Fig. 3: Characterization of free KpDyP.

a Size exclusion chromatogram of free KpDyP (solid red line) compared with KpDyP_Enc (solid blue line) (Superdex 200). The respective main peaks are labeled. Dashed lines indicate heme absorption at 410 nm. b Negative stain TEM micrograph of free KpDyP (16 mL hexamer peak) highlighting homogeneous ca. 10 nm protein complexes. This experiment was repeated independently six times. c Negative stain TEM micrograph of the 18 mL free KpDyP peak (likely dimer). Scale bars: 50 nm. This experiment was repeated independently five times. d Heme loading of purified free KpDyP and encapsulated KpDyP_Enc determined via in vitro heme quantitation assay. Percentages refer to heme binding site occupancy, with one binding site per DyP subunit. Data are shown as mean values. Error bars represent standard deviation of three independent experiments. Source data are provided as a Source Data file. e Saturation kinetics of encapsulated KpDyP_Enc (blue squares) and free KpDyP (red circles). Hydrogen peroxide (H₂O₂) was varied while ABTS was kept constant. Data are shown as mean values. Error bars represent standard deviation of three independent experiments. For KpDyP_Enc, a standard Michaelis-Menten fit is shown as a dashed line while a competitive substrate inhibition fit is shown as a solid line. Source data are provided as a Source Data file. f Representative 2D class averages of free KpDyP. g Model of the KpDyP hexamer viewed down the two-fold (left) and three-fold (right) axes of symmetry as determined via cryo-EM. Individual DyP subunits are shown in transparent surface representation with distinct colors. Heme groups are shown as stick models (tomato red). h Heme group of a DyP subunit shown in stick representation. The proximal histidine (H225) and distal arginine (R242) are shown.