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Trinuclear Copper Biocatalytic Center Forms an Active Site of Thiocyanate Dehydrogenase

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Trinuclear Copper Biocatalytic Center Forms an Active Site of Thiocyanate Dehydrogenase Trinuclear copper biocatalytic center forms an active site of thiocyanate dehydrogenase Tamara V. Tikhonovaa,1, Dimitry Y. Sorokina,b,1, Wilfred R. Hagenb, Maria G. Khrenovaa,c, Gerard Muyzerd, Tatiana V. Rakitinae,f, Ivan G. Shabaling, Anton A. Trofimovh, Stanislav I. Tsallagova, and Vladimir O. Popova,f,2 aResearch Centre of Biotechnology, Russian Academy of Sciences, 119071 Moscow, Russia; bDepartment of Biotechnology, Delft University of Technology, Delft, 2629 HZ The Netherlands; cDepartment of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia; dDepartment of Freshwater and Marine Ecology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, 1098 XH The Netherlands; eShemyakin- Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia; fKurchatov Complex NBICS-Technologies, National Research Center “Kurchatov Institute”, 123182 Moscow, Russia; gDepartment of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22903; and hEngelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia Edited by Edward I. Solomon, Stanford University, Stanford, CA, and approved January 27, 2020 (received for review December 29, 2019) Biocatalytic copper centers are generally involved in the activation via initial formation of (NCS)2Cu, comprises multiple stages, and and reduction of dioxygen, with only few exceptions known. gives sulfate as the final sulfur reaction product. Some sulfur- Here we report the discovery and characterization of a pre- oxidizing bacteria (SOB) known to play an important role in viously undescribed copper center that forms the active site of global geochemical cycles can utilize thiocyanate as the sole a copper-containing enzyme thiocyanate dehydrogenase (sug- source of energy and nitrogen. Until now, the only route of gested EC 1.8.2.7) that was purified from the haloalkaliphilic sul- thiocyanate microbial utilization that has been documented in- fur-oxidizing bacterium of the genus Thioalkalivibrio ubiquitous volved the hydrolytic cleavage of nitrile, leading to formation in saline alkaline soda lakes. The copper cluster is formed by of carbonyl sulfide and ammonia (18–20). However, some pre- three copper ions located at the corners of a near-isosceles tri- vious microbiological evidence showed that thiocyanate may be angle and facilitates a direct thiocyanate conversion into cya- degraded by cell-free extracts of haloalkaliphilic thiocyanate- nate, elemental sulfur, and two reducing equivalents without utilizing SOB in the presence of an electron acceptor via a involvement of molecular oxygen. A molecular mechanism of ca- chemical reaction producing cyanate and elemental sulfur as final talysis is suggested based on high-resolution three-dimensional products (21, 22): structures, electron paramagnetic resonance (EPR) spectroscopy, quantum mechanics/molecular mechanics (QM/MM) simulations, - - 0 + - N ≡ C-S + H2O → N ≡ C-O + S + 2H + 2e [1] kinetic studies, and the results of site-directed mutagenesis. Here we present firm evidence that a specific enzyme from the copper centers | thiocyanate dehydrogenase | crystal structure | EPR | haloalkaliphilic SOB of the genus Thioalkalivibrio can catalyze molecular mechanism this process. The enzyme, thiocyanate dehydrogenase (TcDH), reveals several features special to the chemistry of biological opper is widely utilized in life systems (1, 2). It is incorpo- copper sites. TcDH contains a unique metal site comprising Crated into proteins for the purposes of copper trafficking three copper ions in a configuration that enables two-electron and storage (3, 4) and electron transport and catalysis (5–7). oxidation of thiocyanate ion to cyanate and elemental sulfur Malfunctioning of copper homeostasis leads to a number of disease states (8, 9). Copper enzymes are important in the Significance biosynthesis of natural products, hormones and neurotrans- mitters, proton pumping for adenosine triphosphate synthesis, The bioinorganic chemistry of copper is currently confined to a and the metabolism of iron and copper (1, 2). They are also of few mononuclear and polynuclear clusters in less than two potential technological interest for construction of the anodes dozen different types of enzymes catalyzing diverse biological of (implantable) biofuel cells (10). reactions. Here we present an unpreviously reported type of a Copper enzymes comprise a rather diverse, but not numerous, polynuclear center consisting of three copper ions, which form group of biocatalysts. Over 20 copper-containing enzymes that the active site of a not previously described enzyme, thiocya- catalyze a