The Due Ferri single chain (DFsc) family of proteins was originally created to better understand the characteristics of carboxylate-bridged diiron proteins. It was recently observed that these proteins can hydrolyze phosphate bonds in the presence of metal ions. In this project, we have developed an assay using 31P-NMR spectroscopy to monitor the catalytic activity of DFsc by observing the conversion of bis(4-nitrophenyl) phosphate (BNPP) to para-nitrophenolate (PNP) and para-nitrophenylphosphate (pNPP). Rates of reaction for DFsc and several variants were determined based on peak integrations. Results from 31P-NMR spectroscopy were then compared to results from UV-Vis based assays. Both methods were able to successfully monitor product formation but differed in the relative rates of reactivity. These differences are attributed to the different time scales required for each assay. Further optimization of the NMR assay to achieve more consistent results will be discussed.

Characterization of DFsc phosphatase activity using 31P-NMR

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