Purification and polypeptide characterization of complex III from yeast mitochondria

Area of Science:

• Biochemistry
• Molecular Biology
• Mitochondrial Respiration

Background:

• Mitochondrial Complex III (cytochrome bc1 complex) is crucial for cellular respiration.
• Understanding the structural organization and subunit accessibility of Complex III is key to elucidating its function.

Purpose of the Study:

• To isolate and characterize bakers' yeast Complex III.
• To determine the subunit composition, stoichiometry, and surface accessibility of Complex III.
• To investigate the structural role of core proteins and the iron-sulfur protein.

Main Methods:

• Purification using ammonium sulfate fractionation and Ultrogel AcA 34 chromatography.
• Enzyme activity assays with coenzyme Q analog.
• Electrophoresis (SDS-PAGE) to determine polypeptide molecular weights.
• Protease digestion (trypsin, chymotrypsin) to assess subunit accessibility.
• Isoelectric focusing to determine subunit isoelectric points.
• Antibody production and immunoinhibition studies.

Main Results:

• Purified Complex III exhibited specific activity of 17.1 µmol/min/mg, containing cytochromes b and c1.
• Seven polypeptides (15.5-50 kDa) were identified; lower molecular weight species resulted from proteolysis.
• Core proteins were embedded and inaccessible to proteases, while cytochromes, iron-sulfur protein, and a 17.5 kDa subunit were surface-exposed.
• Iron-sulfur protein showed reduced trypsin susceptibility upon reduction, indicating a conformational change.
• Antibodies confirmed core proteins' embedded antigenic sites.

Conclusions:

• The study elucidated the subunit structure and surface topology of yeast Complex III.
• Core proteins form an internal structural scaffold, while other subunits are more accessible.
• Conformational changes in the iron-sulfur protein during redox cycling influence its accessibility.