scholarly journals Maturation of the unusual single-cysteine (XXXCH) mitochondrial c-type cytochromes found in trypanosomatids must occur through a novel biogenesis pathway

2004 ◽  
Vol 383 (3) ◽  
pp. 537-542 ◽  
Author(s):  
James W. A. ALLEN ◽  
Michael L. GINGER ◽  
Stuart J. FERGUSON
Keyword(s):  
Cytochrome C ◽  
Covalent Attachment ◽  
Binding Motif ◽  
Intermembrane Space ◽  
Cytochrome C Maturation ◽  
E Coli ◽  
Cytochromes C ◽  
Genes Encoding ◽  
Mitochondrial Intermembrane Space ◽  
Mitochondrial Cytochromes

The c-type cytochromes are characterized by the covalent attachment of haem to the polypeptide via thioether bonds formed from haem vinyl groups and, normally, the thiols of two cysteines in a CXXCH motif. Intriguingly, the mitochondrial cytochromes c and c1 from two euglenids and the Trypanosomatidae contain only a single cysteine within the haem-binding motif (XXXCH). There are three known distinct pathways by which c-type cytochromes are matured post-translationally in different organisms. The absence of genes encoding any of these c-type cytochrome biogenesis machineries is established here by analysis of six trypanosomatid genomes, and correlates with the presence of single-cysteine cytochromes c and c1. In contrast, we have identified a comprehensive catalogue of proteins required for a typical mitochondrial oxidative phosphorylation apparatus. Neither spontaneous nor catalysed maturation of the single-cysteine Trypanosoma brucei cytochrome c occurred in Escherichia coli. However, a CXXCH variant was matured by the E. coli cytochrome c maturation machinery, confirming the proposed requirement of the latter for two cysteines in the haem-binding motif and indicating that T. brucei cytochrome c can accommodate a second cysteine in a CXXCH motif. The single-cysteine haem attachment conserved in cytochromes c and c1 of the trypanosomatids is suggested to be related to their cytochrome c maturation machinery, and the environment in the mitochondrial intermembrane space. Our genomic and biochemical studies provide very persuasive evidence that the trypanosomatid mitochondrial cytochromes c are matured by a novel biogenesis system.

scholarly journals Variant c-type cytochromes as probes of the substrate specificity of the E. coli cytochrome c maturation (Ccm) apparatus

2009 ◽  
Vol 419 (1) ◽  
pp. 177-186 ◽  
Author(s):  
James W. A. Allen ◽  
Elizabeth B. Sawyer ◽  
Michael L. Ginger ◽  
Paul D. Barker ◽  
Stuart J. Ferguson
Keyword(s):  
Cytochrome C ◽  
Substrate Specificity ◽  
Cysteine Residue ◽  
Covalent Attachment ◽  
Binding Motif ◽  
Post Translational Modification ◽  
Cytochrome C Maturation ◽  
E Coli ◽  
Cytochrome B562 ◽  
Genome Analyses

c-type cytochromes are normally characterized by covalent attachment of the iron cofactor haem to protein through two thioether bonds between the vinyl groups of the haem and the thiol groups of a CXXCH (Cys–Xaa–Xaa–Cys–His) motif. In cells, the haem attachment is an enzyme-catalysed post-translational modification. We have previously shown that co-expression of a variant of Escherichia coli cytochrome b562 containing a CXXCH haem-binding motif with the E. coli Ccm (cytochrome c maturation) proteins resulted in homogeneous maturation of a correctly formed c-type cytochrome. In contrast, in the absence of the Ccm apparatus, the product holocytochrome was heterogeneous, the main species having haem inverted and attached through only one thioether bond. In the present study we use further variants of cytochrome b562 to investigate the substrate specificity of the E. coli Ccm apparatus. The system can mature c-type cytochromes with CCXXCH, CCXCH, CXCCH and CXXCHC motifs, even though these are not found naturally and the extra cysteine residue might, in principle, disrupt the biogenesis proteins which must interact intricately with disulfide-bond oxidizing and reducing proteins in the E. coli periplasm. The Ccm proteins can also attach haem to motifs of the type CXnCH where n ranges from 2 to 6. For n=3 and 4, the haem attachment was correct and homogeneous, but for higher values of n the holocytochromes displayed oxidative addition of sulfur and/or oxygen atoms associated with the covalent haem-attachment process. The implications of our observations for the haem-attachment reaction, for genome analyses and for the substrate specificity of the Ccm system, are discussed.

scholarly journals Variation of the axial haem ligands and haem-binding motif as a probe of the Escherichia coli c-type cytochrome maturation (Ccm) system

2003 ◽  
Vol 375 (3) ◽  
pp. 721-728 ◽  
Author(s):  
James W. A. ALLEN ◽  
Stuart J. FERGUSON
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Covalent Attachment ◽  
Binding Motif ◽  
Cytochrome C Maturation ◽  
Cytochromes C ◽  
Cytochrome C552 ◽  
Haem Iron ◽  
Made In ◽  
Uncatalysed Reaction

