Active site structure of Rieske-type proteins: Electron nuclear double resonance studies of isotopically labeled phthalate dioxygenase from Pseudomonas cepacia and Rieske protein from Rhodobacter caysulatus and molecular modeling studies of a Rieske cente
Active site structure of Rieske-type proteins: Electron nuclear double resonance studies of isotopically labeled phthalate dioxygenase from Pseudomonas cepacia and Rieske protein from Rhodobacter caysulatus and molecular modeling studies of a Rieske cente
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DOI：
10.1021/bi960380u
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发表时间：
1996-06-18
期刊：
影响因子：
2.9
通讯作者：
Hoffman, BM
中科院分区：
文献类型：
--
作者：
Gurbiel, RJ;Doan, PE;Hoffman, BM
记录了洋葱假单胞菌的[δ-N-15，ε-N-14]-组氨酸标记的邻苯二甲酸酯双加氧酶（PDO）的连续波电子核双共振（CW ENDOR）光谱，发现与以前记录的[δ，E-N-15（2）]组氨酸标记蛋白质的光谱几乎相同[Gurbiel，R. J.，贝西角J.，Sivaraja，M.，没错A E、费，J.A.，霍夫曼，B。M.，& Chaanu，D.（1989）Biochemistry，28，4861-4871]。因此，两个组氨酸残基，先前显示连接簇中的一个铁[参见：Gurbiel等，1989）]，都在其咪唑环的N（δ）位置配位金属。脉冲ENDOR研究表明，在用N-15均匀标记的荚膜红杆菌细胞色素bc（1）复合物样品中，可以从Rieske蛋白中观察到组氨酸咪唑环的“远程”非配位氮，但在用[δ-N-15，ε-N-14]组氨酸标记的PDO样品中却观察不到，但在Rh样品中很容易观察到该原子。这证实了CW ENDOR研究的结论，即PDO中心的两种配体的连接仅通过N（δ）进行。修改以前用于模拟N-14 ENDOR光谱的算法，使我们能够计算光谱，而没有任何约束的超精细和四极张量的相对取向。该新算法用于分析当前和先前公布的光谱，并且给出了N-Fe-N角和咪唑环旋转角的略微不同的值[参见：Gurbiel等人（1989）Gurbiel，R. J.，Ohnishi，T.，Robertson，D. E、Daldal，F.，和霍夫曼，B. M.（1991）Biochemistry 30，11579-11584]。这一分析使我们能够改进[2Fe-2S] Rieske型簇合物的拟议结构，并使这些新中心的一些性质合理化。虽然从Rh，capsulatus细胞色素bc（1）复合物的光谱分辨率略低于PDO样品，我们的分析仍然允许的结论，即集群的几何形状基本上是相同的所有Rieske和Rieske型蛋白质。从光谱结果推断的结构约束使我们能够应用距离几何的原则，以达到可能的三维模型的活性位点结构的Rieske蛋白质从Rh。囊状的从这个测试案例的结果表明，类似的程序应该是普遍有用的金属蛋白质系统。我们还记录了脉冲和CW ENDOR光谱的Fe-57-标记的PDO，并得到的数据被用来推导出完整的超精细张量的Fe（III）和Fe（II）离子，包括它们的取向相对于g张量。三价铁离子的A张量名义上是各向同性的，而亚铁离子的A张量是轴向的，A（平行于）> A（垂直于）;两个张量都与观察到的g张量一致，亚铁离子的A（平行于）沿着最大g值g（1）。对[2Fe-2S]（+）团簇中自旋耦合的现有理论进行了改进，并对实验结果进行了验证，得出结论：现有理论不足以完全描述实验结果。
Continuous wave electron nuclear double resonance (CW ENDOR) spectra of [delta-N-15,epsilon-N-14]-histidine-labeled phthalate dioxygenase (PDO) from Pseudomonas cepacia were recorded and found to be virtually identical to those previously recorded from [delta,E-N-15(2)]histidine-labeled protein [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]. Thus, the two histidine residues, previously shown to ligate one of the irons in the cluster [cf: Gurbiel ct al. 1989)], both coordinate the metal at the N(delta) position of their imidazole rings. Pulsed ENDOR studies showed that the ''remote'', noncoordinating nitrogen of the histidine imidazole ring could be observed from the Rieske protein in a sample of Rhodobacter capsulatus cytochrome bc(1) complex uniformly labeled with N-15 but not in a sample of PDO labeled with [delta-N-15,epsilon-N-14]histidine, but this atom was easily observed with a sample of Rh. capsulatus cytochrome bc(1) complex that had been uniformly labeled with N-15; this confirmed the conclusion from the CW ENDOR studies that ligation is exclusively via N(delta) for both ligands in the PDO center. Modifications in the algorithms previously used to simulate N-14 ENDOR spectra permitted us to compute spectra without any constraints on the relative orientation of hyperfine and quadrupole tensors. This new algorithm was used to analyze current and previously published spectra, and slightly different values for the N-Fe-N angle and imidazole ring rotation angles are presented [cf: Gurbiel et al. (1989) Gurbiel, R. J., Ohnishi, T., Robertson, D. E., Daldal, F., and Hoffman, B. M. (1991) Biochemistry 30, 11579-11584]. This analysis has permitted us to refine the proposed structure of the [2Fe-2S] Rieske-type cluster and rationalize some of the properties of these novel centers. Although the spectra of cytochrome bc(1) complex from Rh, capsulatus are of somewhat lower resolution than those obtained with samples of PDO, our analysis nevertheless permits the conclusion that the geometry of the cluster is essentially the same for all Rieske and Rieske-type proteins. Structural constraints inferred from the spectroscopic results permitted us to apply the principles of distance geometry to arrive at possible three-dimensional models of the active site structure of Rieske protein from Rh. capsulatus. Results from this test case indicate that similar procedures should be generally useful in metalloprotein systems. We also recorded the pulsed and CW ENDOR spectra of Fe-57-labeled PDO, and the resulting data were used to derive the full hyperfine tensors for both Fe(III) and Fe(II) ions, including their orientations relative to the g tenser. The A tensor of the ferric ion is nominally isotropic, while the A tensor of the ferrous ion is axial, having A(parallel to) > A(perpendicular to); both tensors are coincident with the observed g tensor, with A(parallel to) of the ferrous ion lying along the maximum g-value, g(1). These results were examined using refinements of existing theories of spin-coupling in [2Fe-2S](+) clusters, and it is concluded that current theories are not adequate to fully describe the experimental results.