang, J., & Dong, C. (2017). DASH-type cryptochromes regulate fruiting body development and secondary metabolism differently than CmWC-1 in the fungus Cordyceps militaris. Applied Microbiology Biotechnology, 101(11), 4645–4657. https://doi.org/10.1007/s00253-017-8276-7

    Article  CAS  PubMed  Google Scholar 

  17. Castrillo, M., Garcia-Martinez, J., & Avalos, J. (2013). Light-dependent functions of the Fusarium fujikuroi CryD DASH cryptochrome in development and secondary metabolism. Applied Environmental Microbiology, 79(8), 2777–2788. https://doi.org/10.1128/AEM.03110-12

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Cohrs, K. C., & Schumacher, J. (2017). The two cryptochrome/photolyase family proteins fulfill distinct roles in DNA photorepair and regulation of conidiation in the gray mold fungus Botrytis cinerea. Applied Environmental Microbiology 83(17), e00812-00817/00818. https://doi.org/10.1128/AEM.00812-17.

  19. Holub, D., Kubar, T., Mast, T., Elstner, M., & Gillet, N. (2019). What accounts for the different functions in photolyases and cryptochromes: A computational study of proton transfers to FAD. Physical Chemistry Chemical Physics, 21(22), 11956–11966. https://doi.org/10.1039/c9cp00694j

    Article  CAS  PubMed  Google Scholar 

  20. Xu, L., Mu, W., Ding, Y., Luo, Z., Han, Q., Bi, F., et al. (2008). Active site of Escherichia coli DNA photolyase: Asn378 is crucial both for stabilizing the neutral flavin radical cofactor and for DNA repair. Biochemistry, 47(33), 8736–8743. https://doi.org/10.1021/bi800391j

    Article  CAS  PubMed  Google Scholar 

  21. Damiani, M. J., Nostedt, J. J., & O’Neill, M. A. (2011). Impact of the N5-proximal Asn on the thermodynamic and kinetic stability of the semiquinone radical in photolyase. Journal of Biology and Chemistry, 286(6), 4382–4391. https://doi.org/10.1074/jbc.M110.194696

    Article  CAS  Google Scholar 

  22. Muller, P., Brettel, K., Grama, L., Nyitrai, M., & Lukacs, A. (2016). Photochemistry of wild-type and N378D mutant E. coli DNA photolyase with oxidized FAD cofactor studied by transient absorption spectroscopy. ChemPhysChem, 17(9), 1329–1340.

    Article  Google Scholar 

  23. Burney, S., Wenzel, R., Kottke, T., Roussel, T., Hoang, N., Bouly, J.-P., et al. (2012). Single amino acid substitution reveals latent photolyase activity in arabidopsis cry1. Angewandte Chemie International Edition, 51(37), 9356–9360. https://doi.org/10.1002/anie.201203476

    Article  CAS  PubMed  Google Scholar 

  24. Worthington, E. N., Kavakli, I. H., Berrocal-Tito, G., Bondo, B. E., & Sancar, A. (2003). Purification and characterization of three members of the photolyase/cryptochrome family blue-light photoreceptors from Vibrio cholerae. Journal of Biological Chemistry, 278(40), 39143–39154. https://doi.org/10.1074/jbc.M305792200

    Article  CAS  Google Scholar 

  25. Immeln, D., Pokorny, R., Herman, E., Moldt, J., Batschauer, A., & Kottke, T. (2010). Photoreaction of plant and DASH cryptochromes probed by infrared spectroscopy: The neutral radical state of flavoproteins. The Journal of Physical Chemistry B, 114(51), 17155–17161. https://doi.org/10.1021/jp1076388

    Article  CAS  PubMed  Google Scholar 

  26. Berndt, A., Kottke, T., Breitkreuz, H., Dvorsky, R., Hennig, S., Alexander, M., et al. (2007). A novel photoreaction mechanism for the circadian blue light photoreceptor drosophila cryptochrome. Journal of Biological Chemistry., 282(17), 13011–13021. https://doi.org/10.1074/jbc.M608872200

    Article  CAS  Google Scholar 

  27. Paulus, B., Bajzath, C., Melin, F., Heidinger, L., Kromm, V., Herkersdorf, C., et al. (2015). Spectroscopic characterization of radicals and radical pairs in fruit fly cryptochrome—protonated and nonprotonated flavin radical-states. FEBS Journal, 282(16), 3175–3189. https://doi.org/10.1111/febs.13299

