Mechanisms\nof Copper Selectivity and Release by the\nMetallochaperone CusF: Insights from CO-Binding, Rapid-Freeze-Quench\nEXAFS, and Unnatural Amino Acid Substitution

Metallochaperones are small proteins that shuttle essential\nmetal\nions such as Cu selectively to their cellular targets. CusF has unusual\nCu(I) coordination, bound by two methionines, one histidine and a\ncapping tryptophan residue, W44. Here we compare the CO binding reactivity\nof the wild type (WT) protein and its W44A, F, and M variants. Fourier-transform\ninfrared (FTIR) indicates that W44A provides unhindered access for\nCO, while W44M is unreactive. WT is also largely unreactive to CO\nsuggesting that the tryptophan cap is effective in shielding the Cu(I)\ncenter from exogenous adduct formation, while the Phe variant shows\npartial reactivity suggestive of an equilibrium between cap-on and\ncap-off conformers. Rates of metal transfer to the partner CusB are\nconsistent with the π–cation cap providing both selectivity\nand redox protection. Unnatural amino acid substitutions of the W44\nligand with cyano-Phe and Br-Phe underpin the conclusion that the\nPhe ligand is a less effective capping residue. Finally, density functional\ntheory (DFT) calculations validate the CO-binding strategy. Overall,\nthe study suggests that CusF uses the tryptophan cap to protect against\nexogenous ligand (O<sub>2</sub>) attack while the mechanism of protein–protein\ncomplex formation allows the cap to swing out of the way, and thus\nhave minimal effect on the rates of metal transfer.