Ferrozine

Iron content of the purified enzyme was determined using a colorimetric assay with ferrozine (Fish, 1988) using ferrozine (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). The color change was measured at 593 nm. A standard of 0 to 200 µM ammonium iron sulfate hexahydrate was used to calculate the amount of iron in the samples.
The sulfur content of the purified enzyme was determined using a colorimetric assay [52 (link)] using N,N-dimethyl-p-phenylenediamine (DMPD) HCl. The sodium sulfide standard was made freshly every time from an anaerobic 1 mM stock solution and the samples were taken freshly from an anoxically stored solution. The change in color of DMPD was measured after centrifugation at 670 nm. A standard of 0 to 200 µM sodium sulfide was used to calculate the amount of sulfur in the samples.
For the quantification of tungsten and molybdenum in the purified AOR-His, the samples were sent to Spurenanalytisches Laboratorium Dr. Baumann (Maxhütte-Haidhof, Germany) for ICP-MS.

Cells were collected by centrifugation at 10,000 g for 3 min, washed twice in phosphate-buffered saline (PBS), pH 7.2 (http://cshprotocols.cshlp.org), and adjusted to an optical density (OD) at 600 nm (OD_600nm) of 2 in the presence of 2.2 mM ferrihydrite (Schwertmann and Fischer, 1973 (link); Stookey, 2002 (link)) and 2 mM ferrozine (Sigma-Aldrich). Where indicated, 55 mM glucose or mannitol, 20 μg/mL DHNA, and riboflavin were added. After 3 hr incubation at 30 °C, the cells were collected by centrifugation at 10,000 g for 5 min and the absorbance of the supernatant was measured at 562 nm with a Synergy 2 spectrophotometer (BioTek, Winooski, VT, USA). Quantities of ferrihydrite reduced were determined using a standard curve containing a 2-fold range of FeSO₄ (Sigma-Aldrich) (0.25 mM to 0.016 mM) and 2 mM ferrozine. The FeSO₄ was dissolved in 10 mM cysteine-HCl (RPI, Mount Prospect, IL, USA) to prevent environmental re-oxidation of Fe²⁺ to Fe³⁺ in the standard curve. For testing iron reduction activity of cells with a DHNA concentration of 0.01 μg/mL in the medium, iron(III) oxide nanoparticles < 50 nm (Sigma-Aldrich) were used as insoluble iron form (Figure 1—figure supplement 3).

Ferrozine assay was performed by suspending B. circulans (10⁷ CFU) in TSB medium with and without 2% glucose and 0.1 μM roseoflavin total 50 μl, an equal volume of Ferrozine (8 mM) (Sigma) and 100 μl ferric ammonium citrate (100 mM) (Sigma) were added into each well. The mixture was incubated at 37 °C for 1 h in 96-well. The colour change of media was detected from OD at 562 nm.