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Pulsed EPR Spectroscopy of 33S-Labeled Molybdopterin in Sulfite Oxidase

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Klein,  E. L.
Chemistry and Biochemistry, University of Arizona;
Research Department Neese, Max Planck Institute for Chemical Energy Conversion, Max Planck Society;

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Krämer,  T.
Research Department Neese, Max Planck Institute for Chemical Energy Conversion, Max Planck Society;

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Neese,  F.
Research Department Neese, Max Planck Institute for Chemical Energy Conversion, Max Planck Society;

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Citation

Enemark, J. H., Klein, E. L., Klein, E. L., Raitsimring, A. M., Davis, A. C., Astashkin, A. V., et al. (2013). Pulsed EPR Spectroscopy of 33S-Labeled Molybdopterin in Sulfite Oxidase. Talk presented at XVIth International Conference on Biological Inorganic Chemistry. Grenoble, France. 2013-07-22 - 2013-07-26.


Cite as: http://hdl.handle.net/21.11116/0000-0007-A685-7
Abstract
All known molybdenum-containing enzymes, with the exception of nitrogenase, contain a pyranopterin dithiolate (molybdopterin) moiety that coordinates to the metal through the two sulfur atoms of the ene-dithiolate (dithiolene) functionality to form the molybdenum cofactor. To date, molybdenum and tungsten enzymes are the only known examples of dithiolene coordination in biology. During catalysis, molybdenum enzymes pass through the paramagnetic Mo(V) state, and high resolution pulsed electron paramagnetic resonance (EPR) measurements of the hyperfine and nuclear quadrupole interactions of nearby magnetic nuclei (I ≠ 0) provide important experimental data about the structures of these transient Mo(V) states. For sulfite oxidase (SO), pulsed EPR studies of the Mo(V) center to determine the interactions of naturally abundant magnetic nuclei (1H, 14N, 31P) and isotropically enriched reagents (2H2O, H2, 17O, 35Cl-, 37Cl-, [33SO3]2-, [S17O3]2-), in parallel with density functional theory (DFT) calculations, have provided important insights into the structure of the active site and the reaction mechanism of the enzyme. However, extension of pulsed EPR methods to dithiolene S atoms presents major challenges. Naturally abundant 32S has no nuclear spin (I = 0), and isotropic labeling of model Mo-dithiolene compounds with 33S (I = 3/2) has not been feasible. However, the elucidation of the biosynthetic pathway of the molybdenum cofactor has opened up the possibility of direct incorporation of 33S-labeled sulfide into molybdopterin itself using controlled in vitro synthesis with purified proteins. Here we present the biosynthetic reactions for preparing 33S-labeled molybdenum cofactor in a catalytically active SO variant. The 33S hyperfine and nuclear quadrupole parameters for the Mo(V) state of the construct are determined experimentally by pulsed EPR methods and are compared with results from DFT calculations. To our knowledge, this is the first determination of the magnetic resonance parameters for a 33S atom in a dithiolene group.