Cytochromes c are typically characterized by the covalent attachment of haem to polypeptide through two thioether bonds with the cysteine residues of a Cys-Xaa-Xaa-Cys-His peptide motif. In many Gram-negative bacteria, the haem is attached to the polypeptide by the periplasmically functioning cytochrome c maturation (Ccm) proteins. Exceptionally, Hydrogenobacter thermophilus cytochrome c552 can be expressed as a stable holocytochrome both in the cytoplasm of Escherichia coli in an apparently uncatalysed reaction and also in the periplasm in a Ccm-mediated reaction. In the present study we show that a Met60→Ala variant of c552, which does not have the usual distal methionine ligand to the haem iron of the mature cytochrome, can be made in the periplasm by the Ccm system. However, no holocytochrome could be detected when this variant was expressed cytoplasmically. These data highlight differences between the two modes of cytochrome c assembly. In addition, we report investigations of haem attachment to cytochromes altered to have the special Cys-Trp-Ser-Cys-Lys haem-binding motif, and Cys-Trp-Ser-Cys-His and Cys-Trp-Ala-Cys-His analogues, of the active-site haem of nitrite reductase NrfA.

scholarly journals The histidine of the c-type cytochrome CXXCH haem-binding motif is essential for haem attachment by the Escherichia coli cytochrome c maturation (Ccm) apparatus

2005 ◽  
Vol 389 (2) ◽  
pp. 587-592 ◽  
Author(s):  
James W. A. Allen ◽  
Nicholas Leach ◽  
Stuart J. Ferguson
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Covalent Attachment ◽  
Binding Motif ◽  
Binding Motifs ◽  
Cytochrome C Maturation ◽  
E Coli ◽  
Cytochrome B562

c-type cytochromes are characterized by covalent attachment of haem to the protein by two thioether bonds formed between the haem vinyl groups and the cysteine sulphurs in a CXXCH peptide motif. In Escherichia coli and many other Gram-negative bacteria, this post-translational haem attachment is catalysed by the Ccm (cytochrome c maturation) system. The features of the apocytochrome substrate required and recognized by the Ccm apparatus are uncertain. In the present study, we report investigations of maturation of cytochrome b562 variants containing CXXCR, CXXCK or CXXCM haem-binding motifs. None of them showed any evidence for correct maturation by the Ccm system. However, we have determined, for each variant, that the proteins (i) were expressed in large amounts, (ii) could bind haem in vivo and/or in vitro and (iii) were not degraded in the cell. Together with previous observations, these results strongly suggest that the apocytochrome substrate feature recognized by the Ccm system is simply the two cysteine residues and the histidine of the CXXCH haem-binding motif. Using the same experimental approach, we have also investigated a cytochrome b562 variant containing the special CWSCK motif that binds the active-site haem of E. coli nitrite reductase NrfA. Whereas a CWSCH analogue was matured by the Ccm apparatus in large amounts, the CWSCK form was not detectably matured either by the Ccm system or by the dedicated Nrf biogenesis proteins, implying that the substrate recognition features for haem attachment in NrfA may be more extensive than the CWSCK motif.

97 Redox chemistry in the mitochondrial intermembrane space: A novel flavoprotein involved in cytochrome c maturation

Mitochondrion ◽  
2007 ◽  
Vol 7 (6) ◽  
pp. 432-433
Author(s):  
Delphine Bernard ◽  
Sophie Quevillon-Cheruel ◽  
Sabeeha Merchant ◽  
Bernard Guiard ◽  
Patrice Hamel
Keyword(s):  
Cytochrome C ◽  
Redox Chemistry ◽  
Intermembrane Space ◽  
Cytochrome C Maturation ◽  
Mitochondrial Intermembrane Space

What is the substrate specificity of the System I cytochrome c biogenesis apparatus?

2006 ◽  
Vol 34 (1) ◽  
pp. 150-151 ◽  
Author(s):  
J.W.A. Allen ◽  
S.J. Ferguson
Keyword(s):  
Cytochrome C ◽  
Substrate Specificity ◽  
Nitrogen Cycle ◽  
Covalent Attachment ◽  
Binding Motif ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Cytochrome C Maturation ◽  
Cytochrome C Biogenesis

c-Type cytochromes are characterized by covalent attachment of haem to protein through thioether bonds between the vinyl groups of the haem and the thiols of a CXXCH motif. Proteins of this type play crucial roles in the biochemistry of the nitrogen cycle. Many Gram-negative bacteria use the Ccm (cytochrome c maturation) proteins for the post-translational haem attachment to their c-type cytochromes; in the present paper, we discuss the substrate specificity of the Ccm apparatus. The main conclusion is that the feature recognized and required in the apocytochrome is simply the two cysteines and the histidine of the haem-binding motif.

