Role of the metal binding loops in cofactor assembly and electronic structure of a copper site in cytochrome oxidases

ALEJANDRO J. VILA

Salta

Simposio; Latin American Crosstalk in Biophysics; 2015

Sociedad latinoamericana de biofisica

NMR of oxidized copper proteins has been largelyoverlooked, mostly due to the slow electron relaxation times of Cu2+ ion whichinduce extremely fast relaxation rates in nearby nuclei, rendering themundetectable. It has been shown, however, that these unfavorable electronrelaxation features are restricted to T2 copper sites, since T1, T3 and CuAcenters display faster electron relaxation rates which make them amenable toNMR studies. In all these cases, the fast electron relaxation stems from to theavailability of low-lying excited electronic states which is due to theparticular electronic structure of these centers. These features are stronglyrelated to the physiological requirements of these copper centers to performefficient electron transfer or oxidation chemistry. The binuclear copper sites CuA and T3 displayparticularly fast electron relaxation rates which are due to low-lying excitedstates that can be populated at room temperature and contribute to thereactivity of the metal site. Other magnetic techniques, such as EPR, ENDOR andMCD, normally recorded at cryogenic temperatures, are able to monitorexclusively the ground state. NMR in solution, instead can shed light on the availabilityof these invisible electronic states. We have carried on detailed studies inthe CuA site, involved in long range electron transfer in terminal oxidases.NMR discloses the fact that the CuA site canexist in two alternate ground states with different orbital symmetry, which areinvisible to other techniques.  We showthat thermal fluctuations may populate two alternative ground-state electronicwave functions optimized for electron entry and exit, respectively, through twodifferent and nearly perpendicular pathways. These findings suggest a uniquerole for alternative or ?invisible? electronic ground states in directionalelectron transfer. Moreover, it is shown 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 andmembrane potential may optimize and regulate electron?proton energytransduction.