Battistuzzi Gianantonio, Borsari Marco, Dennison Christopher, Li Chan, Ranieri Antonio, Sola Marco, Yanagisawa Sachiko
Department of Chemistry, University of Modena and Reggio Emilia, Via Campi 183, 41100 Modena, Italy.
Biochim Biophys Acta. 2009 Jul;1794(7):995-1000. doi: 10.1016/j.bbapap.2009.02.001. Epub 2009 Feb 20.
The thermodynamics of reduction and His ligand protonation have been determined for a range of loop-contraction variants of the electron transferring type 1 copper protein azurin (AZ). For AZPC, in which the native C-terminal loop containing the Cys, His and Met ligands has been replaced with the shorter sequence from plastocyanin (PC) and AZAMI, in which the even shorter amicyanin (AMI) loop has been inserted, the thermodynamics of reduction match those of the protein whose loop has been introduced which are different to the values for AZ. The enthalpic contribution to His ligand protonation, which is not observed in AZ, is similar in AZAMI and AMI. The thermodynamics of this process in AZPC are more dissimilar to those for PC. In the case of AZAMI-F, a variant possessing the (non natural) minimal loop that can bind a type 1 copper site, the reduction thermodynamics are intermediate between those of AZPC and AZAMI, whilst the thermodynamic data for His ligand protonation are very similar to those for AMI. The results for AZAMI and AZPC are primarily due to protein based enthalpic effects related to the interaction of the metal with permanent protein dipoles from the loop, and to the decreased loop length which favors His ligand protonation in the cuprous proteins. Entropic factors related to loop flexibility have little influence because of constraints imposed by metal coordination and the fact that the introduced loops pack well against the AZ scaffold. Thus, the host scaffold in general plays a minor thermodynamic role in both processes, although for AZAMI-F differences in the first and second coordination spheres influence the thermodynamics of reduction.
已测定了电子传递型1铜蛋白天青蛋白（AZ）一系列环收缩变体的还原热力学以及组氨酸配体质子化情况。对于AZPC，其天然C末端环（包含半胱氨酸、组氨酸和甲硫氨酸配体）已被来自质体蓝素（PC）的较短序列取代；对于AZAMI，其插入了更短的蓝细菌视紫红质（AMI）环。AZPC和AZAMI的还原热力学与引入其环的蛋白质的还原热力学相匹配，这与AZ的值不同。在AZ中未观察到的对组氨酸配体质子化的焓贡献，在AZAMI和AMI中相似。AZPC中该过程的热力学与PC的热力学更不相似。对于AZAMI-F，一种具有能结合1型铜位点的（非天然）最小环的变体，其还原热力学介于AZPC和AZAMI之间，而组氨酸配体质子化的热力学数据与AMI的非常相似。AZAMI和AZPC的结果主要归因于基于蛋白质的焓效应，这与金属与环中永久性蛋白质偶极的相互作用有关，也归因于环长度的减小，这有利于亚铜蛋白中组氨酸配体质子化。由于金属配位所施加的限制以及引入的环能很好地堆积在AZ支架上这一事实，与环柔韧性相关的熵因素影响很小。因此，宿主支架在这两个过程中通常起较小的热力学作用，尽管对于AZAMI-F，第一和第二配位球中的差异影响还原热力学。
