Maruoka, S.; Kametani, Y.; Magome, E.; Setoyama, H.; Kawamoto, M.; Horitani, M.; Teramoto, T.; Kakuta, Y.; Shiota, Y.; Yoshizawa, K.; Watanabe, K.

Efficient catalysis by metalloproteins relies on precise spatial arrangement of metal ions and active-site residues. Family II inorganic pyrophosphatase (PPase) from Shewanella species features a trinuclear metal center and displays higher catalytic activity than binuclear counterparts. Here we elucidate its hydrolytic mechanism using X-ray crystal structure-based extended X-ray absorption fine structure (XCS-EXAFS), site-directed mutagenesis, and density functional theory (DFT) calculations. We identify a catalytic -oxo nucleophile, formed via proton transfer from a bridging -hydroxide to Asp14 and subsequent hydrogen-bond rearrangement to Asp72, as the key species in SN2-type hydrolysis. This conversion defines the rate-limiting step with an activation barrier of 15.5 kcal/mol. Molecular orbital analysis reveals that the trinuclear cluster promotes -oxo formation, aligns the nucleophile for attack, and stabilizes the transition state. The side-chain rotation of the conserved Asp14 is crucial for catalysis. Our results highlight how metalloenzymes exploit geometric and electronic tuning to achieve high reactivity through evolutionarily optimized architectures.