Dehydrogenase versus Oxidase Function: The Interplay between Substrate Binding and Flavin Microenvironment

Redox enzymes, mostly equipped with metal or organic
cofactors,
can vary their reactivity with oxygen by orders of magnitude. Understanding
how oxygen reactivity is controlled by the protein milieu remains
an open issue, with broad implications for mechanistic enzymology
and enzyme design. Here, we address this problem by focusing on a
widespread group of flavoenzymes that oxidize phenolic compounds derived
from microbial lignin degradation, using either oxygen or cytochrome
c as an electron acceptor. A comprehensive phylogenetic analysis revealed
conserved amino acid motifs in the flavin-binding site. Using a combination
of kinetic, mutagenesis, structural, and computational methods, we
examined the role of these residues. Our results demonstrate that
subtle and localized changes in the flavin environment can drastically
impact oxygen reactivity. These effects are afforded through the creation
or blockade of pathways for oxygen diffusion. Substrate binding plays
a crucial role by potentially obstructing oxygen access to the flavin,
thus influencing the enzyme’s reactivity. The switch between
oxidase and dehydrogenase functionalities is thereby achieved through
targeted, site-specific amino acid replacements that finely tune the
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 enzymes with tailored functions.