IDENTIFICATION OF INTERACTION SITE OF PSEUDOAZURIN WITH ITS REDOX PARTNER, COPPER-CONTAINING NITRITE REDUCTASE FROM ALCALIGENES-FAECALIS S-6
IDENTIFICATION OF INTERACTION SITE OF PSEUDOAZURIN WITH ITS REDOX PARTNER, COPPER-CONTAINING NITRITE REDUCTASE FROM ALCALIGENES-FAECALIS S-6
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DOI：
10.1093/protein/8.2.153
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发表时间：
1995-02-01
期刊：
影响因子：
--
通讯作者：
BEPPU, T
中科院分区：
文献类型：
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作者：
KUKIMOTO, M;NISHIYAMA, M;BEPPU, T
假天青蛋白是一种含有单一I型铜的低分子量蛋白质，其功能是作为一种电子供体到一种分解细菌粪产碱杆菌S-6中的含铜亚硝酸盐还原酶（NIR）。为了阐明这两种含铜蛋白之间的蛋白质-蛋白质相互作用，pseudoazurin表面上的13个赖氨酸残基中的9个中的每一个被丙氨酸或天冬氨酸独立地取代，并且分析了突变对与NIR的相互作用以及pseudoazurin的物理化学性质的影响。所有的突变pseudoazurin显示的光谱和氧化还原电位几乎相同的野生型pseudoazurin，这表明没有这些赖氨酸残基的替代影响周围的环境I型铜网站。突变的pseudoazurin和NIR之间的电子转移的动力学分析表明，赖氨酸突变对电子转移到NIR的速率的影响很小，但在残基10，38，57和77处的取代，都接近铜网站，大大降低了pseudoazurin对NIR的亲和力。这表明假天青蛋白通过靠近I型铜位点的区域与NIR相互作用。Lys 38 Asp和Lys 10 Asp/Lys 38 Asp的精细X射线结构表明分子结构确实变化不大。观察到一个新的空间群的Lys 109 Ala突变晶体。Lys 10Asp/Lys 38 Asp突变体的晶体包装相互作用发生变化，但Lys 38 Asp和Lys 59 Ala突变体保持不变。
Pseudoazurin, a low molecular weight protein containing a single type I copper, functions as an electron donor to a copper-containing nitrite reductase (NIR) in a denitrifying bacterium Alcaligenes faecalis S-6. To elucidate the protein-protein interaction between these two copper-containing proteins, each of nine out of 13 lysine residues on the surface of pseudoazurin were independently replaced by alanine or aspartate, and the effects of the mutations on the interaction with NIR, as well as the physicochemical properties of pseudoazurin, were analyzed. All of the mutated pseudoazurins showed optical spectra and oxidation-reduction potentials almost identical to those of wildtype pseudoazurin, suggesting that none of the replacements of these lysine residues affected the environment around the type I copper site. Kinetic analysis of electron transfer between mutated pseudoazurins and NIR reveals that the lysine mutations have very little effect on the rate of electron transfer to NIR, but substitution at residues 10, 38, 57 and 77, all close to the copper site, substantially decreases the affinity of pseudoazurin for NIR. This suggests that pseudoazurin interacts with NIR through the region close to the type I copper site. The refined X-ray structures of Lys38Asp and Lys10Asp/Lys38Asp show that the molecular structure has indeed changed little. A new space group is observed for the Lys109Ala mutant crystal. Crystal packing interactions change for the Lys10Asp/Lys38Asp mutant but remain the same for Lys38Asp and Lys59Ala mutants.