Formation of a μ ₃ -oxo nucleophile enables efficient hydrolysis by a trinuclear metal center in Family II inorganic pyrophosphatase

This is the authors' abstract. We don't add key points for this paper.

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.