Dehydrogenase versus oxidase function: the interplay between substrate binding and flavin microenvironment

Abstract

Redox enzymes, mostly equipped with metal or organiccofactors, can vary their reactivity with oxygen by orders of magnitude.Understanding how oxygen reactivity is controlled by the protein milieuremains an open issue, with broad implications for mechanistic enzymology andenzyme design. Here, we address this problem by focusing on a widespreadgroup of flavoenzymes that oxidize phenolic compounds derived from microbiallignin degradation, using either oxygen or cytochrome c as an electron acceptor.A comprehensive phylogenetic analysis revealed conserved amino acid motifs inthe flavin-binding site. Using a combination of kinetic, mutagenesis, structural,and computational methods, we examined the role of these residues. Our resultsdemonstrate that subtle and localized changes in the flavin environment candrastically impact oxygen reactivity. These effects are afforded through thecreation or blockade of pathways for oxygen diffusion. Substrate binding plays acrucial role by potentially obstructing oxygen access to the flavin, thus influencing the enzyme’s reactivity. The switch betweenoxidase and dehydrogenase functionalities is thereby achieved through targeted, site-specific amino acid replacements that finely tunethe microenvironment around the flavin. Our findings explain how very similar enzymes can exhibit distinct functional properties,operating as oxidases or dehydrogenases. They further provide valuable insights for the rational design and engineering of enzymeswith tailored functions.