Trace nitric acid

The purified MbnH protein concentration was determined using a standard DC assay (Bio-Rad) with equine cytochrome c (Sigma) as the standard curve protein. Metal loading was determined using ICP-MS at the Quantitative Bioelement Imaging Center at Northwestern University. Quantification of Fe was performed after digesting samples in concentrated trace nitric acid (> 69%, Thermo Fisher Scientific) and heating at 65 °C overnight for ~14 h. MilliQ H₂O (18.2 MΩ·cm) was then added to produce a final solution of 3.0% nitric acid (v/v) in a 5 mL sample. A 100 μg/mL Fe element standard (Inorganic Ventures) was used to produce a 100 ng/mL 50 mL quantitative standard in 3.0% nitric acid (v/v). Samples were measured using a Thermo iCapQ ICP-MS (Thermo Fisher Scientific) controlled by the QTEGRA software in the same manner as described previously.^{23 (link)} Quantitative heme loading was achieved in the purified protein as determined by comparison of the Fe content to the protein concentration.