Divergent Cytochrome c Maturation System in Kinetoplastid Protists

ABSTRACT In eukaryotes, heme attachment through two thioether bonds to mitochondrial cytochromes c and c1 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.

Characterization and Comparative Analyses of Mitochondrial Genomes in Single-Celled Eukaryotes to Shed Light on the Diversity and Evolution of Linear Molecular Architecture

Determination and comparisons of complete mitochondrial genomes (mitogenomes) are important to understand the origin and evolution of mitochondria. Mitogenomes of unicellular protists are particularly informative in this regard because they are gene-rich and display high structural diversity. Ciliates are a highly diverse assemblage of protists and their mitogenomes (linear structure with high A+T content in general) were amongst the first from protists to be characterized and have provided important insights into mitogenome evolution. Here, we report novel mitogenome sequences from three representatives (Strombidium sp., Strombidium cf. sulcatum, and Halteria grandinella) in two dominant ciliate lineages. Comparative and phylogenetic analyses of newly sequenced and previously published ciliate mitogenomes were performed and revealed a number of important insights. We found that the mitogenomes of these three species are linear molecules capped with telomeric repeats that differ greatly among known species. The genomes studied here are highly syntenic, but larger in size and more gene-rich than those of other groups. They also all share an AT-rich tandem repeat region which may serve as the replication origin and modulate initiation of bidirectional transcription. More generally we identified a split version of ccmf, a cytochrome c maturation-related gene that might be a derived character uniting taxa in the subclasses Hypotrichia and Euplotia. Finally, our mitogenome comparisons and phylogenetic analyses support to reclassify Halteria grandinella from the subclass Oligotrichia to the subclass Hypotrichia. These results add to the growing literature on the unique features of ciliate mitogenomes, shedding light on the diversity and evolution of their linear molecular architecture.

Complex Oxidation of Apocytochromes c during Bacterial Cytochrome c Maturation

ABSTRACT 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 c. IMPORTANCE 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.

The CcmC–CcmE interaction during cytochrome c maturation by System I is driven by protein–protein and not protein–heme contacts

Cytochromes c are ubiquitous proteins, essential for life in most organisms. Their distinctive characteristic is the covalent attachment of heme to their polypeptide chain. This post-translational modification is performed by a dedicated protein system, which in many Gram-negative bacteria and plant mitochondria is a nine-protein apparatus (CcmA–I) called System I. Despite decades of study, mechanistic understanding of the protein–protein interactions in this highly complex maturation machinery is still lacking. Here, we focused on the interaction of CcmC, the protein that sources the heme cofactor, with CcmE, the pivotal component of System I responsible for the transfer of the heme to the apocytochrome. Using in silico analyses, we identified a putative interaction site between these two proteins (residues Asp47, Gln50, and Arg55 on CcmC; Arg73, Asp101, and Glu105 on CcmE), and we validated our findings by in vivo experiments in Escherichia coli. Moreover, employing NMR spectroscopy, we examined whether a heme-binding site on CcmE contributes to this interaction and found that CcmC and CcmE associate via protein–protein rather than protein–heme contacts. The combination of in vivo site-directed mutagenesis studies and high-resolution structural techniques enabled us to determine at the residue level the mechanism for the formation of one of the key protein complexes for cytochrome c maturation by System I.