Quantification of Cofactors Released from IscS

Cofactors were released from each preparation of IscS and IscS^Q183P as described previously (15 (link), 17 (link), 29 (link)). KOH (30 mM final concentration) was added to 1.5 nmol purified protein in a 100 μL reaction and incubated at room temperature for 10 min. Protein was then precipitated with 10% trifluoroacetic acid (30 μL), resulting in a final volume of 130 μL. The precipitate was removed by centrifugation (17,000 × g for 3 min) and the supernatant was filtered through a 0.45 μm centrifugal tube filter (Costar 8170). Cofactors were separated by HPLC using a Shimadzu HPLC equipped with a Luna C18 column (Phenomenex) using a two-step isocratic method with a flow rate of 0.8 mL/min: 0 to 5 min with 100% buffer A (0.06% vol/vol trifluoroacetic acid) and 5 to 18 min with methanol and buffer A (3:97). The column was washed with methanol and buffer A (60:40) for 10 min between each run. Eluant was monitored at 305 nm using a photodiode array detector (Shimadzu SPD-M20A). Authentic PLP and pyruvate/PLP were used as standards to allow peak assignment. Pyruvate/PLP was synthesized as described previously (46 (link)), purified by HPLC, and concentrated.