pH-dependent transition between delocalized and trapped valence states of a Cu_A center and its possible role in proton-coupled electron transfer

A pH-dependent transition between delocalized and trapped mixed valence states of an engineered Cu_A center in azurin has been investigated by UV-visible absorption and electron paramagnetic resonance spectroscopic techniques. At pH 7.0, the Cu_A azurin displays a typical delocalized mixed valence dinuclear [Cu(1.5)⋯Cu(1.5)] spectra with optical absorptions at 485, 530, and 760 nm, and with a seven-line EPR hyperfine. Upon lowering of the pH from 7.0 to 4.0, the absorption at 760 nm shifted to lower energy toward 810 nm, and a four-line EPR hyperfine, typical of a trapped valence, was observed. The pH-dependent transition is reversible because increasing the pH restores all delocalized spectral features. Lowering the pH resulted in not only a trapped valence state, but also a dramatically increased reduction potential of the Cu center (from 160 mV to 340 mV). Mutation of the titratable residues around the metal-binding site ruled out Glu-114 and identified the C-terminal histidine ligand (His-120) as a site of protonation, because the His120Ala mutation abolished the above pH-dependent transition. The corresponding histidine in cytochrome c oxidases is along a major electron transfer pathway from CUA center to heme a. Because the protonation of this histidine can result in an increased reduction potential that will prevent electron flow from the Cu_A to heme a, the Cu_A and the histidine may play an important role in regulating proton-coupled electron transfer.