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.

CcmI subunit of CcmFHI heme ligation complex functions as an apocytochrome c chaperone during c-type cytochrome maturation.

Cytochrome c maturation (Ccm) is a sophisticated post-translational process. It occurs after translocation of apocytochromes c to the p side of energy transducing membranes and forms stereo-specific thioether bonds between the vinyl groups of heme b (protoporphyrin IX-Fe) and the thiol groups of cysteines at their conserved heme binding sites. In many organisms this process involves up to 10 (CcmABCDEFGHI and CcdA) membrane proteins. One of these proteins is CcmI, which has an N-terminal membrane-embedded domain with two transmembrane helices and a large C-terminal periplasmic domain with protein-protein interaction motifs. Together with CcmF and CcmH, CcmI forms a multisubunit heme ligation complex. How the CcmFHI complex recognizes its apocytochrome c substrates remained unknown. In this study, using Rhodobacter capsulatus apocytochrome c(2) as a Ccm substrate, we demonstrate for the first time that CcmI binds apocytochrome c(2) but not holocytochrome c(2). Mainly the C-terminal portions of both CcmI and apocytochrome c(2) mediate this binding. Other physical interactions via the conserved structural elements in apocytochrome c(2), like the heme ligating cysteines or heme iron axial ligands, are less crucial. Furthermore, we show that the N-terminal domain of CcmI can also weakly bind apocytochrome c(2), but this interaction requires a free thiol group at apocytochrome c(2) heme binding site. We conclude that the CcmI subunit of the CcmFHI complex functions as an apocytochrome c chaperone during the Ccm process used by proteobacteria, archaea, mitochondria of plants and red algae.

Engineering a prokaryotic apocytochrome c as an efficient substrate for Saccharomyces cerevisiae cytochrome c heme lyase.

Cytochromes c are heme proteins that require multiple maturation components, such as heme lyases, for cofactor incorporation. Saccharomyces cerevisiae has two heme lyases that are specific for apocytochromes c (CCHL) or c(1) (CC(1)HL). CCHL can covalently attach heme b groups to apocytochrome c substrates of eukaryotic but not prokaryotic origin. Besides their conserved Cys-Xxx-Xxx-Cys-His heme-binding motifs, the amino-terminal regions of apocytochrome c substrates appear to be important for CCHL function. In this study, we show for the first time that only two amino acid changes in the amino-terminal region of the non-CCHL substrate apocytochrome c(2) from Rhodobacter capsulatus are necessary and sufficient for efficient holocytochrome c formation by CCHL. This finding led us to propose a consensus sequence located at the amino-terminus of apocytochromes c, and critical for substrate recognition and heme ligation by CCHL.

Compensatory thio-redox interactions between DsbA, CcdA and CcmG unveil the apocytochrome c holdase role of CcmG during cytochrome c maturation.

During cytochrome c maturation (Ccm), the DsbA-dependent thio-oxidative protein-folding pathway is thought to introduce a disulphide bond into the haem-binding motif of apocytochromes c. This disulphide bond is believed to be reduced through a thio-reductive pathway involving the Ccm components CcdA (DsbD), CcmG and CcmH. Here, we show in Rhodobacter capsulatus that in the absence of DsbA cytochrome c levels were decreased and CcdA or CcmG or the putative glutathione transporter CydDC was not needed for Ccm. This decrease was not due to overproduction of the periplasmic protease DegP as a secondary effect of DsbA absence. In contrast, CcmH was absolutely necessary regardless of DsbA, indicating that compensatory thio-redox interactions excluded it. Remarkably, the double (DsbA-CcmG) and triple (DsbA-CcmG-CcdA) mutants produced cytochromes c at lower levels than the DsbA-null mutants, unless they contained a CcmG derivative (CcmG*) lacking its thio-reductive activity. Purified CcmG* can bind apocytochrome c in vitro, revealing for the first time a thiol-independent, direct interaction between apocytochrome c and CcmG. Furthermore, elimination of the thio-redox components does not abolish cytochrome c production, restricting the number of Ccm components essential for haem-apocyt c ligation per se during Ccm.

SUMO fusion technology for enhanced protein production in prokaryotic and eukaryotic expression systems.

In eukaryotic cells, the reversible attachment of small ubiquitin-like modifier (SUMO) protein is a post-translational modification that has been demonstrated to play an important role in various cellular processes. Moreover, it has been found that SUMO as an N-terminal fusion partner enhances functional protein production in prokaryotic and eukaryotic expression systems, based upon significantly improved protein stability and solubility. Following the expression and purification of the fusion protein, the SUMO-tag can be cleaved by specific (SUMO) proteases via their endopeptidase activity in vitro to generate the desired N-terminus of the released protein partner. In addition to its physiological relevance in eukaryotes, SUMO can, thus, be used as a powerful biotechnological tool for protein expression in prokaryotic and eukaryotic cell systems.In this chapter, we will describe the construction of a fusion protein with the SUMO-tag, its expression in Escherichia coli, and its purification followed by the removal of the SUMO-tag by a SUMO-specific protease in vitro.

