NMR Studies on Multinuclear Copper Proteins (conferencista invitado)

LUCIANO A. ABRIATA, MA.EUGENIA ZABALLA, GABRIELA N.LEDESMA, LYNN ZIEGLER, ROBERTA PIERATTELLI, DAN KOSMAN AND ALEJANDRO J. VILA

Alghero, Italia

Congreso; 6th International Copper Meeting: Copper and Related Metals in Biology; 2008

Internation Cooper Asociation

NMR is usually exploited as a technique in structural biology to provide 3D structures of proteins. In the case of metalloproteins with paramagnetic centers, such as Cu(II), the unpaired electrons at the metal site may limit the detection of nearby nuclei by inducing unusual chemical shifts and fast relaxation rates. If detected and assigned, the resonances corresponding to these nuclei  can provide information about the electronic properties of the metal site, which can be related to their reactivity. We have studied two copper proteins with multicopper centers, which have been less explored than the mononuclear ones. These are subunit II of T.thermophilusba3 oxidase and the yeast peroxidase Fet3p. Subunit II of the oxidase contains the dinuclear CuA site, which is the electron entry port in most terminal oxidases. Relaxation measurements on the backbone signals reveal a highly rigid structure in both oxidation states, which is in line with the low reorganization energy of this metal center. However, NMR studies on the paramagnetic signals from the metal site ligands reveal a two-state equilibrium. By performing 1H and 13C studies in uniformly and selectively labeled protein samples we have found that this equilibrium correspond to two electronic states which are populated at room temperature. This state is normally not observed by other magnetic spectroscopies, such as EPR or ENDOR, which are performed at cryogenic temperatures. These states are supposed to play a key role in long range electron transfer. Studies on a series of point mutants, where the axial Met160 ligand was replaced by other ligands reveal no changes in the metal-ligand bonding upon the mutations, but in alterations on the energy gap between these two levels, suggesting that axial ligands play a role in modulating the electronic structure of CuA sites (and fine tuning the redox potential). The multicopper oxidase Fet3p from S. cerevisiae contains four copper ions distributed ni a type 1 (T1), a type 2 (T2) and a binuclear type 3 (T3) site. The latter is diamagnetic even in its oxidized form because its two Cu(II) ions are antiferromagnetically coupled through a hydroxide bridge. Besides, T3 is involved in a trinuclear Cu cluster together with the T2 site. Using NMR on wt and some Fet3p mutants, we have identified a set of signals corresponding to the three metal sites, showing that the coupling between the two Cu(II) ions in the binuclear T3 site gives rise to a thermally accessible, paramagnetic, excited state. These spectra allow us to estimate a value of the exchange coupling for this center. We have also seen that removal of the T2 site causes a dramatic change in the T3 site, which normally cannot be monitored by techniques such as EPR, which detect the diamagnetic, ground state. These studies set the ground for a deeper understanding of the role of the electronic structure of metal centers in reactivity studied at physiological temperatures.<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";} @page Section1 {size:595.3pt 841.9pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:35.4pt; mso-footer-margin:35.4pt; mso-paper-source:0;} div.Section1 {page:Section1;} -->