Active site loop dictates the thermodynamics of reduction and ligand protonation in Cupredoxins

The thermodynamics of reduction and His ligand protonation have been determined for a range of loopcontractionvariants of the electron transferring type 1 copper protein azurin (AZ). For AZPC, in which thenative C-terminal loop containing the Cys, His and Met ligands has been replaced with the shorter sequencefrom plastocyanin (PC) and AZAMI, in which the even shorter amicyanin (AMI) loop has been inserted, thethermodynamics of reduction match those of the protein whose loop has been introduced which aredifferent to the values for AZ. The enthalpic contribution to His ligand protonation, which is not observed inAZ, is similar in AZAMI and AMI. The thermodynamics of this process in AZPC are more dissimilar to thosefor PC. In the case of AZAMI-F, a variant possessing the (non natural) minimal loop that can bind a type 1copper site, the reduction thermodynamics are intermediate between those of AZPC and AZAMI, whilst thethermodynamic data for His ligand protonation are very similar to those for AMI. The results for AZAMI andAZPC are primarily due to protein based enthalpic effects related to the interaction of the metal withpermanent protein dipoles from the loop, and to the decreased loop length which favors His ligandprotonation in the cuprous proteins. Entropic factors related to loop flexibility have little influence becauseof constraints imposed by metal coordination and the fact that the introduced loops pack well against theAZ 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 thethermodynamics of reduction