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

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 Cys-45 of CcmH and Cys-34 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.

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

The c-type cytochromes are electron transfer proteins involved in energy transduction. They have heme-binding (CXXCH) sites that covalently ligate heme b via thioether bonds and are classified into different classes based on their protein folds and the locations and properties of their cofactors. Rhodobacter capsulatus produces various c-type cytochromes using the cytochrome c maturation (Ccm) System I, formed from the CcmABCDEFGHI proteins. CcmI, a component of the heme ligation complex CcmFHI, interacts with the heme-handling protein CcmE and chaperones apocytochrome c2 by binding its C-terminal helix. Whether CcmI also chaperones other c-type apocytochromes, and the effects of heme on these interactions were unknown previously. Here, we purified different classes of soluble and membrane-bound c-type apocytochromes (class I, c2 and c1, and class II c') and investigated their interactions with CcmI and apoCcmE. We report that, in the absence of heme, CcmI and apoCcmE recognized different classes of c-type apocytochromes with different affinities (nM to µM KD values). When present, heme induced conformational changes in class I apocytochromes (e.g. c2) and decreased significantly their high affinity for CcmI. Knowing that CcmI does not interact with mature cytochrome c2 and that heme converts apocytochrome c2 into its b-type derivative, these findings indicate that CcmI holds the class I apocytochromes (e.g. c2) tightly until their noncovalent heme-containing b-type cytochrome-like intermediates are formed. We propose that these intermediates are subsequently converted into mature cytochromes following the covalent ligation of heme via the remaining components of the Ccm complex.

Cytochrome c biogenesis System I: an intricate process catalyzed by a maturase supercomplex?

Cytochromes c are ubiquitous heme proteins that are found in most living organisms and are essential for various energy production pathways as well as other cellular processes. Their biosynthesis relies on a complex post-translational process, called cytochrome c biogenesis, responsible for the formation of stereo-specific thioether bonds between the vinyl groups of heme b (protoporphyrin IX-Fe) and the thiol groups of apocytochromes c heme-binding site (C1XXC2H) cysteine residues. In some organisms this process involves up to nine (CcmABCDEFGHI) membrane proteins working together to achieve heme ligation, designated the Cytochrome c maturation (Ccm)-System I. Here, we review recent findings related to the Ccm-System I found in bacteria, archaea and plant mitochondria, with an emphasis on protein interactions between the Ccm components and their substrates (apocytochrome c and heme). We discuss the possibility that the Ccm proteins may form a multi subunit supercomplex (dubbed "Ccm machine"), and based on the currently available data, we present an updated version of a mechanistic model for Ccm. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

High stability of apo-cytochrome c' from thermophilic Hydrogenophilus thermoluteolus.

Apo-cytochomes c without heme are usually unstructured. Here we showed that apo-form of thermophilic Hydrogenophilus thermoluteolus cytochrome c' (PHCP) was a monomeric protein with high helix content. Apo-PHCP was thermally stable, possibly due to the hydrophobic residues and ion pairs. PHCP is the first example of a structured apo-cytochrome c', which will expand our view of hemoprotein structure formation.

Structural insight into an anti-BRIL Fab as a G-protein-coupled receptor crystallization chaperone.

Structure determination of G-protein-coupled receptors (GPCRs) is key for the successful development of efficient drugs targeting GPCRs. BRIL is a thermostabilized apocytochrome b562 (with M7W/H102I/R106L mutations) from Escherichia coli and is often used as a GPCR fusion protein for expression and crystallization. SRP2070Fab, an anti-BRIL antibody Fab fragment, has been reported to facilitate and enhance the crystallization of BRIL-fused GPCRs as a crystallization chaperone. This study was conducted to characterize the high-resolution crystal structure of the BRIL-SRP2070Fab complex. The structure of the BRIL-SRP2070Fab complex was determined at 2.1 Šresolution. This high-resolution structure elucidates the binding interaction between BRIL and SRP2070Fab. When binding to BRIL, SRP2070Fab recognizes conformational epitopes, not linear epitopes, on the surface of BRIL helices III and IV, thereby binding perpendicularly to the helices, which indicates stable binding. Additionally, the packing contacts of the BRIL-SRP2070Fab co-crystal are largely due to SRP2070Fab rather than BRIL. The accumulation of SRP2070Fab molecules by stacking is remarkable and is consistent with the finding that stacking of SRP2070Fab is predominant in known crystal structures of BRIL-fused GPCRs complexed with SRP2070Fab. These findings clarified the mechanism of SRP2070Fab as a crystallization chaperone. Moreover, these data will be useful in the structure-based drug design of membrane-protein drug targets.