Effect of loop mutagenesis on Thermus thermophilus COXII soluble domain

ANDRÉS ESPINOZA-CARA; MARCOS MORGADA; ALEJANDRO J. VILA

Ventura, California

Congreso; Gordon Research Seminar: Bioinorganic Chemistry; 2013

Nature employs metal ions for a wide variety of biological functions. Copper proteins play an important role in electron transfer processes. There are two types of copper electron transfer sites in nature: (a) mononuclear type I centers (T1), also known as blue sites, and (b) the dinuclear CuA center. Proteins with T1 and CuA centers share a conserved fold (cupredoxin fold) despite being from different organisms, revealing a common evolutionary origin2. The functional properties of these redox sites cannot be compared directly because of being in different protein matrixes. The T1 and CuA centers presents and ideal situation for loop changing as they share the same protein fold and most of the metal ligands are present in loops. There are few examples using this strategy, but all of them suggest that the ligand-containing loops gather most of the structural information that regulates the function of the redox sites. In this project we employ loop directed mutagenesis in order to introduce an eukariotic loop and two different T1 loops in the soluble fragment of the subunit II of the cytochrome ba3 oxidase from Thermus thermophilus, naturally harboring a CuA site (TtCuA).The eucariotic chimera two additional loops have been replaced for those of the eucariotic homologous protein. These mutations affect the electronic structure of the CuA center even though they do not change loop length or CuA ligands, only presenting mutations in the second coordination sphere with respect to the thermophilic protein. The two T1 variants were constructed, employing the loops from amicyanin (Ami- TtCuA) and azurin (Az-TtCuA). These proteins have distinct characteristics from the native sites. Using distinct biophysical approaches such as optical spectroscopy, EPR and NMR we determined that both proteins have properties that vary along those of rhombic T1 sites. Both proteins can also bind exogenous imidazole in a reversible way giving rise to a copper site with spectral features similar to those found in nitrosocyanin.