IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
NMR on the Multicopper Oxidase Fet3p
Autor/es:
MARÍA-EUGENIA ZABALLA; LYNN ZIEGLER; DANIEL J. KOSMAN; ALEJANDRO J. VILA
Lugar:
Angra dos Reis, Rio de Janeiro, Brasil
Reunión:
Congreso; 12th Nuclear Magnetic Resonance Users Meeting & 3rd Iberoamerican NMR Meeting; 2009
Institución organizadora:
AUREMN (Asociación de Usuarios de Resonancia Magnética Nuclear)
Resumen:
Multicopper oxidases are an important class of enzymes found in bacteria, fungi, plants, and animals. Well-known members of this class include laccase, ascorbate oxidase, ceruloplasmin, and the ferroxidase Fet3p. Fet3p is a plasma membrane protein that was originally isolated from Saccharomyces cerevisiae. In vivo Fet3p is associated with the iron permease Ftr1p. Together, Fet3p and Ftr1p have been shown to play an integral role in high-affinity iron uptake in yeast; Fet3p appears to oxidize Fe(II) to Fe(III) while Ftr1p is required for the transport of Fe(III) across the plasma membrane.All multicopper oxidases utilize at least four copper ions to couple the four-electron reduction of molecular oxygen to water with four sequential one-electron substrate oxidations. These Cu ions are classified into three types of sites based on their spectroscopic properties in the oxidized state: the type 1 (T1), characterized by an intense Cys-to-Cu charge-transfer transition at ~600 nm and a small parallel hyperfine coupling in electron paramagnetic resonance (EPR); the type 2 (T2), which has distinctive EPR features; and the dinuclear type 3 (T3) site, which is diamagnetic even in its oxidized form because its two Cu(II) ions are antiferromagnetically coupled through a hydroxide bridge. The T2 and T3 Cu centers form a trinuclear cluster (TNC), which is the dioxygen reduction site.Previous work on Fet3p, using different techniques as UV-vis absorption, EPR, MCD, and X-ray absorption, has shown that removal of either the T1 or the T2 site does not alter the spectroscopic features of the remaining sites. Here we have utilized nuclear magnetic resonance focused on the detection of the hyperfine-shifted resonances to get a deeper view on the electronic structure of the copper sites. The 1H NMR spectrum of Fet3p shows many hyperfine-shifted signals, which were assigned to ligands of T1, T2 and T3 sites based on their distinct spectroscopic features. These assignments were confirmed by recording the spectra of mutants in which each of the mononuclear sites was mutated by removal of copper ligands (T1D and T2D Fet3p).  The detection of signals from the type 3 site reveals that this dinuclear center, despite possessing a diamagnetic ground state, displays a thermally accessible excited state with S=1, which gives rise to the observed paramagnetism. The T1 values and chemical shifts of this center are different from analogous T3 in tyrosinases, disclosing distinctive features which may be ascribed to formation of the TNC. The observation of two resonances corresponding to T2 copper ligands is noteworthy since the electron relaxation times of T2 sites give rise to NMR resonances which are broadened beyond detection. Resonances at 50 and 23 ppm in the 1H NMR spectrum of wt Fet3p were assigned to the T2 site, and their features can be explained by assuming that this center is coupled to the fastest relaxing Cu(II) ions of the dinuclear T3 site. The spectroscopic features of the T1 site resemble those reported for the small BCP’s. However, when the T2 site of Fet3p was removed (T2D Fet3p) all the NMR resonances previously assigned to T1 copper ligands could not be detected. Finally, by recording 1H NMR spectra of wt, T1D and T2D Fet3p variants at several temperatures in a 283 – 304 K range, we have been able to get an estimated value for the exchange coupling between the two T3 copper ions.NMR spectroscopy is able to reveal the unexpected electronic structure of these centers, as well as to determine exchange coupling parameters not accessible to other magnetic techniques (EPR, MCD).