Fe(II)/α‐ketoglutarate‐dependent dioxygenases (αKGDs) are versatile biocatalysts whose catalytic activity relies on an efficient supply of O ₂ and αKG. However, in engineering applications, the O ₂ supply is seldom considered, whereas αKG is typically supplied through the oxidation of l ‐glutamate by l ‐glutamate oxidase, which concomitantly produces H ₂ O ₂ that potently inhibits αKGD activity. This study developed a tunnel engineering strategy based on random accelerated molecular dynamics and protein structuromics to precisely modulate the access of small molecules (O ₂ and H ₂ O ₂ ) to isoleucine dioxygenase (IDO), enhancing O ₂ transport while resisting H ₂ O ₂ transport. Notably, mutant 1 (L179C/V225F/I240V) and mutant 2 (N193S/V225I/I240V) displayed markedly higher catalytic activity for five aliphatic amino acid substrates (Leu, Nle, Nva, Met, and Ile), reaching 27.2‐fold greater activity. In addition, mutant 2 exhibited 7.1‐fold higher activity against Ile in the presence of H ₂ O ₂ . This study highlights O ₂ and H ₂ O ₂ tunnel engineering as a new strategy for enhancing αKGDs activity and antioxidative properties.

Research on mixed meals using the INFOGEST protocol has often focused on individual foods, leaving co-ingested matrix associations less well characterized. To address this gap, this study investigated bidirectional associations between steaks cooked to three doneness levels (medium, SM; medium-well, SW; well-done, SD) and red wine during simulated gastrointestinal digestion, focusing on protein oxidation, apparent digestibility, secondary structure, and the apparent bioaccessibility of selected phenolic compounds. Results showed that co-digestion with red wine was associated with lower accumulation of protein oxidation markers across all doneness levels. In the gastrointestinal phase, carbonyl content was 44.5–46.8% lower and free sulfhydryl content was 53.4–59.1% higher than in steak digested alone. SW steak co-digested with wine exhibited the highest mean gastric apparent digestibility (37.7%), and FTIR indicated differences in protein secondary-structure distribution. Furthermore, mixed digests showed higher radical-scavenging activity than when wine was digested alone (an average ABTS increase of 61.4% in the gastrointestinal phase), while the steak matrix altered the digestive recovery and gastric apparent bioaccessibility of several target phenolic compounds. Overall, these findings indicate that steak doneness and red-wine co-digestion are associated with distinct matrix-dependent changes in both protein digestion and apparent phenolic bioaccessibility.