Unambiguous determination of the g-matrix orientation in a neutral flavin radical by pulsed electron-nuclear double resonance at 94 GHz
Unambiguous determination of the g-matrix orientation in a neutral flavin radical by pulsed electron-nuclear double resonance at 94 GHz
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DOI：
10.1021/ja051572s
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发表时间：
2005-08-10
影响因子：
15
通讯作者：
Weber, S
中科院分区：
文献类型：
--
作者：
Kay, CWM;Bittl, R;Weber, S
最近观察到的光诱导自由基对，包括黄素自由基和氧化的氨基酸残基在各种蓝光敏感的蛋白质突出了需要获得一个更完整的了解黄素自由基的电子结构。特别是，通过g张量量化的塞曼相互作用的各向异性的精确知识对于通过电子顺磁共振（EPR）实现黄素自由基的明确识别是必要的。在最近的一项研究中，我们用360 GHz/12.8 T的高频/强磁场EPR确定了蛋白质结合的中性黄素自由基的g张量主值。然而，在这些实验中，g的主轴的取向不能明确地建立相对于异咯嗪部分的分子框架。在本文中，我们通过95 GHz/3.5 T（W波段）的脉冲电子-核双共振（ENDOR）解决了这一模糊性。在这样高的微波频率和磁场值，theganisotropy提供了改进的光谱分辨率相比，ENDOR实验在传统的9.5 GHz/0.35 mT（X波段）。这使得人们能够利用塞曼磁选择从冷冻溶液中的无序样品中获得类似单晶的数据。利用这种取向选择的实验使我们能够利用异咯嗪环C（8α）上甲基质子的超精细耦合来确定分子框架与g主轴之间的角度。令人惊讶的是，FADH·中的g-张量并不像人们对1，3-半苯醌自由基所预期的那样取向。对于后者，g的X轴通常平分沿CO键的两个轴之间的较小角度沿着。在FADH·中，N（5）和C（4a）上的大自旋密度显然有助于g张量轴的显著（44°）重定向。
The recent observation of photoinduced radical pairs comprising a flavin radical and an oxidized amino acid residue in various blue-light-sensitive proteins has highlighted the need to gain a more complete understanding of the electronic structure of flavin radicals. In particular, precise knowledge of the anisotropy of the Zeeman interaction quantified by theg-tensor is necessary for attaining an unambiguous identification of flavin radicals by electron paramagnetic resonance (EPR). In a recent study of a protein-bound neutral flavin radical, we have determined the principal values of theg-tensor using high-frequency/high magnetic field EPR performed at 360 GHz/12.8 T. However, in those experiments, the orientation of the principal axes ofgcould not be unambiguously established with respect to the molecular frame of the isoalloxazine moiety. In this contribution we resolve this ambiguity by pulsed electron−nuclear double resonance (ENDOR) at 95 GHz/3.5 T (W-band). At such high values of the microwave frequency and the magnetic field, theganisotropy provides improved spectral resolution compared to an ENDOR experiment performed at conventional 9.5 GHz/0.35 mT (X-band). This enables one to utilize Zeeman magnetoselection to obtain single-crystal-like data from disordered samples in frozen solution. Experiments exploiting this orientation selection have allowed us to use the hyperfine coupling of the methyl protons at C(8α) of the isoalloxazine ring to determine the angle between the molecular frame and the principal axes ofg. Quite surprisingly, theg-tensor in FADH•is not oriented as one would have expected for a 1,3-semibenzoquinone radical. For the latter, theX-axis ofgcommonly bisects the smaller angle between the two axes along the CO bonds. In FADH•, the large spin density on N(5) and C(4a) apparently contributes to a significant (44°) reorientation of theg-tensor axes.