Invisible states in paramagnetic copper proteins

A.J. VILA; L.A.ABRIATA; M.E.ZABALLA

Florencia

Conferencia; Joint EUROMAR 2010 and 17th ISMAR Conference; 2010

NMR of oxidized copper proteins has been largely overlooked, mostly due to the slow electron relaxation times of Cu2+ ion which induce extremely fast relaxation rates in nearby nuclei, rendering them undetectable. It has been shown, however, that these unfavorable electron relaxation features are restricted to T2 copper sites, since T1, T3 and CuA centers display faster electron relaxation rates which make them amenable to NMR studies. In all these cases, the fastelectron relaxation stems from to the availability of low-lying excited electronic states which is due to the particular electronic structure of these centers. These features are strongly related to the physiological requirements of these copper centers to perform efficient electron transfer or oxidation chemistry.The binuclear copper sites CuA and T3 display particularly fast electron relaxation rates which are due to low-lying excited states that can be populated at room temperature and contribute to the reactivity of the metal site. Other magnetic techniques, such as EPR, ENDOR and MCD, normally recorded at cryogenic temperatures, are able to monitor exclusively the ground state. NMR in solution, instead can shed light on the availability of these invisible electronic states. We have studied different mutants of a native CuA site in which small perturbations are able to tune the energy gap between the ground state and the invisible excited state without perturbing the electronic structure of each of them, thus providing a mechanism to regulate the electronic structure of the metal site at room temperature.We have also studied a multicopper oxidase, Fet3 from yeast, in which signals from the T1, T2 and T3 centers could be identified and assigned toeach metal site. The temperature dependence of the hyperfine shifts reveals the accesibility of the invisible electronic states in the T3 site of this oxidase, which differ from the description for homologous T3 centers present in other enzymes, again suggesting a role of these excited states in regulating the chemistry of the metal binding site.1,2 References:1. Abriata A. L., et al., J.Am.Chem.Soc., 131, 1939 – 1946 (2009)2. Zaballa M. E.,et al., submitted