The structure and function of omega loop A replacements in cytochrome c

Area of Science:

• Biochemistry
• Structural Biology
• Protein Engineering

Background:

• Cytochrome c proteins are crucial electron carriers in cellular respiration.
• Modifications to specific protein loops can significantly impact structure and function.
• Omega-loop A in yeast iso-1-cytochrome c plays a role in protein stability and electron transfer.

Purpose of the Study:

• To investigate the structural and functional effects of replacing omega-loop A in yeast iso-1-cytochrome c.
• To characterize loop replacement mutants using X-ray crystallography and electrochemical methods.
• To understand how alterations in protein structure affect cytochrome c stability and redox potential.

Main Methods:

• X-ray diffraction was used to determine the three-dimensional structures of loop replacement mutants.
• In vivo growth rate studies on nonfermentable media assessed protein function.
• Cyclic voltammetry measured the midpoint reduction potential of the mutant proteins.

Main Results:

• The RepA2 mutant, with phenylalanine at position 20, exhibited altered molecular packing and surface interactions.
• The RepA2(Val 20) mutant restored wild-type hydrophobic core packing.
• RepA2(Val 20) cytochrome c showed wild-type-like growth rates, while RepA2 displayed temperature-dependent stability and function.
• RepA2 mutant had a reduced midpoint reduction potential (271 mV) compared to wild-type and RepA2(Val 20) (290 mV).

Conclusions:

• Replacing omega-loop A in yeast iso-1-cytochrome c with that from Rhodospirillum rubrum cytochrome c2 leads to significant structural changes.
• Restoring the original residue at position 20 (Val 20) rescues protein packing and restores in vivo function.
• Structural disruptions in the RepA2 mutant affect heme pocket interactions, leading to reduced redox potential and enhanced temperature dependence.