@phdthesis{db575f0363b949de99c74edfb45af290, title = From free to fixed: unveiling the impact of covalent FMN attachment in flavoproteins, abstract = Flavoproteins, which use FMN or FAD as cofactors, play vital roles in diverse redox reactions in nature and are indispensable in many biotechnological applications. This thesis explores flavoproteins with a particular focus on covalently attaching flavin mononucleotide (FMN) to proteins using a method known as flavin-fixing. This strategy employs the bacterial flavin transferase ApbE to establish a stable, site-specific covalent bond between FMN and target proteins. By designing specific recognition motifs, this work demonstrates how flavin-fixing can enhance cofactor binding while assessing its impact on enzymatic activity, photochemical properties, and potential applications in protein labeling, ROS generation, and optogenetics. The research further highlights the engineering of light-responsive LOV domains as tools for controlled ROS production in biological imaging and therapeutic applications. Flavin-fixing is applied to a range of proteins, including the phototoxic miniSOG as well as oxidative enzymes such as L-lactate oxidase and dehydrogenase, revealing both the challenges of the approach and strategies to improve enzyme performance while preserving covalent cofactor attachment. Finally, mass photometry is used to investigate how ligand binding can influence the oligomeric state of enzymes. This provides evidence for ligand-induced oligomer dissociation as a potential mechanism underlying the phenomenon of enhanced enzyme diffusion (EED), where enzymes display increased mobility during catalysis., author = Kaya, \{Saniye Gul\}, year = 2025, doi = 10.33612/diss.1402668335, language = English, publisher = University of Groningen, school = University of Groningen, }