Bravo J, Mate M J, Schneider T, Switala J, Wilson K, Loewen P C, Fita I
Departamento de Biología Molecular y Celular, CID (C.S.I.C.), Barcelona, Spain.
Proteins. 1999 Feb 1;34(2):155-66. doi: 10.1002/(sici)1097-0134(19990201)34:2<155::aid-prot1>3.0.co;2-p.
Catalase HPII from Escherichia coli, a homotetramer of subunits with 753 residues, is the largest known catalase. The structure of native HPII has been refined at 1.9 A resolution using X-ray synchrotron data collected from crystals flash-cooled with liquid nitrogen. The crystallographic agreement factors R and R(free) are respectively 16.6% and 21.0%. The asymmetric unit of the crystal contains a whole molecule that shows accurate 222-point group symmetry. The structure of the central part of the HPII subunit gives a root mean square deviation of 1.5 A for 477 equivalencies with beef liver catalase. Most of the additional 276 residues of HPII are located in either an extended N-terminal arm or in a C-terminal domain organized with a flavodoxin-like topology. A small number of mostly hydrophilic interactions stabilize the relative orientation between the C-terminal domain and the core of the enzyme. The heme component of HPII is a cis-hydroxychlorin gamma-spirolactone in an orientation that is flipped 180 degrees with respect to the orientation of the heme found in beef liver catalase. The proximal ligand of the heme is Tyr415 which is joined by a covalent bond between its Cbeta atom and the Ndelta atom of His392. Over 2,700 well-defined solvent molecules have been identified filling a complex network of cavities and channels formed inside the molecule. Two channels lead close to the distal side heme pocket of each subunit suggesting separate inlet and exhaust functions. The longest channel, that begins in an adjacent subunit, is over 50 A in length, and the second channel is about 30 A in length. A third channel reaching the heme proximal side may provide access for the substrate needed to catalyze the heme modification and His-Tyr bond formation. HPII does not bind NADPH and the equivalent region to the NADPH binding pocket of bovine catalase, partially occluded in HPII by residues 585-590, corresponds to the entrance to the second channel. The heme distal pocket contains two solvent molecules, and the one closer to the iron atom appears to exhibit high mobility or low occupancy compatible with weak coordination.
来自大肠杆菌的过氧化氢酶HPII是一种由753个残基组成的亚基同四聚体，是已知最大的过氧化氢酶。利用从液氮快速冷却的晶体收集的X射线同步辐射数据，已将天然HPII的结构精修至1.9埃分辨率。晶体学吻合因子R和R（自由）分别为16.6%和21.0%。晶体的不对称单元包含一个显示精确222点群对称性的完整分子。HPII亚基中心部分的结构与牛肝过氧化氢酶的477个等效位点的均方根偏差为1.5埃。HPII额外的276个残基大多位于一个延伸的N端臂或一个具有类黄素氧还蛋白拓扑结构的C端结构域中。少数主要是亲水的相互作用稳定了C端结构域与酶核心之间的相对取向。HPII的血红素成分是一种顺式羟基二氢卟酚γ-螺内酯，其取向相对于牛肝过氧化氢酶中发现的血红素的取向翻转了180度。血红素的近端配体是Tyr415，它通过其Cβ原子与His392的Nδ原子之间的共价键相连。已鉴定出2700多个定义明确的溶剂分子，它们填充在分子内部形成的复杂腔和通道网络中。两条通道通向每个亚基的血红素远端口袋，表明具有单独的入口和排气功能。最长的通道始于相邻亚基，长度超过50埃，第二条通道长度约为30埃。第三条通向血红素近端的通道可能为催化血红素修饰和His-Tyr键形成所需的底物提供通道。HPII不结合NADPH，牛过氧化氢酶NADPH结合口袋的等效区域在HPII中部分被585 - 590位残基封闭，对应于第二条通道的入口。血红素远端口袋包含两个溶剂分子，靠近铁原子的那个似乎具有高流动性或低占有率，与弱配位兼容。
