In vitro reconstitution reveals major differences between human and bacterial cytochrome c synthases

Cytochromes c are ubiquitous heme proteins in mitochondria and bacteria, all possessing a CXXCH (CysXxxXxxCysHis) motif with covalently attached heme. We describe the first in vitro reconstitution of cytochrome c biogenesis using purified mitochondrial (HCCS) and bacterial (CcsBA) cytochrome c synth...

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Bibliographic Details
Published in:eLife 2021-05, Vol.10
Main Authors: Sutherland, Molly C, Mendez, Deanna L, Babbitt, Shalon E, Tillman, Dustin E, Melnikov, Olga, Tran, Nathan L, Prizant, Noah T, Collier, Andrea L, Kranz, Robert G
Format: Article
Language:English
Subjects:
Attachment
Bacteria
Biochemistry and Chemical Biology
Biosynthesis
Cytochrome
Cytochrome c
cytochrome c biogenesis
Heme
heme attachment
Heme proteins
Histidine
Mitochondria
Peptides
post translational modification
Proteins
Respiration
Standard deviation
synthases
Thiols
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Summary:Cytochromes c are ubiquitous heme proteins in mitochondria and bacteria, all possessing a CXXCH (CysXxxXxxCysHis) motif with covalently attached heme. We describe the first in vitro reconstitution of cytochrome c biogenesis using purified mitochondrial (HCCS) and bacterial (CcsBA) cytochrome c synthases. We employ apocytochrome c and peptide analogs containing CXXCH as substrates, examining recognition determinants, thioether attachment, and subsequent release and folding of cytochrome c. Peptide analogs reveal very different recognition requirements between HCCS and CcsBA. For HCCS, a minimal 16-mer peptide is required, comprised of CXXCH and adjacent alpha helix 1, yet neither thiol is critical for recognition. For bacterial CcsBA, both thiols and histidine are required, but not alpha helix 1. Heme attached peptide analogs are not released from the HCCS active site; thus, folding is important in the release mechanism. Peptide analogs behave as inhibitors of cytochrome c biogenesis, paving the way for targeted control.
ISSN:2050-084X
2050-084X
DOI:10.7554/eLife.64891