Structural studies on mitochondrial NADH dehydrogenase using chemical cross-linking

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Mitochondrial NADH dehydrogenase (Complex I) is crucial for cellular respiration.
• Understanding its complex subunit organization is essential for elucidating its function.

Purpose of the Study:

• To investigate the structural organization and subunit interactions of bovine heart mitochondrial NADH dehydrogenase.
• To identify specific subunit contacts within different domains of the enzyme.

Main Methods:

• Chemical cross-linking using disuccinimidyl tartrate, (ethylene glycol)yl bis(succinimidyl succinate), and dimethyl suberimidate.
• Western blotting with monospecific antisera to identify cross-linked subunits.
• Photolabelling with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine to probe membrane-associated subunits.

Main Results:

• Identified cross-links within flavoprotein, iron-protein, and hydrophobic domains.
• Demonstrated substrate NADH modulates cross-linking between 75 kDa (iron-protein) and 51 kDa (flavoprotein) subunits.
• Showed interactions between iron-protein/flavoprotein domains and the hydrophobic domain, including a 19 kDa subunit.

Conclusions:

• Bovine heart mitochondrial NADH dehydrogenase possesses a complex, multi-domain structure with specific subunit interactions.
• Substrate binding induces conformational changes affecting subunit contacts.
• The hydrophobic domain interacts with both iron-protein and flavoprotein domains, suggesting its role in membrane association and enzyme function.