Fig. S2.

Quantification of 5′-dA, SAH, and iron produced during ChuW reaction(s). Separation and quantification of 5′-dA and SAH using HPLC was performed as described in SI Materials and Methods. In the absence of sodium dithionite (circles), no SAH or 5′-dA was produced (cf. A and B); however, in the absence of only deuteroheme (diamonds) 5′-dA was still produced in a linear manner, whereas SAH was not detected (cf. A and B). In the presence of deuteroheme (triangles), both SAH and 5′-dA were produced in a 1:1 ratio (cf. A and B). Products were quantified using a standard curve, and assays were performed in triplicate. (C) HPLC traces showing the results of a 20-min assay using the physiological electron donor system flavodoxin, NADPH/flavodoxin reductase in the absence of NADPH (trace i), the absence of heme (trace ii), or with the complete assay mix (trace iii). (D) Total labile iron present in the ChuW assay at the start of the reaction and after 30 min.