Avoidance of the cytochrome c biogenesis system by periplasmic CXXCH motifs

Area of Science:

• Microbiology
• Protein Biochemistry
• Molecular Biology

Background:

• The CXXCH motif typically serves as a heme attachment site in bacterial apocytochromes c.
• The Escherichia coli cytochrome c maturation (Ccm) system primarily recognizes the CXXCH sequence.
• Some periplasmic proteins with the CXXCH motif are not c-type cytochromes, suggesting mechanisms to prevent unwanted heme attachment.

Purpose of the Study:

• To investigate how unwanted heme attachment to the CXXCH motif is avoided.
• To determine if heme can be attached to the surface of a non-cytochrome protein.
• To explore substrate recognition by the E. coli Ccm system.

Main Methods:

• Engineered a thioredoxin-like protein (cDsbD) by converting its active-site CXXCK motif to CXXCH.
• Assessed heme attachment to the engineered cDsbD by the E. coli Ccm system.
• Investigated in vivo heme attachment to the periplasmic disulfide isomerase DsbC, which has a native CXXCH motif.

Main Results:

• The E. coli Ccm system catalyzed heme attachment to only a small fraction (approximately 0.2%) of the engineered cDsbD.
• Rapid folding of cDsbD likely prevents efficient recognition by the Ccm system, unlike c-type cytochromes that fold post-heme attachment.
• Low-level covalent heme attachment was observed in vivo for the native CXXCH-containing protein DsbC.

Conclusions:

• Protein folding kinetics play a crucial role in preventing unwanted heme attachment by the Ccm system.
• The Ccm system's substrate recognition is more complex than just the CXXCH sequence.
• These findings expand the understanding of covalent heme attachment requirements and the evolution of c-type cytochromes.