Department of Molecular Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Cracow, Poland.
Phys Chem Chem Phys. 2023 Aug 23;25(33):21935-21943. doi: 10.1039/d3cp02433d.
Quinone redox reactions involve a semiquinone (SQ) intermediate state. The catalytic sites in enzymes stabilize the SQ state various molecular interactions, such as hydrogen bonding to oxygens of the two carbonyls of the benzoquinone ring. To understand how these interactions contribute to SQ stabilization, we examined SQ in the quinone reduction site (Q) of cytochrome using electron paramagnetic resonance (ESEEM, HYSCORE) at the X-band and quantum mechanical (QM) calculations. We compared native enzyme (WT) with a H217R mutant (replacement of histidine that interacts with one carbonyl of the occupant of Q to arginine) in which the SQ stability has previously been shown to markedly increase. The N region of the HYSCORE 2D spectrum for SQ in WT had a shape typical of histidine residue, while in H217R, the spectrum shape changed significantly and appeared similar to the pattern described for SQ liganded natively by arginine in cytochrome . Parametrization of hyperfine and quadrupolar interactions of SQ with surrounding magnetic nuclei (H, N) allowed us to assign specific nitrogens of H217 or R217 as ligands of SQ in WT and H217R, respectively. This was further substantiated by qualitative agreement between the experimental (EPR-derived) and theoretical (QM-derived) parameters. The proton (H) region of the HYSCORE spectrum in both WT and H217R was very similar and indicative of interactions with two protons, which in view of the QM calculations, were identified as directly involved in the formation of a H-bond with the two carbonyl oxygens of SQ (interaction of H217 or R217 with O4 and D252 with O1). In view of these assignments, we explain how different SQ ligands effectively influence SQ stability. We also propose that the characteristic X-band HYSCORE pattern and parameters of H217R are highly specific to the interaction of SQ with the nitrogen of arginine. These features can thus be considered as potential markers of the interaction of arginine with SQ in other proteins.
醌的氧化还原反应涉及半醌（SQ）中间态。酶的催化位点通过各种分子相互作用稳定 SQ 态，例如与苯醌环的两个羰基的氧形成氢键。为了了解这些相互作用如何有助于 SQ 稳定，我们使用电子顺磁共振（ESEEM，HYSCORE）在 X 波段和量子力学（QM）计算研究了细胞色素中的醌还原位点（Q）中的 SQ。我们将天然酶（WT）与 H217R 突变体（取代与占据 Q 位置的羰基之一相互作用的组氨酸突变为精氨酸）进行了比较，先前的研究表明该突变体的 SQ 稳定性显著增加。WT 中 SQ 的 HYSCORE 2D 谱的 N 区域具有典型的组氨酸残基形状，而在 H217R 中，谱形状发生了显著变化，类似于描述的由细胞色素中的精氨酸天然配位的 SQ 的模式。SQ 与周围磁核（H、N）的超精细和四极相互作用的参数化允许我们分别将 WT 和 H217R 中 SQ 的特定 H217 或 R217 氮配体分配。这进一步通过实验（EPR 衍生）和理论（QM 衍生）参数之间的定性一致得到证实。WT 和 H217R 中的 HYSCORE 谱的质子（H）区域非常相似，表明与两个质子相互作用，根据 QM 计算，这两个质子被确定为直接参与与 SQ 的两个羰基氧形成氢键（H217 或 R217 与 O4 和 D252 与 O1 的相互作用）。鉴于这些分配，我们解释了不同的 SQ 配体如何有效影响 SQ 的稳定性。我们还提出，H217R 的特征 X 波段 HYSCORE 模式和参数高度特定于 SQ 与精氨酸氮的相互作用。因此，这些特征可以被认为是其他蛋白质中精氨酸与 SQ 相互作用的潜在标记。