An alternative model of the twin arginine translocation system.

The twin arginine translocation (Tat) system is a machinery which can translocate folded proteins across energy transducing membranes. Currently it is supposed that Tat substrates bind directly to Tat translocon components before a ApH-driven translocation occurs. In this review, an alternative model is presented which proposes that membrane integration could precede Tat-dependent translocation. This idea is mainly supported by the recent observations of Tat-independent membrane insertion of Tat substrates in vivo and in vitro. Membrane insertion may allow i) a quality control of the folded state by membrane bound proteases like FtsH, ii) the recognition of the membrane spanning signal peptide by Tat system components, and iii) a pulling mechanism of translocation. In some cases of folded Tat substrates, the membrane targeting process may require ATP-dependent N-terminal unfolding-steps.

Cytochrome c biogenesis: the Ccm system.

Cytochromes of c-type contain covalently attached hemes that are formed via thioether bonds between the vinyls of heme b and cysteines within C(1)XXC(2)H motifs of apocytochromes. In diverse organisms this post-translational modification relies on membrane-associated specific biogenesis proteins, referred to as cytochrome c maturation (Ccm) systems. A highly complex version of these systems, Ccm or System I, is found in Gram-negative bacteria, archaea and plant mitochondria. We describe emerging functional interactions between the Ccm components categorized into three conserved modules, and present a mechanistic view of the molecular basis of ubiquitous vinyl-2 approximately Cys(1) and vinyl-4 approximately Cys(2) heme b-apocytochrome thioether bonds in c-type cytochromes.

The cytochrome c maturation components CcmF, CcmH, and CcmI form a membrane-integral multisubunit heme ligation complex.

Cytochrome c maturation (Ccm) is a post-translational and post-export protein modification process that involves ten (CcmABCDEFGHI and CcdA or DsbD) components in most Gram-negative bacteria. The absence of any of these components abolishes the ability of cells to form cytochrome c, leading in the case of Rhodobacter capsulatus to the loss of photosynthetic proficiency and respiratory cytochrome oxidase activity. Based on earlier molecular genetic studies, we inferred that R. capsulatus CcmF, CcmH, and CcmI interact with each other to perform heme-apocytochrome c ligation. Here, using functional epitope-tagged derivatives of these components coproduced in appropriate mutant strains, we determined protein-protein interactions between them in detergent-dispersed membranes. Reciprocal affinity purification as well as tandem size exclusion and affinity chromatography analyses provided the first biochemical evidence that CcmF, CcmH, and CcmI associate stably with each other, indicating that these Ccm components form a membrane-integral complex. Under the conditions used, the CcmFHI complex does not contain CcmG, suggesting that the latter thio-reduction component is not always associated with the heme ligation components. The findings are discussed with respect to defining the obligatory components of a minimalistic heme-apocytochrome c ligation complex in R. capsulatus.

Membrane-spanning and periplasmic segments of CcmI have distinct functions during cytochrome c Biogenesis in Rhodobacter capsulatus.

In gram-negative bacteria, like Rhodobacter capsulatus, about 10 membrane-bound components (CcmABCDEFGHI and CcdA) are required for periplasmic maturation of c-type cytochromes. These components perform the chaperoning and thio-oxidoreduction of the apoproteins as well as the delivery and ligation of the heme cofactors. In the absence of any of these components, including CcmI, proposed to act as an apocytochrome c chaperone, R. capsulatus does not have the ability to produce holocytochromes c or consequently to exhibit photosynthetic growth and cytochrome cbb3 oxidase activity. Previously, we have demonstrated that null mutants of CcmI partially overcome cytochrome c deficiency phenotypes upon overproduction of the CcmF-R. capsulatus CcmH (CcmF-CcmH(Rc)) couple in a growth medium-dependent manner and fully bypass these defects by additional overproduction of CcmG. Here, we show that overproduction of the CcmF-CcmH(Rc) couple and overproduction of the N-terminal membrane-spanning segment of CcmI (CcmI-1) have similar suppression effects of cytochrome c maturation defects in CcmI-null mutants. Likewise, additional overproduction of CcmG, the C-terminal periplasmic segment of CcmI (CcmI-2), or even of apocytochrome c2 also provides complementation abilities similar to those of these mutants. These results indicate that the two segments of CcmI have different functions and support our earlier findings that two independent steps are required for full recovery of the loss of CcmI function. We therefore propose that CcmI-1 is part of the CcmF-CcmH(Rc)-dependent heme ligation, while CcmI-2 is involved in the CcdA- and CcmG-dependent apoprotein thioreduction steps, which intersect at the level of CcmI during cytochrome c biogenesis.

