A second-sphere variant of cytochrome P450 PbdA improves turnover and apparent coupling in veratrate oxidation

Efficient use of reducing equivalents is critical for productive turnover in cytochrome P450 redox enzymes, yet improving activity without compromising substrate affinity remains challenging. Here, we engineered the second-sphere region of PbdA, a cytochrome P450 from Rhodococcus jostii RHA1 that catalyzes veratrate oxidation, and identified position 242 as a functionally sensitive site. The hydrophobic D242V variant approximately doubled the catalytic turnover number, k cat , while largely preserving substrate binding affinity. By quantifying NADH consumption and product formation, we found that D242V increased apparent coupling efficiency from 43.88% to 57.30%. Molecular dynamics simulations suggested that V242 remodels active-site hydration, reducing local water occupancy, which may help limit nonproductive uncoupling. Pathways analysis further revealed modest differences in the sampling of putative electron-transfer geometries, which correlated with the observed kinetic improvement but should be interpreted as a relative structural descriptor rather than direct evidence for altered electron-transfer rates. Finally, coupling engineered PbdA with glyoxylate carboligase enabled in situ formaldehyde scavenging and glycolaldehyde synthesis, achieving approximately 90% substrate conversion and 70% glycolaldehyde yield. These results suggest that while the second-sphere generally accommodates a highly restrictive mutational landscape, precise substitutions at isolated sensitive nodes (such as position 242) can occasionally fine-tune turnover and apparent coupling in P450-catalyzed veratrate oxidation, providing a basis for related biocatalytic cascades.