μ₃-Oxo nucleophile formation enables efficient SN2 hydrolysis at the trinuclear metal center in inorganic pyrophosphatase

Abstract Inorganic pyrophosphatases are essential metalloenzymes for phosphate metabolism. Bacterial Family II Inorganic pyrophosphatases utilize a trinuclear metal center and exhibit higher catalytic activity than binuclear counterparts. Here we show the mechanism underlying this enhanced hydrolytic efficiency in the enzyme from Shewanella sp. AS-11 using X-ray absorption spectroscopy, site-directed mutagenesis, and density functional theory calculations. We identify a catalytic μ 3 -oxo nucleophile—generated by proton transfer from a bridging μ 3 -hydroxide to Asp14—as the key reactive species for hydrolysis. Rotation of Asp14 drives this conversion and constitutes the rate-limiting step, with an activation barrier of 15.5 kcal mol -1 . The trinuclear metal center promotes hydrolysis by lowering the p K a of the hydroxide to facilitate μ 3 -oxo formation, stabilizing this intermediate, positioning the nucleophile for optimal in-line attack, and enhancing phosphorus electrophilicity. These findings highlight the importance of reactive species generation and illustrate how metalloenzymes exploit geometric and electronic tuning to achieve high catalytic reactivity.