Control of the Electronic Ground State on CuA Sites by Axial Ligand Mutations and Second-Sphere Perturbations

ALCIDES J. LEGUTO; MARCOS MORGADA; ALEJANDRO J. VILA

Chascomús

Congreso; Latin American Meeting in Biological and Bioinorganic Chemistry LABIC IV; 2014

INTRODUCTION  CuA is a binuclear copper site that acts as an entry port for electrons in cytochrome c oxidases (Fig. 1), among others proteins. It catalyzes two long-range electron transfer (ET) reactions: electrons are shuttled by cytochrome c to the metal site and from there to the heme present in the catalytic site in subunit I where the O2 reduction to water is carried out. These steps involve two long pathways through the non-conducting protein milieu. Despite of that, both ET reactions are highly efficient. This efficiency has been attributed to the unique coordination features of the CuA site, which shows a low reorganization free energy and an electronic structure that enhances superexchange coupling1.  In its oxidized form, CuA is a fully delocalized mixed-valence copper center (Cu1.5+ Cu1.5+) whose electronic structure can be described by two alternative ground states, σu* and πu. σu* ground state has been considered as the only redox-active orbital in ET.  Instead, πu is an alternative ground state with higher energy, which is partially populated at room temperature and interconverts rapidly with the σu* state2.  It has been demonstrated that the identity of the weak axial ligand at position 160 of the CuA-containing domain from Thermus thermophilus ba3 oxidase (TtCuA-WT) shift the fluctuating equilibrium between the σu* and πu ground states, altering the energy difference between them. The substitution of Met160 by glutamine (which has a lower affinity for copper) causes an increase in the energy gap between the states. Conversely, the substitution by histidine diminishes it, then increasing the population of πu ground state3.  We have designed a chimeric protein in the scaffold of TtCuA-WT, in which the three loops surrounding the metal site were replaced by those from a homologous eukaryotic domain. This chimera, named Tt-3L CuA, has only second-sphere perturbations but shows an increase in the population of the πu level4.  In this work, we characterize the geometric and electronic structures of CuA site in Tt-3L CuA after replacing the axial methionine ligand at position 160 by glutamine or histidine. Finally, we compare these effect with those observed for these mutations over TtCuA-WT. Fig. 1. Structure of CuA site highlighting the metal ligands.   EXPERIMENTAL METHODS Production of Protein Samples. Unlabeled and uniformly labeled soluble proteins were expressed in E. coli BL21(DE3) growing in either rich LB medium or M9 minimal medium supplemented with labeled or unlabeled reactants, according to the desired labeling scheme. Purification of proteins from cell lysates was done as described elsewhere5. Nuclear Magnetic Resonance Spectroscopy. This technique was used to characterize the electronic properties of paramagnetic CuA center. NMR experiments were carried out on a Bruker Avance II Spectrometer operating at 600.13MHz. 1H-detected spectra, 1H,13C- and 1H,15N-HMQC experiments, carbon-13 detected spectra, 1H-NOESY and saturation transfer experiments were carried out. The temperature dependence data were fit to a two-state model using the approach described by Shokhirev and Walker6, which calculates the contact contribution to the chemical shift for each data point and fits all data at once yielding the energy gap between the two states. X-Ray Absorption Spectroscopy. Cu K-edge (8.9 keV) EXAFS and X-ray absorption near-edge structure data were collected at the Stanford Synchrotron Radiation Laboratory in order to characterize the geometric structure of CuA sites studied here. Optical Spectroscopy. Absorption and circular dichroism spectra were acquired using thermostatized chambers and quartz cells in order to characterize the electronic structure of CuA sites under study at different temperature or pH values.   RESULTS AND DISCUSSION  Replacement of axial methionine by glutamine or histidine in Tt-3L CuA causes analogous effects to which are observed in TtCuA-WT.  NMR spectroscopy allows to estimate the energy gap between σu* and πu states from a proper fitting of the temperature dependence data. For the Tt-3L CuA mutants, this difference presents similar perturbations that those observed for TtCuA-WT mutants.  Optical spectra are typical of purple CuA sites, showing some distortions that can be correlated with changes in the energy gap between σu* and πu ground states.  We have shown that the relative populations of σu* and πu ground states of CuA sites can be modulated by the identity of the axial ligand of the center and by perturbations on its second coordination sphere. The results presented in this work suggest that both types of effects are additive and independent of each other thus representing two alternative strategies that nature can use in order to modulate the ET function of these proteins.   CONCLUSION  In conclusion, we have produced Met160Gln and Met160His mutants of CuA containing chimeric protein Tt-3L CuA. The substitution of the axial ligand methionine by glutamine increases the energy separation between the ground states of the center, thus leading to perturbations in optical and NMR spectra that can be related with a decreased relative population in the πu ground state. Conversely, the substitution of the axial methionine by histidine diminishes the energy gap, facilitating the population of πu state. The overall observed effects are similar to those shown by the TtCuA-WT mutants.   REFERENCES   1. Beinert H., Eur J Biochem (1997), 245:521-532. 2. Solomon E. I. et al., Chem Soc Rev (2008), 37:623-638. 3. Abriata L. A. et al., Proc Natl Acad Sci U S A (2012), 109:17348-17353. 4. Morgada M. N. et al., Angew Chem Int Ed (2014), doi: 10.1002/anie.201402083 5. Abriata L. A. et al., J Am Chem Soc (2009), 131:1939-1946. 6. Shokhirev, N. V. et al., J Phys Chem (1995), 99:17795?17804.   ACKNOWLEDGMENTS A.J.L. and M.N.M. thank CONICET for doctoral fellowships. A.J.V. is a staff member from CONICET and an HHMI International Research Scholar. This work was supported by Agencia Nacional de Promoción Científica y Tecnológica (PICT2007-00314) grants to A.J.V. The Bruker Avance II 600 MHz NMR spectrometer at Rosario was purchased with funds from ANPCyT (PME2003-0026) and CONICET.