Dataset Information

The CcmFH complex is the system I holocytochrome c synthetase: engineering cytochrome c maturation independent of CcmABCDE.

ABSTRACT: Cytochrome c maturation (ccm) in many bacteria, archaea and plant mitochondria requires eight membrane proteins, CcmABCDEFGH, called system I. This pathway delivers and attaches haem covalently to two cysteines (of Cys-Xxx-Xxx-Cys-His) in the cytochrome c. All models propose that CcmFH facilitates covalent attachment of haem to the apocytochrome; namely, that it is the synthetase. However, holocytochrome c synthetase activity has not been directly demonstrated for CcmFH. We report formation of holocytochromes c by CcmFH and CcmG, a periplasmic thioredoxin, independent of CcmABCDE (we term this activity CcmFGH-only). Cytochrome c produced in the absence of CcmABCDE is indistinguishable from cytochrome c produced by the full system I, with a cleaved signal sequence and two covalent bonds to haem. We engineered increased cytochrome c production by CcmFGH-only, with yields approaching those from the full system I. Three conserved histidines in CcmF (TM-His1, TM-His2 and P-His1) are required for activity, as are the conserved cysteine pairs in CcmG and CcmH. Our findings establish that CcmFH is the system I holocytochrome c synthetase. Although we discuss why this engineering would likely not replace the need for CcmABCDE in nature, these results provide unique mechanistic and evolutionary insights into cytochrome c biosynthesis.

SUBMITTER: San Francisco B

PROVIDER: S-EPMC3959880 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

The CcmFH complex is the system I holocytochrome c synthetase: engineering cytochrome c maturation independent of CcmABCDE.

San Francisco Brian B Sutherland Molly C MC Kranz Robert G RG

Molecular microbiology 20140127 5

Cytochrome c maturation (ccm) in many bacteria, archaea and plant mitochondria requires eight membrane proteins, CcmABCDEFGH, called system I. This pathway delivers and attaches haem covalently to two cysteines (of Cys-Xxx-Xxx-Cys-His) in the cytochrome c. All models propose that CcmFH facilitates covalent attachment of haem to the apocytochrome; namely, that it is the synthetase. However, holocytochrome c synthetase activity has not been directly demonstrated for CcmFH. We report formation of h ...[more]

PMID: 24397552

Similar Datasets

Project description:c-Type cytochromes (cyts c) are proteins that contain covalently bound heme and that thus require posttranslational modification for activity, a process carried out by the cytochrome c (cyt c) maturation system (referred to as the Ccm system) in many Gram-negative bacteria. It has been established that during cyt c maturation (CCM), two cysteine thiols of the heme binding motif (CXXCH) within apocytochromes c (apocyts c) are first oxidized largely by DsbA to form a disulfide bond, which is later reduced through a thio-reductive pathway involving DsbD. However, the physiological impacts of DsbA proteins on CCM in fact vary significantly among bacteria. In this work, we used the cyt c-rich Gram-negative bacterium Shewanella oneidensis as the research model to clarify the roles of DsbA proteins in CCM. We show that in terms of the oxidation of apocyts c, DsbA proteins are an important but not critical factor, and, strikingly, oxygen is not either. By exploiting the DsbD-independent pathway, we identify DsbA1, DsbA2, and DsbA3 as oxidants contributing to the oxidation of apocyts c and reductants, such as cysteine, to be an effective antagonist against DsbA-independent oxidation. We further show that DsbB proteins are partially responsible for the reoxidization of reduced DsbA proteins. Overall, our results indicate that the DsbA-DsbB redox pair has a limited role in CCM, challenging the established notion that it is the main oxidant for apocyts cIMPORTANCE DsbA is a powerful oxidase that functions in the bacterial periplasm to introduce disulfide bonds in many proteins, including apocytochromes c It has been well established that although DsbA is not essential, it plays a primary role in cytochrome c maturation, based on studies in bacteria hosting several cyts c Here, with cyt c-rich S. oneidensis as a research model, we show that this is not always the case. Moreover, we demonstrate that DsbB is also not essential for cytochrome c maturation. These results underscore the need to identify oxidants other than DsbA/DsbB that are crucial in the oxidation of apocyts c in bacteria.

Project description:In eukaryotes, heme attachment through two thioether bonds to mitochondrial cytochromes c and c 1 is catalyzed by either multisubunit cytochrome c maturation system I or holocytochrome c synthetase (HCCS). The former was inherited from the alphaproteobacterial progenitor of mitochondria; the latter is a eukaryotic innovation for which prokaryotic ancestry is not evident. HCCS provides one of a few exemplars of de novo protein innovation in eukaryotes, but structure-function insight of HCCS is limited. Uniquely, euglenozoan protists, which include medically relevant kinetoplastids Trypanosoma and Leishmania parasites, attach heme to mitochondrial c-type cytochromes by a single thioether linkage. Yet the mechanism is unknown, as genes encoding proteins with detectable similarity to any proteins involved in cytochrome c maturation in other taxa are absent. Here, a bioinformatics search for proteins conserved in all hemoprotein-containing kinetoplastids identified kinetoplastid cytochrome c synthetase (KCCS), which we reveal as essential and mitochondrial and catalyzes heme attachment to trypanosome cytochrome c KCCS has no sequence identity to other proteins, apart from a slight resemblance within four short motifs suggesting relatedness to HCCS. Thus, KCCS provides a novel resource for studying eukaryotic cytochrome c maturation, possibly with wider relevance, since mutations in human HCCS leads to disease. Moreover, many examples of mitochondrial biochemistry are different in euglenozoans compared to many other eukaryotes; identification of KCCS thus provides another exemplar of extreme, unusual mitochondrial biochemistry in an evolutionarily divergent group of protists.IMPORTANCE Cytochromes c are essential proteins for respiratory and photosynthetic electron transfer. They are posttranslationally modified by covalent attachment of a heme cofactor. Kinetoplastids include important tropical disease-causing parasites; many aspects of their biology differ from other organisms, including their mammalian or plant hosts. Uniquely, kinetoplastids produce cytochromes c with a type of heme attachment not seen elsewhere in nature and were the only cytochrome c-bearing taxa without evidence of protein machinery to attach heme to the apocytochrome. Using bioinformatics, biochemistry, and molecular genetics, we report how kinetoplastids make their cytochromes c Unexpectedly, they use a highly diverged version of an enzyme used for heme-protein attachment in many eukaryotes. Mutations in the human enzyme lead to genetic disease. Identification of kinetoplastid cytochrome c synthetase, thus, solves an evolutionary unknown, provides a possible target for antiparasite drug development, and an unanticipated resource for studying the mechanistic basis of a human genetic disease.

Dataset Information

The CcmFH complex is the system I holocytochrome c synthetase: engineering cytochrome c maturation independent of CcmABCDE.

Publications

The CcmFH complex is the system I holocytochrome c synthetase: engineering cytochrome c maturation independent of CcmABCDE.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure