Klein Eric L, Belaidi Abdel Ali, Raitsimring Arnold M, Davis Amanda C, Krämer Tobias, Astashkin Andrei V, Neese Frank, Schwarz Günter, Enemark John H
Department of Chemistry and Biochemistry, The University of Arizona , 1306 E. University Boulevard, Tucson, Arizona 85721-0041, United States.
Inorg Chem. 2014 Jan 21;53(2):961-71. doi: 10.1021/ic4023954. Epub 2014 Jan 3.
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) (d(1)) 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 (32)S has no nuclear spin (I = 0). Here we describe direct incorporation of (33)S (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 (33)S-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 (33)S-labeled exchangeable equatorial sulfite ligand [ Klein, E. L., et al. Inorg. Chem. 2012 , 51 , 1408 - 1418 ]. The estimated experimental hfi and nqi parameters for (33)S (aiso = 3 MHz and e(2)Qq/h = 25 MHz) are in good agreement with those predicted by DFT. In addition, the DFT calculations show that the two (33)S atoms are indistinguishable by EPR and reveal a strong intermixing between their out-of-plane pz orbitals and the dxy orbital of Mo(V).
钼酶含有至少一个通过两个二硫醇盐（二硫烯）硫原子与钼配位的吡喃蝶呤二硫醇盐（钼蝶呤，MPT）部分。对于亚硫酸盐氧化酶（SO），已使用高分辨率脉冲电子顺磁共振（EPR）方法测量了Mo(V)（d(1)）中心附近磁性核（I≠0）的超精细相互作用（hfi）和核四极相互作用（nqi），并借助密度泛函理论（DFT）计算进行了解释。这些为酶的活性位点结构和反应机制提供了重要见解。然而，由于天然丰度的(32)S没有核自旋（I = 0），因此无法使用EPR直接探测二硫烯硫。在这里，我们描述了使用纯化蛋白质的体外可控合成将唯一稳定的磁性硫同位素(33)S（I = 3/2）直接掺入MPT。这种具有催化活性的SO变体中(33)S标记的MPT的电子自旋回波包络调制（ESEEM）光谱主要由强核四极相互作用产生的“双二重态间”跃迁主导，(33)S标记的可交换赤道亚硫酸盐配体也是如此[Klein, E. L.,等人。无机化学。2012年，51，1408 - 1418]。(33)S的估计实验hfi和nqi参数（aiso = 3 MHz和e(2)Qq/h = 25 MHz）与DFT预测的参数高度吻合。此外，DFT计算表明，两个(33)S原子在EPR中无法区分，并揭示了它们的面外pz轨道与Mo(V)的dxy轨道之间有强烈的混合。
