Mechanism of thiocyanate dehydrogenase functioning based on structural data

Cover Page

Cite item

Full Text

Open Access Open Access
Restricted Access Access granted
Restricted Access Subscription Access

Abstract

Thiocyanate dehydrogenase is enzyme catalyzing transformation of a thiocyanate ion into a cyanate ion with outcome of two electrons, two protons and a neutral atom of sulphur. Earlier structures of thiocyanate dehydrogenase from Thioalkalivibrio paradoxus were solved. Despite not perfect quality of the structures (twinning and pronounced anisotropy of the crystals, incomplete occupancy of the copper ions, absence of data for complexes with analogues of the substrate), there was suggested a mechanism of the enzyme functioning based on those structures. Recently at atomic resolution there have been solved structures of a gene-modified copy of relative enzyme from Pelomicrobium methylotrophicum for free protein and its complex with thiourea. In the new structures copper ions of the active site possess complete occupancy. In these structures it is possible to reliably identify two conformations of the protein molecule with opened and closed active sites. The new structural high resolution data also allowed us to determine the presence of the superposition of different states of the copper ions for each of the two conformations. In each state the copper ions have different oxidation degrees, different corresponding ligands and partial occupancies. The ion charges were determined according to the ions coordination. In the protein molecule with the closed active site the complexes with inhibitor (thiourea ion) and molecular oxygen are observed. The complex with thiourea allows us to model binding of thiocyanate ion to the enzyme molecule. Taking into account the changes of the structures in the opened and closed conformations, a mechanism of the attacking oxygen ligand activation is suggested. A new scheme of the enzymatic reaction is discussed.

Full Text

Restricted Access

About the authors

K. M. Polyakov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Author for correspondence.
Email: kmpolyakov@gmail.com
Russian Federation, Moscow

S. Gavryushov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: kmpolyakov@gmail.com
Russian Federation, Moscow

References

  1. Tikhonova T.V., Sorokin D.Y., Hagen W.R., Khrenova M.G., Muyzer G., Rakitina T.V., Shabalin I.G., Trofimov A.A., Tsallagov S.I., Popov V.O. (2020) Trinuclear copper biocatalytic center forms an active site of thiocyanate dehydrogenase. Proc. Natl. Acad. Sci. USA. 117, 5280‒5290. https://doi.org/10.1073/pnas.1922133117
  2. Varfolomeeva L.A., Polyakov K.M., Komolov A.S., Rakitina T.V., Dergousova N.I., Dorovatovskii P.V., Boyko K.M., Tikhonova T.V., Popov V.O. (2023) Improvement of the diffraction properties of thiocyanate dehydrogenase crystals. Crystallography Repts. 68, 886−891. https://doi.org/10.1134/s1063774523600990
  3. Haltia T., Brown K., Tegoni M., Cambillau C., Saraste M., Mattila K., Djinovic-Carugo K. (2003) Crystal structure of nitrous oxide reductase from Paracoccus denitrificans at 1.6 Å resolution. Biochem. J. 369, 77−88. https://doi.org/10.1042/BJ20020782
  4. Krissinel E., Henrick K. (2007) Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774−797. https://doi.org/10.1016/j.jmb.2007.05.022
  5. Solomon E.I., Heppner D.E., Johnston E.M., Ginsbach J.W., Cirera J., Qayyum M., Kieber-Emmons M.T., Kjaergaard C.H., Hadt R.G., Tian L. (2014) Copper active sites in biology. Chem. Rev. 114, 3659−3853. https://doi.org/10.1021/cr400327t
  6. Rubino J.T., Frank K.J. (2012) Coordination chemistry of copper proteins: how nature handles a toxic cargo for essential function. J. Inorg. Biochem. 107, 129‒143. https://doi.org/10.1016/j.jinorgbio.2011.11.024
  7. Cotton F.A., Wilkinson G. (1980) Advanced Inorganic Chemistry, 4th ed. New York: John Wiley and Sons, 798–821.
  8. Balamurugan R., Palaniandavar M., Gopalan R.S. (2001) Trigonal planar copper(I) complex: synthesis, structure, and spectra of a redox pair of novel copper(II/I) complexes of tridentate bis(benzimidazol-2’-vl) ligand framework as models for electron-transfer copper proteins. Inorg. Chem. 40, 2246–2255. https://doi.org/10.1021/ic0003372
  9. Reinen D., Friebel C. (1984) Copper(2+) in 5-coordination: a case of a second-order Jahn-Teller effect. 2. Pentachlorocuprate(3-) and other CuIIL5 complexes: trigonal bipyramid or square pyramid? Inorg. Chem. 23, 791–798. https://doi.org/10.1021/ic00175a001
  10. Wansapura C.M., Juyong C., Simpson J.L., Szymanski D., Eaton G.R., Eaton S.S., Fox S. (2003) From planar toward tetrahedral copper(Il) complexes: structural and electron paramagnetic resonance studies of substituent steric effects in an extended class of pyrrolate-imine ligands. J. Coord. Chem. 56, 975–993. https://doi.org/10.1080/00958970310001607752
  11. Hatfield W.E. (1997) Handbook of Copper Compounds and Applications. Ed. Richardson H.W. New York: Marcel Dekker, 13–30.
  12. Raithby P.R., Shields G.P., Allen F.H., Motherwell W.D.S. (2000) Structure correlation study of four-coordinate copper(I) and (II) complexes. Acta Cryst. B56, 444–454. https://doi.org/10.1107/S0108768199016870
  13. Komori H., Sugiyama R., Kataoka K., Miyazaki K., Higuchi Y., Sakurai T. (2014) New insights into the catalytic active-site structure of multicopper oxidases. Acta Cryst. D70, 772–779. https://doi.org/10.1107/S1399004713033051
  14. Polyakov K.M., Gavryushov S., Ivanova S., Fedorova T.V., Glazunova O.A., Popov A.N., Koroleva O.V. (2017) Structural study of the X-ray-induced enzymatic reduction of molecular oxygen to water by Steccherinum murashkinskyi laccase: insights into the reaction mechanism. Acta Cryst. D73, 388–401. https://doi.org/10.1107/S2059798317003667
  15. Derewenda Z.S. (2010) Application of protein engineering to enhance crystallizability and improve crystal properties. Acta Cryst. D66, 604–615. doi: 10.1107/S090744491000644X.
  16. Vagin A., Teplyakov A. (1997) MOLREP: An automated program for molecular replacement. J. Appl. Сryst. 66, 22–25. https://doi.org/10.1107/S0021889897006766
  17. Murshudov G.N., Vagin A.A., Dodson E.J. (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Cryst. D53, 240–255. https://doi.org/10.1107/S0907444996012255
  18. McNicholas S., Potterton E., Wilson K.S., Noble M.E.M. (2011) Presenting your structures: the CCP4mg molecular-graphics software. Acta Cryst. D67, 386–394. https://doi.org/10.1107/S0907444910045749