wide-ranging group of chemical reactions have been nate dehydrogenase. Enzymatic thiocyanate degradation characterized so far (1, 2). With just a few exceptions [enzymes resides on the copper cluster and proceeds via a chemical re- participating in nitrogen metabolism, nitrite reductase (11) and action unmatched in the chemistry of thiocyanate. Our findings nitrous oxide reductase (12), and lytic polysaccharide mono- expand the knowledge on copper-containing enzymes and oxygenase (13) thought to utilize peroxide as an oxidant], all of reveal a catalytic machinery employing a trinuclear copper the other Cu-based biocatalysts use dioxygen as a reactant. With center. the help of copper proteins, dioxygen is activated for the mono- oxygenation, dioxygenation, or oxidation of substrate molecules, Author contributions: T.V.T., D.Y.S., and V.O.P. designed research; W.R.H., M.G.K., G.M., and can also undergo four-electron reduction to water. T.V.R., I.G.S., A.A.T., and S.I.T. performed research; T.V.T., D.Y.S., W.R.H., M.G.K., S.I.T., Copper-dependent catalysis resides on the redox properties of and V.O.P. analyzed data; and V.O.P. wrote the paper. + + the Cu2 /Cu couple and geometry and electronic structures of The authors declare no competing interest. the individual Cu-sites (1, 2). Quite a few Cu-centers that differ This article is a PNAS Direct Submission. in the number and geometry of the constituent copper atoms are Published under the PNAS license. known to date. Enzyme active centers were reported to contain Data deposition: The atomic coordinates and structure factors have been deposited in the metal clusters comprising one to four copper ions as well as other Protein Data Bank, http://www.wwpdb.org/ (PDB ID codes 6I3Q, 6UWE, 6SJI, and 6G50). moieties such as heme groups (14–16). 1T.V.T. and D.Y.S. contributed equally to this work. Thiocyanate is a relatively stable compound and is oxidized by 2To whom correspondence may be addressed. Email: [email protected]. dioxygen only under elevated temperatures and pressures (up to This article contains supporting information online at https://www.pnas.org/lookup/suppl/ 200 °C and 100 bar) in the presence of Cu(II) catalyst (17). doi:10.1073/pnas.1922133117/-/DCSupplemental. According to a suggested mechanism (17), the process proceeds First published February 24, 2020. 5280–5290 | PNAS | March 10, 2020 | vol. 117 | no. 10 www.pnas.org/cgi/doi/10.1073/pnas.1922133117 Downloaded by guest on September 27, 2021 without utilizing oxygen as a cosubstrate. We describe physi- K3Fe(CN)6. Low-potential electron acceptors, such as 2,3,5-tri- cochemical and catalytic properties of TcDH, solve and character- phenyltetrazolium chloride and methylene blue, were not reduced. ize its three-dimensional structure, provide data on the electronic Direct oxidation of thiocyanate by molecular oxygen was not state of the copper cluster, and propose and verify a possible observed. molecular mechanism of action. The degradation of thiocyanate was accompanied by the for- mation of one equivalent of cyanate and the reduction of two Results and Discussion equivalents of one-electron acceptors (ferricyanide, c550; SI Isolation and Characterization of TcDH. Appendix, Fig. S3). The formation of elemental sulfur as the Thiocyanate-utilizing potential within the genus Thioalkalivibrio. second product of the thiocyanate oxidation was confirmed by Haloalkaliphilic sulfur-oxidizing bacteria of the genus Thioalkalivibrio reversed-phase high-performance liquid chromatography (HPLC) can utilize sulfide, sulfur, or thiosulfate as energy sources during using biosulfur from Tv. paradoxus ARh1 as a marker (SI Ap- aerobic growth. The strains used in this study, Thioalkalivibrio pendix,Fig.S4)(25). paradoxus (ARh1) and Thioalkalivibrio thiocyanoxidans (ARh2 These results show that the thiocyanate-degrading enzyme and ARh4) (21, 22), can grow using thiocyanate as the sole source catalyzes the two-electron oxidation of thiocyanate to yield cyanate of energy and nitrogen. and elemental sulfur, coupled with electron transfer to an external For all of the three strains tested, polypeptide profiles of cells electron acceptor (cytochromes), and is thus a thiocyanate: cyto- grown with either thiosulfate or thiocyanate as an energy source chrome с oxidoreductase (or thiocyanate dehydrogenase, TсDH). showed a major protein with a molecular weight of ∼52 to 55 kDa According to the Enzyme Commission (EC) classification, TcDH unique to thiocyanate-grown cells (SI Appendix,Fig.S1A). An can be assigned to the ENZYME class 1.8.2., which includes oxi- increase in the copper concentration in the growth medium doreductases acting on a sulfur group of donors using cytochrome correlated both with an increase in the accumulation of this as an acceptor. protein in cell extracts
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