Table 2.

Methods for Purification of K. pneumoniae or M. extorquens PqqE

	K. pneumoniae (Wecksler et al., 2009)	M. extorquens (Barr et al., 2016; Latham et al., 2015)
Cell pellet disruption	Lysis with BugBuster (Novagen) in 50mM Tris pH 7.9, 1 mM DTT, 300mM NaCl, 10mM imidazole, 5μL benzonase nuclease; centrifugation 15,000 × g, 20 min, 4°C	Lysis with BugBuster (Novagen) in 50 mM Tris pH 7.9, 300mM NaCl, 1mM TCEP, 30mM imidazole, 5μL benzonase nuclease; 15,000 × g, 20min, 4°C
Chromatography	(i) Ni-NTA column (12 in. long, 2.5 in wide) equilibrated with 50 mM Tris pH 7.9, 1mM DTT, 300mM NaCl, 10mM imidazole. Washed with 100 mL of the same buffer, then with 100mL of 25 mM imidazole in buffer, followed by 100mL of 50 mM imidazole in buffer. Eluted with 200mM imidazole in buffer. Fractions selected based on color, pooled and concentrated to 5 mL by ultrafiltration (Amicon Ultra 30K membrane) (ii) Gel filtration in PD-10 column equilibrated with 50 mM Tris pH 7.9, 1mM DTT, 300 mM NaCl to remove imidazole. Protein collected off the column and concentrated to ~10 mg/mL by ultrafiltration (Amicon Ultra 30K membrane)	(i) His-Trap FF column (Novagen) equilibrated with 50 mM Tris pH 7.9, 300mM NaCl, 1mM TCEP, 30 mM imidazole buffer. Washed with the same buffer and eluted with 300 mM imidazole in buffer. Fractions pooled and concentrated to 2.5 mL by ultrafiltration (Amicon Ultra 30K membrane) (ii) Gel filtration in PD-10 columns equilibrated with 50mM Tris pH 7.9, 300mM NaCl, 10% (v/v) glycerol
Yield	0.5–1.5mg/L LB medium	18mg/L TB medium
Reconstitution	Not possible, the protein precipitates	Chemical reconstitution with Na2S and ammonium Fe(III) citrate
Fe and S content	10.4 ± 0.9 mol Fe and 7.0 ± 1.0mol S/monomer	7–10 mol Fe/monomer (as-purified) 13 mol Fe and 12.2 mol S/monomer (reconstituted)

All procedures are done in anaerobic conditions, in a glovebox.