Towards new chemistry by redesign of electron transfer copper proteins

LEGUTO, AJ; REBECHI, JP; VILA, AJ

San Miguel de Tucuman

Congreso; III LAFeBS; 2016

Sociedad Argentina de Biofísica

Copper-containing proteins that contain type 1 (T1) and CuA centersconform a group that shares a cupredoxin fold and whose only functionin biological systems is related to the exceptionally important electrontransfer (ET) process(1).The structural similarity between proteins bearing CuA and T1 centers,and the fact that most of the ligands in these metallic sites are in a sin-gle loop, has allowed the design of mutants with new functionalitiesby exchanging the ligand loop between these copper proteins. A num-ber of these redesigns have been reported(2), particularly those whereCuA loops are inserted in T1 scaffolds. These chimeras showed spec-troscopic and functional features similar to those from which the loopwas obtained.In order to gain more insight into these mutants, and taking advantageof our expertise in loop engineering over the Thermus thermophilusCuA scaffold (TtCuA)(3), we generated and characterized two chimeras(amicyanin-TtCuA and azurin-TtCuA) bearing two different T1 loopsin the TtCuA scaffold, and found that they showed perturbed T1 siteswith interesting features regarding redox catalysis: binding of exoge-nous ligands and higher redox potentials. As these features could befurther optimized to develop new chemistry, our current aim is to evalu-ate them in a greater number of cases. For that purpose, we have gener-ated six more mutants of the same class, inserting a variety of T1 loopsin the TtCuA scaffold. These new mutants showed T1 centers with dif-ferent degrees of perturbation according to their UV-vis spectra and canalso bind exogenous ligands in a reversible manner. This is a promisingfeature that will allow us to explore catalytic functions and develop newchemistry not observed in native cupredoxins