The pre-steady-state kinetics was used to determine the pH optimum of reductive half-reaction (RHR) and oxidative half-reaction (OHR) catalysed by AcmB. The following buffers were used: 50 mM Tris-HCl, 150 mM NaCl, 5% (w/v) glycerol (pH 7.5-8.5) and 50 mM glycine-NaOH, 150 mM NaCl, 5% (w/v) glycerol (pH 9.0-10.0). The enzyme purified with Ni-affinity chromatography was transferred to the respective buffer using Econo-Pac® 10DG desalting columns (BioRad) according to the instruction manual. Due to the rapid aggregation of the enzyme at low pH observed under the concentration required for the stopped-flow experiment, it was impossible to measure the reduction rate below pH 7.5.