RESONANCE RAMAN EVIDENCE FOR AN FE-O-FE CENTER IN STEAROYL-ACP DESATURASE - PRIMARY SEQUENCE IDENTITY WITH OTHER DIIRON-OXO PROTEINS
RESONANCE RAMAN EVIDENCE FOR AN FE-O-FE CENTER IN STEAROYL-ACP DESATURASE - PRIMARY SEQUENCE IDENTITY WITH OTHER DIIRON-OXO PROTEINS
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DOI：
10.1021/bi00209a008
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发表时间：
1994-11-01
期刊：
影响因子：
2.9
通讯作者：
SANDERSLOEHR, J
中科院分区：
文献类型：
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作者：
FOX, BG;SHANKLIN, J;SANDERSLOEHR, J
植物中的硬脂酰-ACP Delta(9)去饱和酶是越来越多的含有氧桥联或羟桥联双铁簇的蛋白质的新例子。根据提供簇配体的初级序列基序的不同，以及X射线结晶学阐明的结构差异，我们现在提出，目前已知的可溶性双铁氧合蛋白可分为两类，I类包括三氯氰菊酯、肌红素，可能还有紫色酸性磷酸酶。第二类包括核糖核苷酸还原酶、细菌碳氢化合物羟基酶(甲烷单加氧酶、甲苯-4-单加氧酶和苯酚羟基酶)、红蛋白和硬脂酰-ACP去饱和酶。通过共振拉曼光谱，我们在Caor-stearoyl-ACP Delta(9)脱饱和度酶中检测到了对称(v(S)=519 cm(-1))和不对称(v(As)=747 cm(-1))振动模式，这是典型的矿桥联双铁簇合物。在(H2O)-O-18中，这两个频率分别移动了-18和-34 cm(-1)，证明桥联配体可以很容易地与溶剂(t(1/2)=7min)交换。从v(S)和v(As)的位置计算出类似123度的Fe-O-Fe角，并从这些频率上依赖于O-18的位移计算表明，去饱和酶中的二铁-氧簇在二铁状态下是三桥联的。从穆斯堡尔四极分裂的温差关系可以看出，在亚铁状态下，团簇的两个铁位在结构上是不等价的。对于II类双铁氧代蛋白，一级序列比对显示保守的氨基酸残基作为铁簇配体，参与氢键网络，并可能参与O-2的结合和激活。基于这一保守性，提出了硬脂酰-ACP Delta(9)脱饱和酶活性中心的结构模型，该模型与核糖核苷酸还原酶和甲烷单加氧酶都有很强的相似性。然而，在与O-18(2)的二亚铁状态单次翻转后，在蓖麻脱饱和酶的氧桥中没有检测到O-18。这与观察到的核糖核苷酸还原酶的结果相反，这表明去饱和酶和核糖核苷酸还原酶在各自的O-2激活反应的某些方面不同。
The stearoyl-ACP Delta(9) desaturase from plants is a new example of a growing number of proteins that contain oxo- or hydroxo-bridged diiron clusters. On the basis of differences in primary sequence motifs providing the cluster ligands and upon structural differences elucidated by X-ray crystallography, we now propose that the presently known, soluble diiron-oxo proteins can be grouped into two classes, I and II. Class I contains hemerythrin, myohemerythrin, and, possibly, purple acid phosphatase. Class II contains ribonucleotide reductases, bacterial hydrocarbon hydroxylases (methane monooxygenase, toluene-4-monooxygenase, and phenol hydroxylase), rubrerythrin, and stearoyl-ACP desaturases. Through the use of resonance Raman spectroscopy, we have detected symmetric (v(s) = 519 cm(-1)) and asymmetric (v(as) = 747 cm(-1)) vibrational modes in the castor stearoyl-ACP Delta(9) desaturase, which are typical of ore-bridged diiron clusters. These frequencies shift by -18 and -34 cm(-1), respectively, in (H2O)-O-18, proving that the bridging ligand is readily exchangeable with solvent (t(1/2) = 7 min). Calculation of an similar to 123 degrees Fe-O-Fe angle from the position of v(s) and v(as) and from the O-18-dependent shift in these frequencies suggests that the diiron-oxo cluster in the desaturase is triply bridged in the diferric state. In the diferrous state, the two iron sites of the cluster are structurally inequivalent, as shown by differential temperature dependence of the Mossbauer quadrupole splittings. For the class II diiron-oxo proteins, primary sequence alignments reveal conserved amino acid residues which act as iron cluster ligands, participate in a hydrogen-bonding network, and are potentially involved in O-2 binding and activation. Based on this conservation, a structural model for the stearoyl-ACP Delta(9) desaturase active site is proposed that has strong similarity to both ribonucleotide reductase and methane monooxygenase. However, after single turnover of the diferrous state with O-18(2), O-18 is not detected in the oxo bridge of the castor desaturase. This is in contrast to the outcome observed for ribonucleotide reductase, suggesting the desaturase and ribonucleotide reductase differ in certain aspects of their respective O-2-activation reactions.