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The Cu(A) site of cytochrome c oxidase is a redox hub that participates in rapid electron transfer at low driving forces with two redox cofactors in nearly perpendicular orientations. Spectroscopic and electrochemical characterizations performed on first and second-sphere mutants have allowed us to experimentally detect the reversible switching between two alternative electronic states that confer different directionalities to the redox reaction. Specifically, the M160H variant of a native Cu(A) shows a reversible pH transition that allows to functionally probe both states in the same protein species. Alternation between states exerts a dramatic impact on the kinetic redox parameters, thereby suggesting this effect as the mechanism underlying the efficiency and directionality of Cu(A) electron transfer in vivo. These findings may also prove useful for the development of molecular electronics., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The Cu(A) center is a dinuclear copper site that serves as an optimized hub for long-range electron transfer in heme-copper terminal oxidases. Its electronic structure can be described in terms of a σ(u)* ground-state wavefunction with an alternative, less populated ground state of π(u) symmetry, which is thermally accessible. It is now shown that second-sphere mutations in the Cu(A) containing subunit of Thermus thermophilus ba3 oxidase perturb the electronic structure, which leads to a substantial increase in the population of the π(u) state, as shown by different spectroscopic methods. This perturbation does not affect the redox potential of the metal site, and despite an increase in the reorganization energy, it is not detrimental to the electron-transfer kinetics. The mutations were achieved by replacing the loops that are involved in protein-protein interactions with cytochrome c, suggesting that transient protein binding could also elicit ground-state switching in the oxidase, which enables alternative electron-transfer pathways., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)