Structural Superimposition

Abstact

Flavoenzymes primarily function in a light-independent manner, yet their intrinsic photophysical properties offer potential for nonnatural photocatalytic applications. Herein, we focus on three physiologically "photoinactive" flavoprotein amine oxidases, namely, monomeric sarcosine oxidase (MSOX), N-methyltryptophan oxidase (MTOX), and glycine oxidase (GOX), which can bind various carboxylate ligands in the active sites in a manner analogous to the binding of fatty acid substrates in the natural flavin-dependent photoenzyme, fatty acid photodecarboxylase (FAP), in the ground state. Using ultrafast spectroscopy, protein mutagenesis with natural and noncanonical amino acids, X-ray crystallography, and classical and quantum chemical calculations, we systematically characterize the photochemistry of these amine oxidases in ligand-free and ligand-bound states. We demonstrate that the binding of carboxylate ligands does not lead to productive photochemical transformation such as photodecarboxylation; instead, the ligands either alter the dynamics of photoinduced electron transfer (ET) between the flavin and nearby aromatic residue(s) or participate in a reversible photoswitching reaction. This allows us to utilize the dynamics of the ultrafast photochemical processes as a probe, and the ligands as surrogates for substrates, to characterize the active-site conformational properties and structure-function relationships of the proteins. Furthermore, we explore why the carboxylate ligands do not undergo photodecarboxylation in GOX by comparing the active-site features and excited-state properties of GOX with those of FAP. The results indicate that the presence of intrinsic quenchers, improper positioning of the ligands, and high energy barriers for ligand-to-flavin ET prevent FAP-like reactivity. Our findings provide critical insights into the active-site conformational landscapes of flavoprotein amine oxidases and offer design principles for engineering new flavin-based photobiocatalysts.