Fig. 7: Analysis of CO’s impact on heme coordination and utilization by RnHO-1Δ22.
From: Systemic engineering and global regulation enabling high-level bilirubin biosynthesis
a Reaction progress from heme to bilirubin following the elimination of Fe²⁺ interference, data are presented as means ± SD with error bars connected sequentially to form error bands. (n = 3 biological replicates). b Relative activity of RnHO-1Δ22 using CO-pretreated heme as the substrate, data are presented as means ± SD (n = 5 biological replicates), a two-tailed unpaired Student’s t-test was used for statistical analysis. c Visualization of the CO release channel (blue) and the substrate-binding pocket in RnHO-1Δ22, with hydrophobicity increasing from white (low hydrophobicity) to red (high hydrophobicity), the green, red, brown, and blue spheres in the zoomed image represent C, O, Fe, and N atoms, respectively. Referenced the crystal structure provided by PDB code: 1DVE (doi.org/10.2210/pdb1DVE/pdb). d Quantum mechanics (QM) calculations of the coordination energies of heme with O₂ and CO. e Binding energies of RnHO-1Δ22 with heme-O₂ and heme-CO. The box plot shows the mean and the upper and lower quartiles, boxes show the 25th and 75th percentile around the median line, whiskers extend to ± 1.5 × IQR (n = 600, from 3 independent replicates). f Substrate-binding pocket of RnHO-1Δ22 (shown as a cartoon loop) complexed with heme-O₂. Ligand and key residue were displayed as sticks with van der Waals surfaces. Residue color (blue to white to red) represents the hindrance to ligand binding, from low to high. g Substrate-binding pocket of RnHO-1Δ22 (cartoon loop) complexed with heme-CO. Ligand and key residue were displayed as sticks with van der Waals surfaces. Residue color (blue to white to red) represents the hindrance to ligand binding, from low to high. h Binding energy contributions of individual residues within the substrate pocket to heme-CO versus heme-O₂, data are presented as means ± SD (n = 3 independent replicates). i Free energy landscape of the heme-O₂–RnHO-1Δ22 complex, the red to blue rainbow gradient represents the probability of conformations from low to high during the simulation. j, Free energy landscape of the heme-CO–RnHO-1Δ22 complex, the red to blue rainbow gradient represents the probability of conformations from low to high during the simulation. Source data are provided as a Source Data file.
