Directional proton transport along ‘wires’ that feed biochemical reactions in proteins is poorly understood. Amino-acid residues with high pKa 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 Å fully accounts for the rates at all temperatures, including the unexpectedly large value (2.5 × 109 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-pKa residues within a proton wire can act as a ‘tunnel diode’ to kinetically trap protons and control the direction of proton flow.
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This work was supported by National Science Foundation awards CHE-1243948 (P.M.C) and CHE-1026369 (P.M.C. and J.T.S.) and EPSRC award EP/I003304/1 (J.v.T.). The authors thank M. Bearpark and L. Thompson for providing the ONIOM files associated with the GFP normal mode calculation and A. Fitzpatrick for preparing the E222D mutant. A. McClelland and B. Kellner are also thanked for early contributions to this work.
The authors declare no competing financial interests.
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Salna, B., Benabbas, A., Sage, J. et al. Wide-dynamic-range kinetic investigations of deep proton tunnelling in proteins. Nature Chem 8, 874–880 (2016). https://doi.org/10.1038/nchem.2527
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