    Article  CAS  Google Scholar 

  28. Einholz, C., Nohr, D., Rodriguez, R., Topitsch, A., Kern, M., Goldmann, J., et al. (2021). pH-dependence of signaling-state formation in Drosophila cryptochrome. Archives of Biochemistry and Biophysics, 700, 108787. https://doi.org/10.1016/j.abb.2021.108787

    Article  CAS  PubMed  Google Scholar 

  29. Iwata, T., Zhang, Y., Hitomi, K., Getzoff, E. D., & Kandori, H. (2010). Key dynamics of conserved asparagine in a cryptochrome/photolyase family protein by fourier transform infrared spectroscopy. Biochemistry, 49(41), 8882–8891. https://doi.org/10.1021/bi1009979

    Article  CAS  PubMed  Google Scholar 

  30. Xu, L., Wen, B., Shao, W., Yao, P., Zheng, W., Zhou, Z., et al. (2019). Impacts of Cys392, Asp393, and ATP on the FAD binding, photoreduction, and the stability of the radical state of Chlamydomonas reinhardtii cryptochrome. ChemBioChem, 20(7), 940–948. https://doi.org/10.1002/cbic.201800660

    Article  CAS  PubMed  Google Scholar 

  31. Gindt, Y. M., Schelvis, J. P. M., Thoren, K. L., & Huang, T. H. (2005). Substrate binding modulates the reduction potential of DNA photolyase. Journal of the American Chemical Society, 127(30), 10472–10473. https://doi.org/10.1021/ja051441r

    Article  CAS  PubMed  Google Scholar 

  32. Schelvis, J. P. M., Ramsey, M., Sokolova, O., Tavares, C., Cecala, C., Connell, K., et al. (2003). Resonance Raman and UV–Vis spectroscopic characterization of FADH.bul. in the complex of photolyase with UV-damaged DNA. The Journal of Physical Chemistry B, 107(44), 12352–12362. https://doi.org/10.1021/jp034209l

    Article  CAS  Google Scholar 

  33. Zieba, A. A., Richardson, C., Lucero, C., Dieng, S. D., Gindt, Y. M., & Schelvis, J. P. M. (2011). Evidence for concerted electron proton transfer in charge recombination between FADH- and 306Trp· in Escherichia coli photolyase. Journal of the American Chemical Society, 133(20), 7824–7836. https://doi.org/10.1021/ja2001488

    Article  CAS  PubMed  Google Scholar 

  34. Balland, V., Byrdin, M., Eker, A. P. M., Ahmad, M., & Brettel, K. (2009). What makes the difference between a cryptochrome and DNA photolyase? A spectroelectrochemical comparison of the flavin redox transitions. Journal of the American Chemical Society, 131(2), 426–427. https://doi.org/10.1021/ja806540j

    Article  CAS  PubMed  Google Scholar 

  35. Muller, P., Bouly, J.-P., Hitomi, K., Balland, V., Getzoff, E. D., Ritz, T., et al. (2014). ATP Binding turns plant cryptochrome into an efficient natural photoswitch. Scientific Reports, 4, 5175. https://doi.org/10.1038/srep05175

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Gindt, Y. M., Messyasz, A., & Jumbo, P. I. (2015). Binding of substrate locks the electrochemistry of CRY-DASH into DNA repair. Biochemistry, 54(18), 2802–2805. https://doi.org/10.1021/acs.biochem.5b00307

    Article  CAS  PubMed  Google Scholar 

  37. Murphy, A. K., Tammaro, M., Cortazar, F., Gindt, Y. M., & Schelvis, J. P. M. (2008). Effect of the cyclobutane cytidine dimer on the properties of escherichia coli dna photolyase. The Journal of Physical Chemistry B, 112(47), 15217–15226. https://doi.org/10.1021/jp806526y

    Article  CAS  PubMed  Google Scholar 

  38. Li, J., Uchida, T., Todo, T., & Kitagawa, T. (2006). Similarities and differences between cyclobutane pyrimidine dimer photolyase and (6–4) photolyase as revealed by resonance Raman spectroscopy: Electron transfer from the FAD cofactor to ultraviolet-damaged DNA. Journal of Biological Chemistry, 281(35), 25551–25559. https://doi.org/10.1074/jbc.M604483200

