Busse S C, La Mar G N, Yu L P, Howard J B, Smith E T, Zhou Z H, Adams M W
Department of Chemistry, University of California, Davis 95616.
Biochemistry. 1992 Dec 1;31(47):11952-62. doi: 10.1021/bi00162a038.
The 3Fe forms of ferredoxins (Fd) from the hyperthermophilic archaebacteria Pyrococcus furiosus (Pf) and Thermococcus litoralis (Tl) have been investigated by 1H NMR. A combination of one-dimensional nuclear Overhauser and two-dimensional NOESY and bond correlation spectroscopy provides the assignment of the aromatic residues, one conserved valine, and the location of the signals for each of the three cysteines coordinated to the clusters. Dipolar contacts between the Trp 2 and Tyr 46 in Pf Fd and from an invariant phenylalanine to an invariant valine and a cluster cysteine in both Fd confirm a folding pattern for these proteins that is very similar to that of the crystallographically characterized ferredoxin from the mesophile Desulfovibro gigas. The sequence-specific assignment of the buried cysteine near the invariant phenylalanine has been made. The temperature dependence of the contact-shifted cysteinyl residues reveals a distinct 2:1 asymmetry in the magnetic coupling among the three high-spin ferric ions, in that one cysteine exhibits Curie behavior, while the other two cysteines display anti-Curie behavior. These magnetic properties are rationalized qualitatively on the basis of a magnetic coupling scheme where two iron couple to yield an intermediate spin of 2 which couples to the remaining S = 5/2 iron to yield the total cluster spin 1/2. This magnetic asymmetry appears to be a characteristic feature of oxidized 3 Fe clusters. Pf Fd also undergoes a dynamic equilibrium between two alternate forms that differ slightly in the environment of two of the coordinated cysteines. Analysis of the pattern of the contact shifts for the three cysteines in the two ferredoxins suggests that the cysteine coordinated to the unique iron does not have the same sequence origin.
利用核磁共振氢谱（1H NMR）对嗜热古细菌激烈火球菌（Pf）和嗜热栖热放线菌（Tl）中的3Fe形式铁氧化还原蛋白（Fd）进行了研究。通过一维核Overhauser效应、二维NOESY和键相关光谱的组合，确定了芳香族残基、一个保守缬氨酸以及与簇配位的三个半胱氨酸中每个半胱氨酸信号的位置。Pf Fd中Trp 2和Tyr 46之间以及两种Fd中一个不变苯丙氨酸与一个不变缬氨酸和一个簇半胱氨酸之间的偶极接触，证实了这些蛋白质的折叠模式与中温嗜热栖热放线菌中晶体学表征的铁氧化还原蛋白非常相似。已对不变苯丙氨酸附近埋藏的半胱氨酸进行了序列特异性归属。接触位移的半胱氨酸残基的温度依赖性揭示了三个高自旋铁离子之间磁耦合的明显2:1不对称性，即一个半胱氨酸表现出居里行为，而其他两个半胱氨酸表现出反居里行为。基于一种磁耦合方案对这些磁性性质进行了定性解释，其中两个铁耦合产生中间自旋2，该中间自旋与剩余的S = 5/2铁耦合产生总簇自旋1/2。这种磁不对称似乎是氧化3Fe簇的一个特征。Pf Fd还在两种交替形式之间经历动态平衡，这两种形式在两个配位半胱氨酸的环境中略有不同。对两种铁氧化还原蛋白中三个半胱氨酸的接触位移模式的分析表明，与独特铁配位的半胱氨酸并非来自相同的序列起源。
