Farrar J A, Lappalainen P, Zumft W G, Saraste M, Thomson A J
Centre for Metalloprotein Spectroscopy and Biology, School of Chemical Sciences, University of East Anglia, Norwich, England.
Eur J Biochem. 1995 Aug 15;232(1):294-303. doi: 10.1111/j.1432-1033.1995.tb20811.x.
Cytochrome-c oxidase contains an unusual copper centre (CuA) located in subunit II. This centre mediates one-electron transfer from cytochrome c to low-spin heme a. Recent spectroscopic and biochemical studies have shown that this centre is a valence delocalised dinuclear [Cu(+1.5)-Cu(+1.5)] centre. We have measured the absorption, EPR and variable-temperature magnetic circular dichroism spectra of the CuA-binding domain isolated from Paracoccus denitrificans cytochrome aa3. The EPR spectrum showed the following signals: gparallel = 2.18; gperpendicular = 2.03. gparallel exhibited a seven-line hyperfine splitting pattern, with an intensity ratio showing that the single unpaired electron interacted equally with two copper nuclei. The magnetic circular dichroism spectrum was identical to those from CuA in bovine heart cytochrome-c oxidase and centre A of nitrous-oxide reductase, showing the close structural similarity between the three centres. To identify the ligands of CuA, all the conserved putative ligands in the P. denitrificans CuA domain were substituted. Only five residues, Cys244, Cys248, His209, His252, and Met255, were required for correct assembly of the CuA centre. Replacement of Met255 caused protein misfolding. Hence, methionine may have a structural role for the folding of the protein rather than being a CuA ligand. Given that both copper ions must have identical coordination geometries, the number of possible structures is limited. Two models are proposed: one involves the thiolate side-chains of Cys244 and Cys248 bridging a pair of copper ions with one histidine coordinating each copper ion, and the other has terminal ligation of each copper ion by one cysteine and one histidine residue. In both models, the metal-metal distance can be sufficiently short to permit direct d-orbital overlap of the copper ions. The magnetic circular dichroism transitions at 475 nm and 525 nm are assigned to thiolate-to-copper charge-transfer processes polarised perpendicular to one another, although the magnetic circular dichroism intensities show that the excited states were heavily mixed with copper d-orbitals. These intensities can be interpreted in the thiolate bridged model in terms of transitions within a Cu2(SR)2 rhomb. In the model involving terminal cysteine ligation, exciton coupling of two thiolate-to-copper charge-transfer transitions of similar energy, polarised along the Cu-S bonds, would contribute two transitions perpendicular to one another. This requires that the cysteine ligands have a cis orientation relative to one another.(ABSTRACT TRUNCATED AT 400 WORDS)
细胞色素c氧化酶含有一个位于亚基II中的特殊铜中心（CuA）。该中心介导从细胞色素c到低自旋血红素a的单电子转移。最近的光谱学和生物化学研究表明，这个中心是一个价态离域的双核[Cu(+1.5)-Cu(+1.5)]中心。我们测量了从反硝化副球菌细胞色素aa3中分离出的CuA结合结构域的吸收光谱、电子顺磁共振（EPR）光谱和变温磁圆二色性光谱。EPR光谱显示出以下信号：g平行 = 2.18；g垂直 = 2.03。g平行呈现出七线超精细分裂模式，强度比表明单个未成对电子与两个铜核的相互作用程度相同。磁圆二色性光谱与牛心细胞色素c氧化酶中的CuA以及一氧化二氮还原酶的中心A的光谱相同，表明这三个中心在结构上非常相似。为了确定CuA的配体，对反硝化副球菌CuA结构域中所有保守的假定配体进行了替换。只有五个残基，即半胱氨酸244、半胱氨酸248、组氨酸209、组氨酸252和甲硫氨酸255，是CuA中心正确组装所必需的。甲硫氨酸255的替换导致蛋白质错误折叠。因此，甲硫氨酸可能对蛋白质折叠具有结构作用，而不是作为CuA的配体。鉴于两个铜离子必须具有相同的配位几何结构，可能的结构数量有限。提出了两种模型：一种模型涉及半胱氨酸244和半胱氨酸半胱氨酸248的硫醇盐侧链桥接一对铜离子，每个铜离子由一个组氨酸配位；另一种模型是每个铜离子由一个半胱氨酸和一个组氨酸残基进行末端配位。在这两种模型中，金属 - 金属距离都可以足够短，以允许铜离子的d轨道直接重叠。475纳米和525纳米处的磁圆二色性跃迁被归因于相互垂直极化的硫醇盐到铜的电荷转移过程，尽管磁圆二色性强度表明激发态与铜d轨道有大量混合。在硫醇盐桥接模型中，这些强度可以根据Cu2(SR)2菱形内的跃迁来解释。在涉及末端半胱氨酸配位的模型中，两个能量相似、沿Cu - S键极化的硫醇盐到铜的电荷转移跃迁的激子耦合将产生两个相互垂直的跃迁。这要求半胱氨酸配体彼此具有顺式取向。（摘要截取自400字）
