During Cytochrome c Maturation CcmI Chaperones the Class I Apocytochromes until the Formation of Their b-Type Cytochrome Intermediates

Verissimo, Andreia F.; Shroff, Namita P.; Daldal, Fevzi

doi:10.1074/jbc.m115.652818

Cited by 1 publication

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“…3F). Our earlier studies established that CcmI is an apocyt c chaperone that binds tightly the C terminus of class I apocyts (29,30,38) and that CcmG has oxidoreductase and chaperone (holdase) activities to assist the apocyts c and enhance cyt c maturation (14). Similar cooperation of CcmG with the heme ligation complex was reported with an engineered E. coli CcmFGH complex (equivalent of R. capsulatus CcmFGHI) that can carry out Ccm in the absence of CcmABCDE (39).…”

Section: Thioreduction Branch Of the Ccm Pathwaysupporting

confidence: 53%

“…capsulatus apocyt c 1 mutants were produced using the QuikChange site-directed mutagenesis kit and the plasmid pMAM1 as a template. pMAM1 encodes a variant of apocyt c 1 missing its last C-terminal 39 amino acids that constitute the TM helix and lacking the non-heme ligating Cys-144 and Cys-167 that form a structural disulfide bridge (Strep-apocyt c 1 WT ) (30). The plasmids pMAM1C37S, pMAM1C34S, and pMAM1C34SC37S obtained by site-directed mutagenesis produced the single and double (indicated by *) Cys mutant derivatives Strep-apocyt c 1…”

Section: Methodsmentioning

confidence: 99%

“…Because the use of apocyt c 1 WT for these assays was not suitable due to its high tendency to dimerize in the presence of oxygen, we used biolayer interferometry to study these interactions. Real-time binding kinetics between purified CcmG WT and apocyt c 1 WT were determined, as done earlier (30). The association (k on of 9.97 Ϯ 0.11 ϫ 10 2 M Ϫ1 s Ϫ1 ) and dissociation (k off of 7.31 Ϯ 1.16 ϫ 10 Ϫ3 s Ϫ1 ) rates thus determined yielded a K D value of 7.2 Ϯ 1.8 M using a 1:1 homogeneous kinetic model describing bimolecular interactions (30).…”

Section: Thioreduction Branch Of the Ccm Pathwaymentioning

confidence: 99%

“…Real-time binding kinetics between purified CcmG WT and apocyt c 1 WT were determined, as done earlier (30). The association (k on of 9.97 Ϯ 0.11 ϫ 10 2 M Ϫ1 s Ϫ1 ) and dissociation (k off of 7.31 Ϯ 1.16 ϫ 10 Ϫ3 s Ϫ1 ) rates thus determined yielded a K D value of 7.2 Ϯ 1.8 M using a 1:1 homogeneous kinetic model describing bimolecular interactions (30). Similarly, when native apocyt c 2 WT (another class I cyt c, known to interact with CcmI and CcmE (29 -31)) was used instead of apocyt c 1 WT , its co-purification with CcmG WT was readily seen using specific antibodies (Fig.…”

Section: Thioreduction Branch Of the Ccm Pathwaymentioning

confidence: 99%

“…Binding kinetics of CcmG and apocyt c 1 were monitored quantitatively in real time by biolayer interferometry using an Octet RED96 instrument (ForteBio, Inc.) as described elsewhere (30). Briefly, streptavidin (SA)-coated biosensors were loaded with biotinylated Strep-apocyt c 1 WT (400 nM) in 50 mM Tris, pH 8.0, 100 mM NaCl, 0.05% DDM, 1% BSA buffer at 30°C and at 1000 rpm.…”

Section: Protein-protein Interactions Determined By Biolayer Interfermentioning

confidence: 99%

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

Verissimo

Khalfaoui-Hassani

Hwang

et al. 2017

Journal of Biological Chemistry

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In many Gram-negative bacteria, including cytochrome 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 heme-binding site (CCH). 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 and the identity of their active Cys residues are unknown. In this work, we first investigated protein-protein interactions among the apocytochrome CcmG, and the heme-ligation components CcmF, CcmH, and CcmI. We found that they all interact with each other, forming a CcmFGHI-apocytochrome complex. Using purified wild-type CcmG, CcmH, and apocytochrome 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 and also resolve a mixed disulfide bond formed between apocytochrome and CcmH. We further show that Cys-45 of CcmH and Cys-34 of apocytochrome are most likely to form this mixed disulfide bond, which is consistent with the stereo-specificity of the heme-apocytochrome 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 ligation.

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