Wide-dynamic-range kinetic investigations of deep proton tunnelling in proteins

Directional proton transport along 'wires' that feed biochemical reactions in proteins is poorly understood. Amino-acid residues with high pK(a) are seldom considered as active transport elements in such wires because of their large classical barrier for proton dissociation. Here, we use the light-triggered proton wire of the green fluorescent protein to study its ground-electronic-state proton-transport kinetics, revealing a large temperature-dependent kinetic isotope effect. We show that 'deep' proton tunnelling between hydrogen-bonded oxygen atoms with a typical donor-acceptor distance of 2.7-2.8 angstrom fully accounts for the rates at all temperatures, including the unexpectedly large value (2.5 x 10(9) s(-1)) found at room temperature. The rate-limiting step in green fluorescent protein is assigned to tunnelling of the ionization-resistant serine hydroxyl proton. This suggests how high-pK(a) residues within a proton wire can act as a 'tunnel diode' to kinetically trap protons and control the direction of proton flow.