Pyridine Hemochrome Assay for MmcA

This assay was performed as described before^{52 (link),53 (link)}. Briefly, a 0.2 M NaOH with 40% pyridine solution was made fresh using a 1 M NaOH stock and 100% pyridine solution (Sigma–Aldrich, St. Louis, MO). 5 µL (i.e., 1/200) of 0.1 M potassium ferricyanide stock solution was added to 495 µL of the aforementioned NaOH + pyridine mix to generate the pyridine hemochrome assay solution. 50 µL of the assay solution was mixed with 50 µL of TBS buffer (50 mM Tris-HCl, 150 mM NaCl, pH = 7.4) and used as a blank. Next, 50 µL of the assay solution was mixed with 50 µL of MmcA in TBS buffer, and UV-vis scans were immediately performed using a Shimadzu 1900i (Shimadzu, Torrance, CA, USA) to record the oxidized spectra. A 10 mM stock solution of sodium dithionate was added to the protein assay mixture and UV-vis scans were performed using a Shimadzu 1900i (Shimadzu, Torrance, CA, USA) to record the fully reduced pyridine hemochrome spectra.