Direct Observation of Nanosecond Water Exchange Dynamics at a Protein Metal Site

Nanosecond ligand exchange dynamics at metal sites within proteins is essential in catalysis, metal ion transport, and regulatory metallobiochemistry. Herein we present direct observation of the exchange dynamics of water at a Cd²⁺ binding site within two de novo designed metalloprotein constructs using ^111mCd perturbed angular correlation (PAC) of γ-rays and ¹¹³Cd NMR spectroscopy. The residence time of the Cd²⁺-bound water molecule is tens of nanoseconds at 20 °C in both proteins. This constitutes the first direct experimental observation of the residence time of Cd²⁺ coordinated water in any system, including the simple aqua ion. A Leu to Ala amino acid substitution ∼10 Å from the Cd²⁺ site affects both the equilibrium constant and the residence time of water, while, surprisingly, the metal site structure, as probed by PAC spectroscopy, remains essentially unaltered. This implies that remote mutations may affect metal site dynamics, even when structure is conserved.