Heme-Proximal Loop Dynamics Modulate Mn(II) Oxidation in Pseudomonas putida MET94 DyP-type peroxidase

Enzymatic Mn(II) oxidation is relevant to both natural lignin degradation and the development of new industrial biocatalysts. DyP-type peroxidases are versatile microbial biocatalysts with potential applications in lignocellulose valorization, but the structural features influencing their Mn(II)-oxidizing activity remain incompletely understood. We determined the X-ray crystal structure of PpDyP from Pseudomonas putida bound to Mn(II), identifying a heme-proximal Mn-binding environment defined by E135, E188, and E204 in loop regions surrounding the heme pocket. Mutagenesis and steady-state kinetics indicate that these residues cooperate to shape the Mn(II)-oxidation environment and support catalysis. We then examined the E209A variant, in which the non-coordinating residue E209, located near E204, was replaced by alanine. E209A increased apparent Mn(II)-oxidation turnover approximately fivefold. Structural analysis showed that E209A displaces E204 and rearranges Q203-mediated inter-loop contacts, supporting E204's structural contribution to organizing the heme-proximal loop environment. This rearrangement was associated with increased loop flexibility and greater heme exposure, supporting a more accessible and dynamically permissive heme-proximal architecture. A distal Mn(II)-binding site involving E237 and E240 was also observed in E209A, but mutational analysis suggests that it contributes mainly to residual activity when the heme-proximal site is perturbed. Finally, we show that Mn(II) oxidation by PpDyP markedly enhances the oxidation of lignin-relevant substrates, including 2,6-dimethoxyphenol, guaiacol, guaiacylglycerol-β-guaiacyl ether, and alkali lignin, by up to 100-fold. Together, these findings support loop-network modulation as a strategy to tune bacterial DyP peroxidases for lignin valorization and oxidative biotransformations.