REFINED CRYSTAL-STRUCTURE OF ASCORBATE OXIDASE AT 1.9 A RESOLUTION
REFINED CRYSTAL-STRUCTURE OF ASCORBATE OXIDASE AT 1.9 A RESOLUTION
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DOI：
10.1016/0022-2836(92)90583-6
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发表时间：
1992-03-05
影响因子：
5.6
通讯作者：
FINAZZIAGRO, A
中科院分区：
文献类型：
--
作者：
MESSERSCHMIDT, A;LADENSTEIN, R;FINAZZIAGRO, A
采用能量约束最小二乘法对西葫芦中抗坏血酸氧化酶（EC 1.10.3.3）的全氧化型晶体结构进行了精细化处理，其分辨率为1·90 nm（1 nm = 0·1 nm）。改进的模型包括8764个蛋白质原子、9个铜原子和970个溶剂分子，在8到1.90 nm分辨率之间的85，252个反射中，晶体学R因子为20.3%。键长和键角与理想值的均方根偏差分别为0.011 °和2.99 °，由552个残基（70，000 Mr）组成的亚基以D2对称性排列成四聚体。其中一个二联体是由平行于轴的晶轴实现的，在不对称单元中产生一个二聚体。与该晶轴相关的二聚体被认为是溶液中存在的二聚体。Asn 92是两个N-连接糖基之一的连接位点，其具有N-连接的N-乙酰葡糖胺环的确定的电子密度。每个亚基由三个结构域组成，在多肽链上顺序排列，并在空间上紧密结合。所有三个结构域的折叠都是类似的β-桶型，并且与质体蓝素和天青蛋白相关。分析了四聚体内和四聚体间的氢键和货车德瓦尔斯相互作用，每个亚基有四个铜原子以单核和三核形式结合。单核铜具有两个组氨酸、一个半胱氨酸和一个甲硫氨酸配体，代表1型铜。它位于域3中。1型铜中心的键长与氧化质体蓝素的值相当。三核簇有八个组氨酸配体对称地从结构域1和3提供。它可以被细分为一对铜原子与组氨酸配体，其连接N-原子（5个NE 2原子和一个ND 1原子）排列为三角棱柱。这对是公认的3型铜。其余的铜有两个组氨酸配体，是推定的光谱2型铜。两个氧原子以OH−或O2−的形式与三核物种结合，并桥接假定的3型铜对，以OH−或H2O的形式与假定的2型铜对结合。三核铜中心内的键长与双核模型化合物相似，推测的还原底物结合位点接近1型铜。两个通道提供访问从溶剂到三核铜网站，推定的结合位点的双氧，可以确定。基于已有的动力学数据，提出了抗坏血酸氧化酶的催化机理，并讨论了三维结构和相关的电子转移过程。
The crystal structure of the fully oxidized form of ascorbate oxidase (EC 1.10.3.3) from Zucchini has been refined at 1·90 Å (1 Å = 0·1 nm) resolution, using an energy-restrained least-squares refinement procedure. The refined model, which includes 8764 protein atoms, 9 copper atoms and 970 solvent molecules, has a crystallographicR-factor of 20·3% for 85,252 reflections between 8 and 1·90 Å resolution. The root-mean-square deviation in bond lengths and bond angles from ideal values is 0·011 Å and 2·99 °, respectively.The subunits of 552 residues (70,000Mr) are arranged as tetramers withD2 symmetry. One of the dyads is realized by the crystallographic axis parallel to thec-axis giving one dimer in the asymmetric unit. The dimer related about this crystallographic axis is suggested as the dimer present in solution. Asn92 is the attachment site for one of the two N-linked sugar moieties, which has defined electron density for the N-linkedN-acetylglucosamine ring.Each subunit is built up by three domains arranged sequentially on the polypeptide chain and tightly associated in space. The folding of all three domains is of a similar β-barrel type and related to plastocyanin and azurin. An analysis of intra-and intertetramer hydrogen bond and van der Waals interactions is presented.Each subunit has four copper atoms bound as mononuclear and trinuclear species. The mononuclear copper has two histidine, a cysteine and a methionine ligand and represents the type-1 copper. It is located in domain 3. The bond lengths of the type-1 copper centre are comparable to the values for oxidized plastocyanin. The trinuclear cluster has eight histidine ligands symmetrically supplied from domain 1 and 3. It may be subdivided into a pair of copper atoms with histidine ligands whose ligating N-atoms (5 NE2 atoms and one ND1 atom) are arranged trigonal prismatic. The pair is the putative type-3 copper. The remaining copper has two histidine ligands and is the putative spectroscopic type-2 copper. Two oxygen atoms are bound to the trinuclear species as OH−or O2−and bridging the putative type-3 copper pair and as OH−or H2O bound to the putative type-2 coppertransto the copper pair. The bond lengths within the trinuclear copper site are similar to comparable binuclear model compounds.The putative binding site for the reducing substrate is close to the type-1 copper. Two channels providing access from the solvent to the trinuclear copper site, the putative binding site of the dioxygen, could be identified. A catalytic mechanism of ascorbate oxidase is proposed based on the available kinetic data, and the three-dimensional structure and the associated electron transfer processes are discussed.