Anaerobic Reconstitution.

Samples were reconstituted in an anaerobic chamber (Coy Laboratory Products, Grass Lake, MI) containing a N₂/H₂ atmosphere and operating at < 7 mg/m³ (5 ppm) O₂. Briefly, protein was brought into the anaerobic chamber and allowed to equilibrate with the anaerobic chamber’s atmosphere overnight at 6 °C with shaking. Protein was then diluted to 100 µmol/L in reconstitution buffer comprising 50 mmol/L MOPS, pH 7.5, 100 mmol/L NaCl, 1 mmol/L DTT, 0.7 mol/L glycerol (5% (v/v)). 10 mmol/L stock FeCl₃ was first titrated into the apo protein until up to 6 mole equivalents had been added with 10 min shaking at 6 °C between the addition of each mole equivalent of Fe⁺³. 10 mmol/L stock Na₂S was then titrated into the iron-bound protein in the same manner. Afterwards, protein was equilibrated with FeCl₃ and Na₂S for ≈ 2 h at 6 °C with shaking. Particulate matter was removed by first centrifuging at 14000×g anaerobically for 10 min at 4 °C and then by filtration through a filter with a 0.22 μm pore size. Excess iron and sulfide were removed by buffer exchanging using a 0.5 mL Amicon 3 kg/mol (3 kDa) MWCO spin concentrator at least four times into fresh 50 mmol/L MOPS, pH 7.5, 100 mmol/L NaCl, 1 mmol/L DTT, 0.7 mol/L glycerol (5% (v/v)). Iron contents were determined as described below.