Electron-transferring flavoprotein from pig kidney: flavin analogue studies

Area of Science:

• Biochemistry
• Enzyme kinetics
• Protein structure

Background:

• Electron-transferring flavoprotein (ETF) is crucial in fatty acid metabolism.
• Understanding the flavin binding site is key to elucidating ETF's function.
• Previous studies have hinted at the accessibility of the flavin's 8-position.

Purpose of the Study:

• To probe the flavin binding site of apo-ETF using FAD analogues.
• To investigate the role of the 8-position of FAD in ETF's function.
• To explore the mechanism of electron transfer between ETF and dehydrogenases.

Main Methods:

• Preparation and reconstitution of apo-ETF with FAD analogues.
• Enzymatic assays using octanoyl-CoA and medium-chain acyl-CoA dehydrogenase.
• Spectroscopic analysis and photoaffinity labeling.
• Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Main Results:

• The 8-position of bound flavin is accessible to solvent.
• Bulky 8-alkylmercapto-FAD analogues bind tightly but significantly slow electron transfer rates.
• ETF anion radical intermediates accumulate during reduction of 8-substituted derivatives.
• Photoaffinity labeling identified preferential labeling of the smaller ETF subunit.

Conclusions:

• The 8-position substituents influence ETF's interaction with dehydrogenases and electron transfer kinetics.
• Results support a model of obligatory 1-electron transfer involving the dimethylbenzene edge of ETF.
• The smaller ETF subunit appears to be the primary site of flavin interaction.