Decoding and Reprogramming Redox Partner Specificity in Rieske Oxygenases for Enhanced Catalytic Activity
Year: 2026
Type: preprint
Abstract: ABSTRACT Multicomponent Rieske oxygenases catalyze diverse oxidative transformations but require precisely matched redox partners to sustain efficient electron transfer, severely limiting their modularity and biocatalytic application. Yet, the molecular logic underlying this specificity remains poorly defined. Here we decode the molecular principles governing redox partner specificity in representative three-component Rieske oxygenase systems. Through systematic mutagenesis analysis and cross-component reconstitution assays, we identify a single ferredoxin residue that acts as a class-defining determinant of oxygenase recognition. Guided by this insight, we reprogram electron transfer between non-cognate components by complementary engineering of the oxygenase interface, creating an unnatural redox chain with substantially enhanced catalytic turnover compared to the native system. Spectroscopic, binding and computational analyses reveal that productive electron transfer arises from optimized electrostatic complementarity and redox potential alignment rather than maximal binding affinity. Extending this strategy to another oxygenase system demonstrates its generality. Together, these results establish transferable design rules for rationally engineering electron transfer pathways in multicomponent oxygenases, enabling their predictable adaptation as customizable biocatalysts.
Institutions: University of Groningen, Delft University of Technology
Language: English
Subfield: Inorganic Chemistry
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
Awards: G3760283037, G470823024
OpenAlex ID: W7151602006
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