Fig. 4: Superoxide from Cpd III mediates the cooperative catalysis of two distant pockets.

a, Detailed inhibitory data of superoxide scavenger tempol towards EasC_Cf activity. The reaction system generally consisted of 20 μM enzyme, 0.5 mM PCC and 2 mM NADPH. A triplicate assay was conducted. The enzyme activity of each sample was calculated as a percentage of the control sample. The individual (n = 3) and average values along with error bars (s.d.) of each group in one representative experiment among three independent experiments are shown. b, High-performance liquid chromatography (HPLC) profiles of the restoration of SOTS-1 of the tempol-inhibited EasC_Cf activity. HPLC profiles were recorded at λ = 320 nm. SOTS-1 (8 mM) was directedly added to reaction mixtures, which were incubated with the superoxide scavenger tempol (10 mM) in advance. c, HPLC profiles of the Xox system (312 μM per 5 mU) restoration of tempol-inhibited EasC_Cf activity. HPLC profiles were recorded at λ = 320 nm. The Xox system was directedly added to the EasC reaction mixtures, which were incubated with the superoxide scavenger tempol (10 mM) in advance. d, HRMS of CC isolated from the competitive incorporation experiment with ¹⁸O-superoxide. ¹⁸O-labelled CC [M + H]⁺ m/z: calculated 259.1696 and observed 259.1689. The blue line displays the data for the control group without the addition of ¹⁸O-superoxide, whereas the red line displays the data for the experimental group with the addition of O¹⁸-superoxide. This diagram shows the relative abundance of each component, with the molecular weight of 257 set as 100%. EasC noted in the figure is EasC_Cf.