Shergill J K, Joannou C L, Mason J R, Cammack R
Center for the Study of Metals in Biology and Medicine, King's College, University of London, U.K.
Biochemistry. 1995 Dec 26;34(51):16533-42. doi: 10.1021/bi00051a001.
One- and two-dimensional (1D and 2D) electron spin-echo envelope modulation (ESEEM) spectroscopy has been used to investigate the ligand environment of the [2Fe-2S] cluster from the terminal dioxygenase (ISPBED) of the Pseudomonas putida benzene dioxygenase complex. The modulation frequencies observed in the 0.5-8.5 MHz region of the Fourier transforms of 1D and 2D ESEEM spectra measured across the electron paramagnetic resonance (EPR) absorbance envelope (from gz through to gx) are consistent with their assignment to two 14N nuclei. Using hyperfine sublevel correlation spectroscopy (HYSCORE), two sets of correlated double quantum transitions sharing the same hyperfine coupling were observed and were identified as being due to the same two 14N nuclei. On the basis of the isotropic hyperfine and quadrupolar couplings estimated for these 14N nuclei [N(1), Aiso = 3.6 MHz and e2qQ = 2.2-2.8 MHz; N(2), Aiso = 4.8 MHz and e2qQ = 2.2-2.4 MHz], the ESEEM pattern of ISPBED is assigned to two histidine nitrogens which are directly coordinated to the reduced iron-sulfur cluster. Bonding parameters of the two [14N]histidine ligands were calculated from these hyperfine couplings. The primary covalent contributions to the hyperfine interaction arise from 14N-to-Fe2+ sigma bonds. For N(1), our analysis of the percentage of unpaired 2s and 2p electrons gave f2s approximately 1.3% and f2p approximately 0.2%, while values of f2s approximately 1.7% and f2p approximately 1.4% were estimated for N(2). Comparison of these values with those determined from electron nuclear double resonance (ENDOR) data of the Rieske-type [2Fe-2S] center of Pseudomonas cepacia phthalate dioxygenase [Gurbiel, R. J., Batie, C. J., Sivaraja, M., True, A. E., Fee, J. A., Hoffman, B. M., & Ballou, D. P. (1989) Biochemistry 28, 4861-4871] indicates an apparent reduction in unpaired electron spin density residing on the two 14N ligands of ISPBED. Analysis of slices of the HYSCORE spectrum has provided evidence for another 14N nucleus (A approximately 1.1 MHz, e2qQ = 3.3 MHz), which we have attributed to a weakly coupled peptide nitrogen, similar to those observed for ferredoxin-type [2Fe-2S] clusters. This type of weak interaction has not been previously described by the detailed ENDOR and ESEEM studies of Rieske-type centers. The resolution of the spectra demonstrates the effectiveness of 2D ESEEM for the disentanglement of multiple hyperfine interactions to metalloprotein centers.
一维和二维（1D和2D）电子自旋回波包络调制（ESEEM）光谱已被用于研究恶臭假单胞菌苯双加氧酶复合物末端双加氧酶（ISPBED）中[2Fe-2S]簇的配体环境。在横跨电子顺磁共振（EPR）吸收包络（从gz到gx）测量的1D和2D ESEEM光谱的傅里叶变换的0.5 - 8.5 MHz区域中观察到的调制频率与它们归属于两个14N核一致。使用超精细亚能级相关光谱（HYSCORE），观察到两组共享相同超精细耦合的相关双量子跃迁，并被确定为归因于相同的两个14N核。基于为这些14N核估计的各向同性超精细和四极耦合[N(1)，Aiso = 3.6 MHz且e2qQ = 2.2 - 2.8 MHz；N(2)，Aiso = 4.8 MHz且e2qQ = 2.2 - 2.4 MHz]，ISPBED的ESEEM模式被归因于两个直接与还原态铁硫簇配位的组氨酸氮。从这些超精细耦合计算出两个[14N]组氨酸配体的键合参数。对超精细相互作用的主要共价贡献来自14N到Fe2+的σ键。对于N(1)，我们对未配对2s和2p电子百分比的分析得出f2s约为1.3%且f2p约为0.2%，而对于N(2)估计f2s约为1.7%且f2p约为1.4%。将这些值与从洋葱假单胞菌邻苯二甲酸双加氧酶的Rieske型[2Fe-2S]中心的电子核双共振（ENDOR）数据确定的值进行比较[Gurbiel, R. J., Batie, C. J., Sivaraja, M., True, A. E., Fee, J. A., Hoffman, B. M., & Ballou, D. P. (1989) Biochemistry 28, 4861 - 4871]表明，ISPBED的两个14N配体上未配对电子自旋密度明显降低。对HYSCORE光谱切片的分析提供了另一个14N核（A约为1.1 MHz，e2qQ = 3.3 MHz）的证据，我们将其归因于一个弱耦合的肽氮，类似于在铁氧化还原蛋白型[2Fe-2S]簇中观察到的情况。这种弱相互作用类型以前在Rieske型中心的详细ENDOR和ESEEM研究中未被描述。光谱的分辨率证明了2D ESEEM在解开与金属蛋白中心的多个超精细相互作用方面的有效性。
