Abstract
Copper is a ubiquitous metal in biology that, among other functions, is implicated in enzymatic redox catalysis and in protein electron transfer (ET). When it comes to ET, copper sites are found in two main forms, mononuclear type 1 (T1) and binuclear CuA sites, which share a common cupredoxin fold. Other relevant copper sites are the so-called type 2 (T2), which are more resilient to undergo direct electrochemistry and are usually involved in catalysis. Here we report the electrochemical and spectroscopic characterization of a novel T2-like copper site engineered following the loop swapping strategy. The ligand loop sequence of the newly discovered T1 copper site from Nitrosopumilus maritimus was introduced into the CuA scaffold from Thermus thermophilus yielding a chimeric protein that shows spectroscopic features different from both parental proteins, and resemble those of red T2 copper sites, albeit with a shorter Cu-S(Cys) bond length. The novel T2 site undergoes efficient direct electrochemistry, which allows performing temperature-dependent cyclic voltammetry studies. The obtained results reveal that this chimera constitutes the first example of a copper protein with entropically controlled reduction potential, thereby contrasting the enthalpic supremacy observed for all other copper sites reported so far. The underlying bases for this entropic control are critically discussed.
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