NMR Spectroscopy reveals alternative Ground States in Copper Proteins

ALEJANDRO J. VILA; LUCIANO A. ABRIATA; ANDRES EPINOZA CARA; MARCOS N. MORGADA; MARÍA-EUGENIA ZABALLA

Rio de janeiro

Congreso; 18th ISMAR Conference; 2013

NMR Spectroscopy reveals alternative Ground States in Copper Proteins Alejandro J. Vila, Luciano A. Abriata, Andrés Espinoza- Cara, Marcos N. Morgada, María-Eugenia Zaballa Institute for Molecular and Celular Biology (IBR), University of Rosario, Argentina NMR of oxidized copper proteins has been hampered by the unfavorable electron relaxation times of the Cu2+ ion which induce fast relaxation rates in nearby nuclei, rendering them undetectable. However, this limitation applies only to T2 copper sites, while T1, T3 and CuA centers are amenable to NMR studies due to the availability of low-lying excited electronic states. 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 carried on detailed studies in the CuA site, involved in long range electron transfer in terminal oxidases. NMR discloses the fact that the CuA site can exist in two alternate ground states with different orbital symmetry, which are invisible to other techniques. We show that thermal fluctuations may populate two alternative ground-state electronic wave functions optimized for electron entry and exit, respectively, through two different and nearly perpendicular pathways. These findings suggest a unique role for alternative or "invisible" electronic ground states in directional electron transfer. Moreover, we show that this energy gap and, therefore, the equilibrium between ground states can be fine-tuned by minor perturbations, suggesting alternative ways through which protein?protein interactions and membrane potential may optimize and regulate electron?proton energy transduction. References: [1] Abriata et al., J.Am.Chem.Soc., 131, 1939?1946 (2009). [2] Zaballa et al., J.Am.Chem.Soc., 132, 11191?11196 (2010). [3] K. Lancaster et al., J.Am.Chem.Soc., 134, 8241-53 (2012). [4] M.E.Zaballa et al., Proc.Natl.Acad.Sci USA, 109, 9254-9 (2012). [5] L.A.Abriata et al., Proc.Natl.Acad.Sci USA, 109, 17348- 53 (2012).