A π-stacking gate for redox control in flavin ferredoxin-thioredoxin reductases

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Flavin ferredoxin-thioredoxin reductases (FFTRs) are vital enzymes in electron transfer pathways across bacteria.
• Cyanobacterial FFTRs possess a unique C-terminal tail with a conserved tryptophan, distinct from clostridial homologs.
• This tail interacts with the flavin cofactor, influencing enzyme activity.

Purpose of the Study:

• To investigate the functional role of the C-terminal tail and conserved tryptophan in cyanobacterial FFTRs.
• To elucidate how these features modulate the flavin's electronic environment and electron transfer dynamics.
• To understand the impact on ferredoxin donor interactions and enzyme specificity.

Main Methods:

• Utilized the FFTR from Gloeobacter violaceus as a model system.
• Generated mutants lacking the C-terminal tail or the conserved tryptophan.
• Analyzed changes in flavin solvent exposure, redox potential, reduction kinetics, and ferredoxin binding specificity.

Main Results:

• Mutants showed increased flavin solvent exposure and a significant shift in redox potential.
• Electron transfer efficiency and reduction kinetics were altered in the absence of the tail or tryptophan.
• Ferredoxin binding specificity was reduced, indicating the tail's role in donor interaction.

Conclusions:

• The C-terminal tail plays a dual role: facilitating productive donor binding and optimizing the flavin environment for electron transfer.
• FAD-aromatic π-stacking interactions are critical for regulating flavin reactivity, donor specificity, and redox behavior in cyanobacterial FFTRs.
• Findings provide mechanistic insights for engineering FFTRs as biocatalysts in synthetic biology.