A Rationally Designed CuA Containing Protein Shows Degenerate Alternative Ground States

LEGUTO, ALCIDES JOSÉ; MORGADA, MARCOS NICOLÁS; VILA, ALEJANDRO JOSÉ

Workshop; III Workshop de Resonancia Magnética "NMR and EPR at the Forefront of Research"; 2016

Dpto. de Física - Facultad de Bioquímica y Cs. Biológicas - Univ. Nac. del Litoral

CuA is a binuclear copper center found naturally in terminal electron acceptors where it acts as the electron entry port, participating in highly efficient long-range electron transfer (ET) processes. High efficiency has been attributed to an evolutionarily optimized elec-tronic structure. In CuA centers, a rigid Cu2S2 ?diamond core structure? yields a low reorganization free energy (λ) which increase ET efficiency. Additionally, each copper ion is coordinated by equatorial histidines and distal axial ligands: one is weakly bound to a methionine (Met160), as the other is bound to a backbone carbonyl. In the oxidized state, CuA center is a mixed valence pair with an unpaired electron delocal-ized between the two ions. The electronic structure of CuA sites can be described by two alternative ground state wavefunctions which involves an antibonding  interaction (u* ground-state) or a bonding π interaction (πu ground-state) between the two copper ions. πu ground-state has higher energy and is partially populated at room temperature, rapidly interconverting with u* state. We have demonstrated that πu ground state is redox-active in ET, compensating its larger λ with an increase in superexchange coupling by allowing an alternative pathway. Moreover, replacing the native Met axial ligand both redox poten-tial and u*/ πu energy gap can be tuned.2 πu level population can be increased from 5% in WT protein to almost 30% when a His residue replace axial Met. We have also demon-strated that second sphere perturbations can elicit significant changes in electronic struc-ture of CuA sites. Tt3L CuA chimeric protein was created replacing the three loops defining the nearby environment of CuA site by their eukaryotic homologous. Ligand set and loop lengths were conserved in this chimera, however showing an increase in the πu level population to 25%. Herein we report the design of a point mutant of chimeric protein Tt3L CuA in which axial methionine is replaced by a histidine residue. Tt3L CuA M160H shows notably perturbed spectroscopic features, consistent with a large increase in the population of πu level, even higher than TtCuA M160H or Tt3L CuA variants themselves. After precise estimations using paramagnetic NMR, both alternative ground states in Tt3L CuA M160H mutant appear to be degenerate. This is the first soluble and stable CuA variant reported with equal popula-tions in its alternative ground states at room temperature.