Abstract
Glucosinolates are plant thioglucosides, which act as chemical defenses. Upon tissue damage, their myrosinase-catalyzed hydrolysis yields aglucones that rearrange to toxic isothiocyanates. Specifier proteins such as thiocyanate-forming protein from Thlaspi arvense (TaTFP) are non-heme iron proteins, which capture the aglucone to form alternative products, e.g. nitriles or thiocyanates. To resolve the electronic state of the bound iron cofactor in TaTFP, we applied continuous wave electron paramagnetic resonance (CW EPR) spectroscopy at X-and Q-band frequencies (∼9.4 and ∼34 GHz). We found characteristic features of high spin and low spin states of a d5 electronic configuration and local rhombic symmetry during catalysis. We monitored the oxidation states of bound iron during conversion of allylglucosinolate by myrosinase and TaTFP in presence and absence of supplemented Fe2+. Without added Fe2+, most high spin features of bound Fe3+ were preserved, while different g’-values of the low spin part indicated slight rearrangements in the coordination sphere and/or structural geometry. We also examined involvement of the redox pair Fe3+/Fe2 in samples with supplemented Fe2+. The absence of any EPR signal related to Fe3+ or Fe2+ using an iron-binding deficient TaTFP variant allowed us to conclude that recorded EPR signals originated from the bound iron cofactor.
Acknowledgments
We thank Heike Schimm (MLU, Halle) for technical support with the EPR measurements and Anita Backenköhler (TU Braunschweig) for activitiy tests.
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Author contributions: HHH: investigation, measurements, methodology, writing original draft, review and editing. NS: protein expression and purification, sample preparation, writing and editing. DEB: Docking experiments and writing. WB: conceptualization. UW: supervision, conceptualization, methodology, writing, review and editing. DH: supervision, conceptualization, methodology, funding acquisition, writing, review and editing.
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Research funding: This work was financially supported by the European Union and the State of Saxony-Anhalt under grant number ZS/2016/06/79740.
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Conflict of interest statement: The Authors declare no conflict of interests.
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