    Article  CAS  Google Scholar 

  39. Damiani, M. J., Yalloway, G. N., Lu, J., McLeod, N. R., & O’Neill, M. A. (2009). Kinetic stability of the flavin semiquinone in photolyase and cryptochrome-DASH. Biochemistry, 48(48), 11399–11411. https://doi.org/10.1021/bi901371s

    Article  CAS  PubMed  Google Scholar 

  40. Klar, T., Pokorny, R., Moldt, J., Bratschauer, A., & Essen, L.-O. (2007). Cryptochrome 3 from Arabidopsis Thaliana: Structural and functional analysis of its complex with a folate light antenna. Journal of Molecular Biology, 366(3), 954–964

    Article  CAS  Google Scholar 

  41. Mees, A., Klar, T., Gnau, P., Hennecke, U., Eker, A. P. M., Carell, T., et al. (2004). Crystal structure of a photolyase bound to a CPD-like DNA lesion after in situ repair. Science (Washington, DC, United States), 306(5702), 1789–1793. https://doi.org/10.1126/science.1101598

    Article  CAS  Google Scholar 

  42. Wijaya, I. M. M., Iwata, T., Yamamoto, J., Hitomi, K., Iwai, S., Getzoff, E. D., et al. (2014). Flavin adenine dinucleotide chromophore charge controls the conformation of cyclobutane pyrimidine dimer photolyase α-helices. Biochemistry, 53(37), 5864–5875. https://doi.org/10.1021/bi500638b

    Article  CAS  PubMed  Google Scholar 

  43. Park, H.-W., Kim, S.-T., Sancar, A., & Deisenhofer, J. (1995). Crystal structure of DNA photolyase from Escherichia coli. Science (Washington, D. C.), 268(5219), 1866–1872. https://doi.org/10.1126/science.7604260

    Article  CAS  Google Scholar 

  44. Kapetanaki, S. M., Ramsey, M., Gindt, Y. M., & Schelvis, J. P. M. (2004). Substrate electric dipole moment exerts a pH-dependent effect on electron transfer in Escherichia coli photolyase. Journal of the American Chemical Society, 126(20), 6214–6215. https://doi.org/10.1021/ja049226i

    Article  CAS  PubMed  Google Scholar 

  45. Jordan, S. P., & Jorns, M. S. (1988). Evidence for a singlet intermediate in catalysis by Escherichia coli DNA photolyase and evaluation of substrate binding determinants. Biochemistry, 27(25), 8915–8923. https://doi.org/10.1021/bi00425a007

    Article  CAS  PubMed  Google Scholar 

  46. Cohen, J., & Schulten, K. (2007). O2 migration pathways are not conserved across proteins of a similar fold. Biophysical Journal, 93(10), 3591–3600. https://doi.org/10.1529/biophysj.107.108712

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Y.M.G. and J.P.M.S. acknowledge funding by the Department of Chemistry and Biochemistry, the CSAM Dean’s Office and the Provost’s Office at Montclair State University.

Funding

Department of Chemistry and Biochemistry, the CSAM Dean’s Office and the Provost’s Office at Montclair State University.

Author information

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Montclair State University, Montclair, NJ, 07043, USA

    Yvonne M. Gindt, Gabrielle Connolly, Amy L. Vonder Haar, Miryam Kikhwa & Johannes P. M. Schelvis

Authors
  1. Yvonne M. Gindt
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gabrielle Connolly
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Amy L. Vonder Haar
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Miryam Kikhwa
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Johannes P. M. Schelvis
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

YMG and JPMS designed experiments, YMG overexpressed, isolated and purified protein, ALVH and YMG performed stopped-flow experiments, GC, YMG and MK performed hand-mixing experiments, JPMS performed resonance Raman experiments, YMG and JPMS interpreted data and wrote manuscript.

Corresponding author

Correspondence to Johannes P. M. Schelvis.

Ethics declarations

Conflict of interest

The authors declare no conflicts or competing interests.

Additional information

Pushing the limits of flash photolysis to unravel the secrets of biological electron and proton transfer - a topical issue in honour of Klaus Brettel.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gindt, Y.M., Connolly, G., Vonder Haar, A.L. et al. Investigation of the pH-dependence of the oxidation of FAD in VcCRY-1, a member of the cryptochrome-DASH family. Photochem Photobiol Sci 20, 831–841 (2021). https://doi.org/10.1007/s43630-021-00063-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s43630-021-00063-5

Keywords

Search

Navigation