Phenylalanine Modification in Plasma-Driven Biocatalysis Revealed by Solvent Accessibility and Reactive Dynamics in Combination with Protein Mass Spectrometry

Biocatalysis is an emerging field that provides an environmentally friendly alternative to conventional catalysis but still faces some challenges. One of the major difficulties for biocatalysts that require reactive species like H₂O₂ as cosubstrates lies in the concentration of these reactive species. On the one hand, they are used as reactants; on the other hand, they inactivate the enzymes at high concentrations. When utilizing nonthermal plasma to deliver H₂O₂ for biocatalysis, it is essential to understand the potential interactions between plasma-generated species (PGS) and enzymes. This is particularly important because, alongside H₂O₂, other reactive species such as hydroxyl radicals, atomic oxygen, superoxide, and nitric oxide are also produced. The investigation of the localized reactivity of the solvent accessible surface area (SASA) of an enzyme, with certain species, is an important tool for predicting these interactions. In combination with reactive molecular dynamics (MD) simulations, this enabled us to identify amino acid residues that are likely targets for modifications by the PGS. A subset of the theoretical predictions made in the present study was confirmed experimentally by mass spectrometry, leading to the discovery of plasma-mediated phenylalanine modifications. This result underlines the utility of the SASA and MD-based screening approach to direct time-consuming experiments and assist their interpretation.