Formation of a μ _₃ -oxo nucleophile enables efficient hydrolysis by a trinuclear metal center in Family II inorganic pyrophosphatase

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 μ₃-oxo nucleophile, formed via proton transfer from a bridging μ₃-hydroxide to Asp14 and subsequent hydrogen-bond rearrangement to Asp72, as the key species in S _N 2-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 μ₃-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.