Proton-coupled electron transfer at a mis-metalated zinc site detected with protein charge ladders.

Abstract

Distinguishing proton-coupled electron transfer (PCET) from uncoupled electron transfer (ET) in proteins can be challenging. A recent investigation [J. C. Koone, M. Simmang, D. L. Saenger, M. L. Hunsicker-Wang and B. F. Shaw, J. Am. Chem. Soc., 145, 16488-16497] reported that protein charge ladders and capillary electrophoresis can distinguish between single ET, PCET, and two-proton coupled ET (2PCET) by directly measuring the change in protein net charge upon reduction/oxidation (ΔZET). The current study used similar methods to assess PCET in zinc-free, "double copper" superoxide dismutase-1 (4Cu-SOD1), where one copper is bound at the copper site of each monomer and one copper is bound at the bridging zinc site, resulting in a quasi-type III Cu center. At pH 7.4, the net charge (Z) of the 4Cu-SOD1 dimer was unaffected by reduction of all four Cu2+ ions, i.e., ΔZ4ET = -0.09 ± 0.05 per dimer (-0.02 ± 0.01 per copper atom). These values suggest that PCET is taking place at all four Cu atoms of the homodimer. Molecular dynamics and Poisson-Boltzmann calculations suggest that a metal-coordinating histidine at the zinc site (His71) is the proton acceptor. These data show how ligands of a naturally occurring zinc site can help facilitate PCET when the right redox metal is bound.