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Title: The thioreduction component CcmG confers efficiency and the heme ligation component CcmH ensures stereo-specificity during cytochrome c maturation

Abstract

In many Gram-negative bacteria, including Rhodobacter capsulatus, cytochrome c maturation (Ccm) is carried out by a membrane-integral machinery composed of nine proteins (CcmA to I). During this process, the periplasmic thiol-disulfide oxidoreductase DsbA is thought to catalyze the formation of a disulfide bond between the Cys residues at the apocytochrome c heme binding site (CXXCH). Subsequently, a Ccm-specific thioreductive pathway involving CcmG and CcmH reduces this disulfide bond to allow covalent heme ligation. Currently, the sequence of thioredox reactions occurring between these components and apocytochrome c, and the identity of their active Cys residues are unknown. In this work, we first investigated protein-protein interactions among the apocytochrome c, CcmG, and the heme ligation components CcmF, CcmH, and CcmI. We found that they all interact with each other, forming a CcmFGHI-apocytochrome c complex. Using purified wild-type CcmG, CcmH, and apocytochrome c, as well as their respective Cys mutant variants we determined the rates of thiol-disulfide exchange reactions between selected pairs of Cys residues from these proteins. We established that CcmG can efficiently reduce the disulfide bond of apocytochrome c and also resolve a mixed disulfide bond formed between apocytochrome c and CcmH. We further show that C45 of CcmH and C34more » of apocytochrome c are most likely to form this mixed disulfide bond, which is consistent with the stereo-specificity of the heme-apocytochrome c ligation reaction. We conclude that CcmG confers efficiency, and CcmH ensures stereo-specificity during Ccm, and present a comprehensive model for thioreduction reactions that lead to heme-apocytochrome c ligation.« less

Authors:
; ; ; ; ; ; ;
Publication Date:
Research Org.:
Univ. of Pennsylvania, Philadelphia, PA (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1535361
Grant/Contract Number:  
FG02-91ER20052
Resource Type:
Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 292; Journal Issue: 32; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Country of Publication:
United States
Language:
English
Subject:
Biochemistry & Molecular Biology

Citation Formats

Verissimo, Andreia F., Khalfaoui-Hassani, Bahia, Hwang, Josephine, Steimle, Stefan, Selamoglu, Nur, Sanders, Carsten, Khatchikian, Camilo E., and Daldal, Fevzi. The thioreduction component CcmG confers efficiency and the heme ligation component CcmH ensures stereo-specificity during cytochrome c maturation. United States: N. p., 2017. Web. doi:10.1074/jbc.m117.794586.
Verissimo, Andreia F., Khalfaoui-Hassani, Bahia, Hwang, Josephine, Steimle, Stefan, Selamoglu, Nur, Sanders, Carsten, Khatchikian, Camilo E., & Daldal, Fevzi. The thioreduction component CcmG confers efficiency and the heme ligation component CcmH ensures stereo-specificity during cytochrome c maturation. United States. doi:10.1074/jbc.m117.794586.
Verissimo, Andreia F., Khalfaoui-Hassani, Bahia, Hwang, Josephine, Steimle, Stefan, Selamoglu, Nur, Sanders, Carsten, Khatchikian, Camilo E., and Daldal, Fevzi. Tue . "The thioreduction component CcmG confers efficiency and the heme ligation component CcmH ensures stereo-specificity during cytochrome c maturation". United States. doi:10.1074/jbc.m117.794586. https://www.osti.gov/servlets/purl/1535361.
@article{osti_1535361,
title = {The thioreduction component CcmG confers efficiency and the heme ligation component CcmH ensures stereo-specificity during cytochrome c maturation},
author = {Verissimo, Andreia F. and Khalfaoui-Hassani, Bahia and Hwang, Josephine and Steimle, Stefan and Selamoglu, Nur and Sanders, Carsten and Khatchikian, Camilo E. and Daldal, Fevzi},
abstractNote = {In many Gram-negative bacteria, including Rhodobacter capsulatus, cytochrome c maturation (Ccm) is carried out by a membrane-integral machinery composed of nine proteins (CcmA to I). During this process, the periplasmic thiol-disulfide oxidoreductase DsbA is thought to catalyze the formation of a disulfide bond between the Cys residues at the apocytochrome c heme binding site (CXXCH). Subsequently, a Ccm-specific thioreductive pathway involving CcmG and CcmH reduces this disulfide bond to allow covalent heme ligation. Currently, the sequence of thioredox reactions occurring between these components and apocytochrome c, and the identity of their active Cys residues are unknown. In this work, we first investigated protein-protein interactions among the apocytochrome c, CcmG, and the heme ligation components CcmF, CcmH, and CcmI. We found that they all interact with each other, forming a CcmFGHI-apocytochrome c complex. Using purified wild-type CcmG, CcmH, and apocytochrome c, as well as their respective Cys mutant variants we determined the rates of thiol-disulfide exchange reactions between selected pairs of Cys residues from these proteins. We established that CcmG can efficiently reduce the disulfide bond of apocytochrome c and also resolve a mixed disulfide bond formed between apocytochrome c and CcmH. We further show that C45 of CcmH and C34 of apocytochrome c are most likely to form this mixed disulfide bond, which is consistent with the stereo-specificity of the heme-apocytochrome c ligation reaction. We conclude that CcmG confers efficiency, and CcmH ensures stereo-specificity during Ccm, and present a comprehensive model for thioreduction reactions that lead to heme-apocytochrome c ligation.},
doi = {10.1074/jbc.m117.794586},
journal = {Journal of Biological Chemistry},
number = 32,
volume = 292,
place = {United States},
year = {2017},
month = {6}
}

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Works referenced in this record:

Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4
journal, August 1970