Klein, Eric L. and Belaidi, Abdel Ali and Raitsimring, Arnold M. and Davis, Amanda C. and Krämer, Tobias and Astashkin, Andrei V. and Neese, Frank and Schwarz, Günter and Enemark, John H. (2014) Pulsed Electron Paramagnetic Resonance Spectroscopy of 33S-Labeled Molybdenum Cofactor in Catalytically Active Bioengineered Sulfite Oxidase. Inorganic Chemistry, 53 (2). pp. 961-971. ISSN 0020-1669

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Abstract

Molybdenum enzymes contain at least one pyranopterin dithiolate (molybdopterin, MPT) moiety that coordinates Mo through two dithiolate (dithiolene) sulfur atoms. For sulfite oxidase (SO), hyperfine interactions (hfi) and nuclear quadrupole interactions (nqi) of magnetic nuclei (I ≠ 0) near the Mo(V) (d1) center have been measured using high-resolution pulsed electron paramagnetic resonance (EPR) methods and interpreted with the help of density functional theory (DFT) calculations. These have provided important insights about the active site structure and the reaction mechanism of the enzyme. However, it has not been possible to use EPR to probe the dithiolene sulfurs directly since naturally abundant 32S has no nuclear spin (I = 0). Here we describe direct incorporation of 33S (I = 3/2), the only stable magnetic sulfur isotope, into MPT using controlled in vitro synthesis with purified proteins. The electron spin echo envelope modulation (ESEEM) spectra from 33S-labeled MPT in this catalytically active SO variant are dominated by the “interdoublet” transition arising from the strong nuclear quadrupole interaction, as also occurs for the 33S-labeled exchangeable equatorial sulfite ligand [Klein, E. L., et al. Inorg. Chem. 2012, 51, 1408−1418]. The estimated experimental hfi and nqi parameters for 33S (aiso = 3 MHz and e2Qq/h = 25 MHz) are in good agreement with those predicted by DFT. In addition, the DFT calculations show that the two 33S atoms are indistinguishable by EPR and reveal a strong intermixing between their out-of-plane pz orbitals and the dxy orbital of Mo(V).

Item Type:	Article
Keywords:	Pulsed Electron; Paramagnetic Resonance Spectroscopy; 33S-Labeled Molybdenum Cofactor; Catalytically Active; Bioengineered Sulfite Oxidase;
Academic Unit:	Faculty of Science and Engineering > Computer Science Faculty of Science and Engineering > Research Institutes > Hamilton Institute
Item ID:	15501
Identification Number:	https://doi.org/10.1021/ic4023954
Depositing User:	Tobias Kraemer
Date Deposited:	15 Feb 2022 12:42
Journal or Publication Title:	Inorganic Chemistry
Publisher:	ACS Publications
Refereed:	Yes
URI:	Publisher
Use Licence:	This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here

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