Extracytoplasmic prosthetic group ligation to apoproteins: maturation of c-type cytochromes.

In all organisms, haem is post-translationally and covalently attached to c apocytochromes to produce c holocytochromes via a process called c-type cytochromes maturation, which involves numerous components. In bacteria it was not clear which of these components catalyses the extracytoplasmic haem-apocytochrome ligation per se. In this issue of Molecular Microbiology, Feissner and colleagues report that a single polypeptide from Helicobacter pylori, corresponding to the fusion of two proteins found in other organisms, performs haem ligation to a coexpressed Bordetella pertussis apocytochrome c in an Escherichia coli mutant lacking its own cytochrome c maturation proteins. This simple experimental system pinpoints the components catalysing extracytoplasmic covalent haem ligation and raises intriguing issues about the requirements for delivery of haem and apocytochrome c substrates to produce c holocytochromes.

sacB-5-Fluoroorotic acid-pyrE-based bidirectional selection for integration of unmarked alleles into the chromosome of Rhodobacter capsulatus.

The gram-negative, purple nonsulfur, facultative photosynthetic bacterium Rhodobacter capsulatus is a widely used model organism and has well-developed molecular genetics. In particular, interposon mutagenesis using selectable gene cartridges is frequently employed for construction of a variety of chromosomal knockout mutants. However, as the gene cartridges are often derived from antibiotic resistance-conferring genes, their numbers are limited, which restricts the construction of multiple knockout mutants. In this report, sacB-5-fluoroorotic acid (5FOA)--pyrE-based bidirectional selection that facilitates construction of unmarked chromosomal knockout mutations is described. The R. capsulatus pyrE gene encoding orotate phosphoribosyl transferase, a key enzyme of the de novo pyrimidine nucleotide biosynthesis pathway, was used as an interposon in a genetic background that is auxotrophic for uracil (Ura-) and hence resistant to 5FOA (5FOA(r)). Although Ura+ selection readily yielded chromosomal allele replacements via homologous recombination, selection for 5FOA(r) to replace pyrE with unmarked alleles was inefficient. To improve the latter step, 5FOA(r) selection was combined with sucrose tolerance selection using a suicide plasmid carrying the Bacillus subtilis sacB gene encoding levansucrase that induces lethality upon exposure to 5% (wt/vol) sucrose in the growth medium. Sucrose-tolerant, 5FOA(r) colonies that were obtained carried chromosomal unmarked mutant alleles of the target gene via double crossovers between the resident pyrE-marked and incoming unmarked alleles. The effectiveness of this double selection was proven by seeking insertion and deletion alleles of helC involved in R. capsulatus cytochrome c biogenesis, which illustrated the usefulness of this system as a genetic means for facile construction of R. capsulatus unmarked chromosomal mutants.

Overproduction of CcmG and CcmFH(Rc) fully suppresses the c-type cytochrome biogenesis defect of Rhodobacter capsulatus CcmI-null mutants.

Gram-negative bacteria like Rhodobacter capsulatus use intertwined pathways to carry out the posttranslational maturation of c-type cytochromes (Cyts). This periplasmic process requires at least 10 essential components for apo-Cyt c chaperoning, thio-oxidoreduction, and the delivery of heme and its covalent ligation. One of these components, CcmI (also called CycH), is thought to act as an apo-Cyt c chaperone. In R. capsulatus, CcmI-null mutants are unable to produce c-type Cyts and thus sustain photosynthetic (Ps) growth. Previously, we have shown that overproduction of the putative heme ligation components CcmF and CcmH(Rc) (also called Ccl1 and Ccl2) can partially bypass the function of CcmI on minimal, but not on enriched, media. Here, we demonstrate that either additional overproduction of CcmG (also called HelX) or hyperproduction of CcmF-CcmH(Rc) is needed to completely overcome the role of CcmI during the biogenesis of c-type Cyts on both minimal and enriched media. These findings indicate that, in the absence of CcmI, interactions between the heme ligation and thioreduction pathways become restricted for sufficient Cyt c production. We therefore suggest that CcmI, along with its apo-Cyt chaperoning function, is also critical for the efficacy of holo-Cyt c formation, possibly via its close interactions with other components performing the final heme ligation steps during Cyt c biogenesis.