Pulsed Electron Paramagnetic Resonance Spectroscopy of 33S-Labeled Molybdenum Cofactor in Catalytically Active Bioengineered Sulfite Oxidase

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• 作者：Eric L. Klein ; Abdel Ali Belaidi ; Arnold M. Raitsimring ; Amanda C. Davis ; Tobias Kr?mer ; Andrei V. Astashkin ; Frank Neese ; Günter Schwarz ; John H. Enemark
• 刊名：Inorganic Chemistry
• 出版年：2014
• 出版时间：January 21, 2014
• 年：2014
• 卷：53
• 期：2
• 页码：961-971
• 全文大小：585K
• ISSN：1520-510X

文摘

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 鈮?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 鈥渋nterdoublet鈥?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鈭?418]. The estimated experimental hfi and nqi parameters for ³³S (a_iso = 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 p_z orbitals and the d_xy orbital of Mo(V).