Measurement and Control of Crossed Potentials in a Flavoprotein.

Area of Science:

• Biochemistry
• Structural Biology
• Computational Chemistry

Background:

• Flavoproteins are essential redox-active proteins utilizing flavin cofactors.
• Protein scaffolds tune flavin cofactor redox potentials, but mechanisms remain unclear.
• Understanding flavin tuning is crucial for interpreting flavoprotein function.

Purpose of the Study:

• Investigate how the flavin N5 environment affects flavin mononucleotide redox properties in the iLOV sensor.
• Elucidate the molecular mechanisms of redox potential modulation.
• Provide benchmarks for theoretical models of flavoprotein tuning.

Main Methods:

• Site-directed mutagenesis of the iLOV sensor.
• Redox titrations to determine reduction potentials.
• Hybrid quantum mechanical/molecular mechanical (QM/MM) and alchemical free energy simulations.

Main Results:

• Mutating residue Q103 near flavin N5/O4 altered potential crossing by up to 168 mV.
• Water penetration into the flavin-binding pocket explained observed redox potential trends.
• Hydrogen bonding to the neutral semiquinone significantly impacts potential crossing.

Conclusions:

• Flavin tuning involves modulation of hydrogen bonding interactions at the N5/O4 site.
• Protein-penetrating water molecules play a key role in tuning flavin redox potentials.
• The study advances a molecular mechanism for redox tuning in flavoproteins.