Expression and characterization of two pathogenic mutations in human electron transfer flavoprotein

Area of Science:

• Biochemistry
• Molecular Biology
• Human Genetics

Background:

• Glutaric acidemia type II is a human inherited metabolic disease caused by defects in electron transfer flavoprotein (ETF) or ETF-ubiquinone oxidoreductase (ETF-QO).
• This impairment disrupts electron transfer from primary flavoprotein dehydrogenases, including those in fatty acid beta-oxidation, to the main respiratory chain.
• Specific mutations in the ETF alpha subunit are implicated in disease pathogenesis.

Purpose of the Study:

• To investigate the functional consequences of two patient-identified mutations (alphaT266M and alphaG116R) in the human electron transfer flavoprotein (ETF).
• To characterize the structural and catalytic effects of the alphaT266M mutation on ETF function and its interaction with ETF-QO.

Main Methods:

• Expression of wild-type and mutant ETF alpha subunits in Escherichia coli.
• Coexpression with chaperonins (GroEL and GroES) for stabilization of mutant ETF.
• Purification and characterization of mutant ETF proteins using spectroscopy (absorption, circular dichroism) and enzymatic assays.
• Assessment of ETF-QO activity using a coupled acyl-CoA:ubiquinone reductase assay.

Main Results:

• The alphaG116R mutant ETF required chaperonin coexpression, exhibited altered folding, and was catalytically inactive and unstable.
• The alphaT266M mutant ETF, though globally structurally unchanged, showed an altered flavin environment and increased flavin semiquinone stability.
• The alphaT266M mutation minimally affected ETF reduction but significantly reduced ETF-QO activity (kcat/Km decreased 33-fold), primarily due to impaired semiquinone disproportionation.

Conclusions:

• The alphaT266M mutation in ETF disrupts the flavin binding environment, leading to altered redox properties and significantly impaired electron transfer to ETF-QO.
• This impairment in ETF-QO activity likely contributes to the metabolic dysfunction observed in glutaric acidemia type II patients with this mutation.
• Understanding these molecular mechanisms is crucial for comprehending the pathophysiology of ETF-related metabolic disorders.