Interdomain contacts in flavocytochrome b(2), a mutational analysis

Area of Science:

• Biochemistry
• Enzyme kinetics
• Protein structure and dynamics

Background:

• Flavocytochrome b(2) (l-lactate cytochrome c oxidoreductase) has a modular structure with N-terminal heme and C-terminal flavodehydrogenase (FDH) domains.
• Enzyme mobility, particularly of the heme domain, is implicated in its catalytic cycle and electron transfer efficiency.
• Previous studies suggested antibody binding at the interdomain interface affects flavin-to-heme electron transfer.

Purpose of the Study:

• To investigate the role of interdomain interactions in flavocytochrome b(2) function.
• To identify specific amino acid residues and interactions critical for heme domain docking and electron transfer.
• To characterize the dynamics of electron transfer using advanced kinetic and spectroscopic methods.

Main Methods:

• Site-directed mutagenesis of heme domain residues at the interdomain interface.
• Steady-state and pre-steady-state kinetic analyses.
• Redox potential measurements and Electron Paramagnetic Resonance (EPR) spectroscopy.

Main Results:

• Several hydrophobic and van der Waals contacts at the heme-FDH domain interface were identified as essential for catalytically competent docking.
• Mutations significantly impacting electron transfer rates were generated, providing insights into the mechanism.
• An isosbestic wavelength for monitoring flavin radical formation was proposed.

Conclusions:

• Specific interdomain interactions stabilize the heme domain for efficient electron transfer.
• The study elucidates the structural basis for flavin-to-heme electron transfer in flavocytochrome b(2).
• The findings contribute to understanding enzyme dynamics and catalytic mechanisms.