STRUCTURAL DETERMINATION OF PARAMAGNETIC CENTER IN PUTIDAREDOXIN

固相法测定顺磁性蛋白质的结构 而溶液核磁共振技术受到严重的谱线展宽的限制 原子核和原子核之间的强超精细相互作用 顺磁中心。这阻止了标准核磁共振结构的使用 方法在金属中心的几埃范围内。在这方面， 高频(140 GHz)脉冲Endor技术非常适合于 提取顺磁附近的超精细相互作用 中间。我们一直在调查一种2-铁-2-S铁氧还蛋白 恶臭还蛋白(PDX)，其中金属中心显示有效自旋 S=1/2的还原形式(Fe“-Fe”)，该簇通过 四个半胱氨酸硫醚。我们能够记录到140 GHz的自旋回波 在低温(10K)下检测到的光谱具有出色的 信噪比(&gt；50)，并确定g-各向异性到g，，=2.02 G_L=1.94。测量了Fe原子团簇的钛弛豫 在9K和40K下，我们发现了50GS的短T，在9K和更高的温度下 15-40K之间的复杂双指数行为，可能是由于 较高激发自旋态的热布居。一种特殊的恩多 研制了用于低伽马原子核高效辐照的谐振器 并在10K下记录了初步的C-Mims-Endor谱。 与之前文献中报道的Endor数据形成对比 EPR频率(35 GHz)，5特斯拉时的‘3C Larmor频率(53 MHz)与其他原子核很好地分开，我们观察到对称的 恩多线的形状围绕着拉莫尔频率。在“C”中统一使用 标记的蛋白质，观察到两个明显的超精细分裂， 我们将其归因于半胱氨酸亚甲基。进一步 选择性标记蛋白质的实验正在进行中，并将 允许对观察到的超精细耦合进行明确的分配。

Structural determination of paramagnetic proteins by solid state and solution NMR techniques is limited by severe line broadening due to the strong hyperfine interaction between the nuclei and the paramagnetic center. This prevents the use of standard NMR structural methods within a few angstroms of the metal center. In this context, high frequency (140 GHz) pulsed ENDOR techniques are ideally suited to extract hyperfine interactions in the vicinity of the paramagnetic center. We have been investigating a 2-Fe 2-S ferredoxin putidaredoxin (Pdx), where the metal center displays an effective spin S = 1/2 in the reduced form (Fe" - Fe") and the cluster is ligated by four cysteinyl sulfurs. We were able to record 140 GHz spin-echo detected spectra at low temperatures (10 K) with excellent signal-to-noise (> 50) and determine the g-anisotropy to g,, = 2.02 and g_L = 1.94. TI-relaxation of the Fe-cluster was measured between 9 and 40 K. We found short T,- values of 50 gs at 9 K and a more complex biexponential behavior between 15 - 40 K, likely due to the thermal population of higher excited spin states. A special ENDOR resonator for efficient irradiation of low-gamma nuclei was developed and preliminary "C-Mims-ENDOR spectra were recorded at 10 K. In contrast to ENDOR data previously reported in the literature at low EPR frequencies (35 GHz), the ' 3C larmor frequency at 5 Tesla (53 MHz) is well separated from other nuclei and we observe symmetric ENDOR lineshapes around the larmor frequency. In a "C uniformly labeled protein, two distinct hyperfine splitting have been observed, which we attribute to the cystein methylene groups. Further experiments with selectively labeled proteins are in progress and will permit unambiguous assignments of the observed hyperfine couplings.