Supplementary files

Supplementary Files
Action
1. JATS XML
Download
2. Fig. 1. Dimer pmTcDH from the structure of the complex with thiourea. Subunits in the “closed” and “open” conformations are shown as a ribbon model on the right and left, respectively. Copper ions of pmTcDH are shown as spheres. The thiourea molecule is represented as a ball-and-stick model.

Download (942KB)
Indexing metadata
3. Fig. 2. Superposition of the structures of pmTcDH subunits in the “closed” and “open” conformations. The light tone shows the course of the polypeptide chain of the subunit in the “closed” conformation. The dark tones show the loops of the subunit in the “open” conformation, the position of which differs in the subunits in the “closed” and “open” conformations. The spheres show the positions of copper ions for the subunit in the “closed” conformation. The thiourea molecule is shown as a ball-and-stick model. View from the side of the entrance to the active center.

Download (893KB)
Indexing metadata
4. Fig. 3. The active site of the pmTcDH subunit complex with thiourea in the “closed” conformation. a — Model of the complex and electron density (2Fobs–Fcalc) for the 1σ and 5σ levels. The first state (b) and the second state (c) of the active site of the pmTcDH subunit in the “closed” conformation. The amino acid residues of the active site and thiourea are presented as a ball-and-stick model. Water molecules are shown as small spheres. Copper ions in oxidation states II and I are shown as large spheres. The “attacking” water molecule is designated by W. Coordination bonds and hydrogen bonds with residues H101 and K68 are shown as dotted lines.

Download (889KB)
Indexing metadata
5. Fig. 4. The first state (a) and the second state (b) of the active site of the pmTcDH subunit in the “open” conformation of the structure of the complex with thiourea. The amino acid residues of the active site are presented as a ball-and-stick model. Water molecules are shown as small spheres. Copper ions in oxidation states II and I are shown as large spheres. The “attacking” water molecule is designated W. Coordination bonds and hydrogen bonds with the water molecule W are shown as dotted lines.

Download (724KB)
Indexing metadata
6. Fig. 5. The active site of the free pmTcDH subunit in the “closed” conformation. The amino acid residues of the active site and the water molecules are shown as a ball-and-stick model. Copper ions in oxidation states II and I are shown as large spheres. The “attacking” water molecule is designated W. Coordination bonds and hydrogen bonds with residues H101 and K68 are shown as dotted lines.

Download (469KB)
Indexing metadata
7. Fig. 6. Superposition of the active centers of the structure of the complex with thiourea in the “closed” and “open” conformations. The structure in the “open” conformation is shown in light tone. The fragment of the structure corresponding to the “closed” conformation is highlighted in dark tone. Copper ions in the structures in the “open” and “closed” conformations in oxidation states II and I are shown as large spheres (dark and light tone, respectively). The thiourea molecule is shown as a ball-and-stick model. The “attacking” water molecule is designated by W. Hydrogen bonds are shown as dotted lines. View from the side of the entrance to the active center. The side chain of Pro256 in the closed conformation blocks access to the active center of the enzyme.

Download (414KB)
Indexing metadata
8. Fig. 7. Proposed mechanism of the pmTcDH catalytic cycle in the reaction NCS– + H2O → NCO– + S0 + 2H+ + 2e–. Key coordination bonds are shown as light lines. Covalent bonds are shown as black lines. Dashed lines represent hydrogen bonds. Oxidized and reduced copper ions are shown as large spheres. Small short arrows show the displacement of copper ion Cu2 with the change in oxidation state. Long arrows show electron transfer as well as the movement of molecules in the active site. Open and closed boxes represent the “open” and “closed” conformations of the hbTcDH molecule, respectively.

Download (766KB)
Indexing metadata

Copyright (c) 2025 Russian Academy of Sciences