Creating efficient and residue-directed artificial proteases is ac hallenging task due to the extreme inertness of the peptideb ond, combined with the difficulty of achieving specific interactions between the catalysts and the protein side chains. Herein we report strictly site-selective hydrolysis of am ulti-subunit globular protein, hemoglobin (Hb) from bovine blood, by ar ange of Zr IV -substituted polyoxometalates (Zr-POMs) in mildly acidic and physiological pH solutions. Among5 70 peptideb onds in Hb, selective cleavage was observed at only eleven sites, each occurring at AspÀXp eptideb onds located in the positive patcheso n the protein surface. The molecular origins of the observed AspÀXs electivity were rationalized by means of molecular docking, DFT-based binding, and mechanistic studies on model peptides. The proposed mechanism of hydrolysis involves coordination of the amide oxygen to Zr IV followed by ad irect nucleophilic attack of the side chain carboxylate group on the C-terminal amide carbon atom with formation of ac yclic anhydride, which is furtherh ydrolyzed to give the reactionp roducts. The activation energy for the cleavage of the structurally relatedG luÀXs equence compared to AspÀX was calculated to be higherb y1 .4 kcal mol À1 ,w hich corresponds to ad ifference of about one order of magnitude in the rates of hydrolysis. The highera ctivatione nergy is attributed to the higher strain presenti nt he six-membered ring of glutaric anhydride (GluÀX), as compared to the five-membered ring of the succinic anhydride (AspÀX) intermediate. Similarly,t he cleavage at XÀAsp and XÀGlu bonds are predicted to be kinetically less likely as the corresponding activation energies were 6kcal mol À1 higher , explaining the experimentally observeds electivity.T he synergy between the negativelyc harged polyoxometalate cluster, which binds at positive patches on protein surfaces, ands elective activation of AspÀXp eptideb onds located in these regions by Zr IV ions, resultsi nanovel class of artificial proteases with aspartate-directed reactivity,w hich is very rare among naturally occurring proteases.[a] Dr.