HPLC chromatograms monitoring the reaction of WT MBP-SO3491 with different nucleotides. The x-axis is the elution time, and the y-axis is the absorbance at 254 nm. All reactions were performed with 5 μM protein (diiron concentration) and 50 μM substrate analogues under O2-free conditions. The trace label corresponds to the substrate nucleotide. p1 corresponds to 5′-pGpG, p2 to 5′-pApG,38 p3 to 3′-AMP, p4 to 2′-AMP, and p5 to 3′-GMP.