Abstract
Metallochaperones are small proteins that shuttle essential metal ions such as Cu selectively to their cellular targets. CusF has unusual Cu(I) coordination, bound by two methionines, one histidine and a capping tryptophan residue, W44. Here we compare the CO binding reactivity of the wild type (WT) protein and its W44A, F, and M variants. Fourier-transform infrared (FTIR) indicates that W44A provides unhindered access for CO, while W44M is unreactive. WT is also largely unreactive to CO suggesting that the tryptophan cap is effective in shielding the Cu(I) center from exogenous adduct formation, while the Phe variant shows partial reactivity suggestive of an equilibrium between cap-on and cap-off conformers. Rates of metal transfer to the partner CusB are consistent with the π-cation cap providing both selectivity and redox protection. Unnatural amino acid substitutions of the W44 ligand with cyano-Phe and Br-Phe underpin the conclusion that the Phe ligand is a less effective capping residue. Finally, density functional theory (DFT) calculations validate the CO-binding strategy. Overall, the study suggests that CusF uses the tryptophan cap to protect against exogenous ligand (O(2)) attack while the mechanism of protein-protein complex formation allows the cap to swing out of the way, and thus have minimal effect on the rates of metal transfer.