IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Electronic structure of CuA sites explored by NMR spectroscopy
Autor/es:
LUCIANO A. ABRIATA; GABRIELA N. LEDESMA; ALEJANDRO J. VILA
Lugar:
Hotel do Frade, Angra dos Reis, Brasil
Reunión:
Congreso; 12th Nuclear Magnetic Resonance Users Meeting; 2009
Institución organizadora:
AUREMN
Resumen:
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Cytochrome c oxidases (COX) couple oxygen
reduction with proton translocation across the inner membrane of mitochondria
and bacteria, contributing to the accumulation of energy in the form of an
electrochemical gradient.1,2 Subunit II of cytochrome c oxidase contains a soluble subunit
exposed to the outer part, which bears a dinuclear copper termed the CuA
site, which functions as the electron entry port in this oxidase as well as in most
terminal oxidases. Electrons arriving from docked cytochrome c molecules are shuttled through the CuA
center to the catalytic O2-reducing center located inside subunit I,
nearly 20Å away. This process takes place with high efficiency (k = 105 s-1)
even if the driving force is minimum.
The CuA site features two copper ions
strongly bound to two bridging cysteins, two terminal histidine ligands and two weakly bound
axial ligands (a methionine and a backbone carbonyl).3,4 The Cu2S2
moiety forms a highly covalent core that delocalizes the unpaired
electron in the oxidized protein giving raise to a mixed-valence cluster, as evidenced
by 1H-NMR, 1H-ENDOR, XAS and EPR experiments together
with DFT calculations.5-8
Figure 1. The 3D structure of Thermus thermophilus ba3
oxidase, its three subunits and cofactors. The CuA center is zoomed
in the inset.
According to Marcus' semiclassical theory, high efficiency in long-range
electron transfer processes with low driving force requires a low
reorganization energy and a high electronic coupling between the donor and
acceptor sites. We have employed NMR to study both backbone dynamics in the
soluble fragment of COX that bears the CuA site, as well as the
electronic structure of the center in the wild type protein and in mutants
where the axial Met160 ligand has been replaced by His or Gln resulting in fine
tuning of the redox potential.9
Standard T1, T2, NOE and T1r
relaxation experiments were measured in the wild type protein in order to probe
backbone dynamics in the ps-ms time scales. These studies revealed a highly
rigid structure in both oxidation states, in agreement with the low
reorganization energy of this metal center.
The electronic structure of the CuA center was studied by
analyzing the paramagnetically shifted 1H, 15N and 13C resonances from
nuclei close to it. A combined strategy of direct and indirect detection
experiments allowed us to assign all resonances from 1H, 13C and 15N
nuclei of the Cys and His ligands in the wild type protein and in the two
mutants. Our results confirm the large degree of delocalization
in the wild type protein.10 The similar amount of unpaired spin
density found at the Cb nuclei of the Cys residues indicates
that the extent of delocalization is similar in the three proteins, even though
the shifts of the Cys Ca suggest that the geometry of the
site is somewhat perturbed in the mutants. Having
all ligand resonances assigned, we can quantify the unpaired spin densities on
all the nuclei of the ligands. Completion of the 1H assignment
allowed us to find that delocalization also occurs through a hydrogen-bond from
one of the cysteine S atoms to a backbone proton conserved in all known CuA
sites, a situation similar to that observed for blue copper proteins but
previously unnoticed.
Temperature dependence studies allowed us to determine the value of the
energy gap between the ground and first excited states and to map the electron
density in each state for the three isoforms. In the wild type protein, ground
and excited state spin densities for atoms at dihedral positions of the sulfur
p orbitals are in agreement with the symmetry proposed by theoretical
calculations. The energy gap separating the ground and excited states is 600 cm-1
for the wild type protein10 and is influenced by the identity of the
axial ligand.
These results show NMR is a valuable tool which can yield information that
is unretrievable by other spectroscopies, and that can be linked to the functioning
of electron transfer centers.
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Olsson, M.; Ryde, U. J. Am. Chem. Soc. 2001, 123, 7866-7876.
10-Abriata, L.A.; Ledesma, G.N.; Pierattelli, R.; Vila, A.J.; J. Am. Chem. Soc. 2009, 131(5), 1939-1946.
Funding: CONICET,
ANPCYT, NIH