Atomically defined mechanism for proton transfer to a buried redox centre in a protein

The basis of the chemiosmotic theory is that energy from light or respiration is used to generate a trans-membrane proton gradient(1). This is largely achieved by membrane-spanning enzymes known as 'proton pumps'(2-5). There is intense interest in experiments which reveal, at the molecular level, how protons are drawn through proteins(6-13). Here we report the mechanism, at atomic resolution, for a single long-range electron-coupled proton transfer. In Azotobacter vinelandii ferredoxin I, reduction of a buried iron-sulphur cluster draws in a solvent proton, whereas re-oxidation is 'gated' by proton release to the solvent. Studies of this 'proton-transferring module' by fast-scan protein film voltammetry, high-resolution crystallography, site-directed mutagenesis and molecular dynamics, reveal that proton transfer is exquisitely sensitive to the position and pK of a single amino acid. The proton is delivered through the protein matrix by rapid penetrative excursions of the side-chain carboxylate of a surface residue (Asp 15), whose pK shifts in response to the electrostatic charge on the iron-sulphur cluster. Our analysis defines the structural, dynamic and energetic requirements for proton courier groups in redox-driven proton-pumping enzymes.