Probing the redox properties of the alternative ground states in native CuA centers

ALVAREZ PAGGI, D.; ZITARE U; ABRIATA L; VILA A; MURGIDA D

Bochum

Conferencia; • 12th Topical Meeting of the International Society of Electrochemistry & XXII International Symposium on Bioelectrochemistry and Bioenergetics of the Bioelectrochemical; 2013

International Society of Electrochemistry

Cytochrome c oxidase (CcO) is a transmembrane multimeric enzyme that contains several redox sites and is a component of the electron transport chain. Electrons shuttled by a soluble cytochrome c (Cyt) are delivered to the CcO’s primary acceptor, the CuA site of subunit II and from there to the catalytic site embedded in subunit I where O2 is reduced to water. These steps involve two long, nearly perpendicular pathways through the protein milieu. Despite the low driving forces, electron transfer (ET) takes place with high rates along these two pathways. CuA is a binuclear copper site, the two copper ions being bridged by two cysteine ligands, forming a nearly planar Cu2S2 diamond core characterized by a short Cu-Cu distance. The coordination sphere of the metal site is completed by two terminal histidine residues and two weakly coordinated axial ligands provided by a methionine sulfur and a backbone carbonyl. It has been reported that the electronic ground state of the CuA site is of σu* symmetry, while a πu state could be achieved by elongation of the Cu-Cu distance. The latter state has been deemed redox inactive. Here we present the spectroscopical, electrochemical and computational characterization of several weak axial ligand and second sphere mutants that perturb the electronic structure of the metal site while retaining its native fold. Said perturbations modify the populations between the two alternative electronic ground states of the CuA site, allowing us to probe the redox and spectroscopic features of each one individually. In the present work we show evidence that conversion between both states may be achieved by several different small structural fluctuations. In addition, we show that both states are redox active and they present distinct electronic properties that allow for efficient electron (πu) entry and exit (σu*), respectively, by means of a fine-tuning of the reorganization energy and electronic coupling. Moreover, our results allow us to rationalize previous and new evidence that show how other perturbations like pH, Cyt-CuA complex formation and the strength of the electric field generated by the membrane potential impact on the population of both ground states, thus acting as possible modulators of the ET reaction.