scholarly journals Divergent Cytochrome c Maturation System in Kinetoplastid Protists

mBio ◽  
2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Asma Belbelazi ◽  
Rachel Neish ◽  
Martin Carr ◽  
Jeremy C. Mottram ◽  
Michael L. Ginger
Keyword(s):  
Genetic Disease ◽  
De Novo ◽  
Covalent Attachment ◽  
Human Genetic Disease ◽  
Cytochrome C Maturation ◽  
Genes Encoding ◽  
Mechanistic Basis ◽  
Protein Attachment ◽  
Heme Cofactor ◽  
Mitochondrial Cytochromes

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.

scholarly journals Biosynthesis of the Cyanobacterial Light-Harvesting Polypeptide Phycoerythrocyanin Holo-α Subunit in a Heterologous Host

2002 ◽  
Vol 184 (17) ◽  
pp. 4666-4671 ◽  
Author(s):  
Aaron J. Tooley ◽  
Alexander N. Glazer
Keyword(s):  
Ternary Complexes ◽  
Covalent Attachment ◽  
Heme Oxygenase 1 ◽  
Α Subunit ◽  
Ionization Time ◽  
E Coli ◽  
Metal Affinity ◽  
Flight Mass Spectrometry ◽  
Genes Encoding ◽  
Cyanobacterial Genes

ABSTRACT The entire pathway for the biosynthesis of the phycobiliviolin-bearing His-tagged holo-α subunit of the cyanobacterial photosynthetic accessory protein phycoerythrocyanin was reconstituted in Escherichia coli. Cyanobacterial genes encoding enzymes required for the conversion of heme to 3Z-phycocyanobilin, a precursor of phycobiliviolin (namely, heme oxygenase 1 and 3Z-phycocyanobilin:ferredoxin oxidoreductase), were expressed from a plasmid under the control of the hybrid trp-lac (trc) promoter. Genes for the apo-phycoerythrocyanin α subunit (pecA) and the heterodimeric lyase/isomerase (pecE and pecF), which catalyzes both the covalent attachment of phycocyanobilin and its concurrent isomerization to phycobiliviolin, were expressed from the trc promoter on a second plasmid. Upon induction, recombinant E. coli used endogenous heme to produce holo-PecA with absorbance and fluorescence properties similar to those of the same protein produced in cyanobacteria. About two-thirds of the apo-PecA was converted to holo-PecA. No significant bilin addition took place in a similarly engineered E. coli strain that lacks pecE and pecF. By using immobilized metal affinity chromatography, both apo-PecA and holo-PecA were isolated as ternary complexes with PecE and PecF. The identities of all three components in the ternary complexes were established unambiguously by protein and tryptic peptide analyses performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

scholarly journals Physicochemical properties of two atypical cytochromes c, Crithidia cytochrome c-557 and Euglena cytochrome c-558

1975 ◽  
Vol 149 (1) ◽  
pp. 155-167 ◽  
Author(s):  
G W Pettigrew ◽  
I Aviram ◽  
A Schejter
Keyword(s):  
Cytochrome C ◽  
Physicochemical Properties ◽  
High Spin ◽  
Visible Spectrum ◽  
Prosthetic Group ◽  
Ph Values ◽  
Cytochromes C ◽  
Kinetics Of ◽  
Alkaline Isomerization ◽  
Mitochondrial Cytochromes

Cytochrome c-557 from Crithidia oncopelti and cytochrome c-558 from Euglena gracilis are mitochondrial cytochromes c that have an atypical haem-binding site. It was of interest to know whether the loss of one thioether bond affected the physicochemical properties of these cytochromes. The thermodynamic parameters of the redox potential were measured. The reaction with imidazole, the kinetics and thermodynamics of the alkaline isomerization and the effect of heating on the visible spectrum are described for the ferricytochromes. The kinetics of the loss of cyanide, the spectral changes occurring on reduction with dithionite at alkaline pH values and the reactivity with CO are described for the ferrocytochromes. In many respects the cytochromes of the two protozoans are very similar to the cytochromes of horse and yeast. The ferricytochromes do, however, undergo a reversible transition to high-spin species on heating, which may be due to the more flexible attachment of the prosthetic group. Similarly the alkaline isomers of cytochromes c-557 and c-558 give rise to high-spin proteins above pH 11. The alkaline isomerization of cytochrome c-558, involves a pKobs. of 10 and kinetics which do not obey the model of Davis et al. [(1974) J. Biol. Chem.249, 2624-2632] for horse cytochrome c. It is proposed that a model involving two ionizations, followed by a conformation change, may fit the data. Both cytochromes c-557 and c-558 combine slowly with CO at neutral pH values.

Avoidance of the cytochrome c biogenesis system by periplasmic CXXCH motifs

2008 ◽  
Vol 36 (6) ◽  
pp. 1124-1128 ◽  
Author(s):  
Despoina A.I. Mavridou ◽  
Martin Braun ◽  
Linda Thöny-Meyer ◽  
Julie M. Stevens ◽  
Stuart J. Ferguson
Keyword(s):  
Cytochrome C ◽  
Tertiary Structure ◽  
Evolutionary Relationship ◽  
Attachment Site ◽  
Cytochrome C Maturation ◽  
E Coli ◽  
Cytochrome C Biogenesis ◽  
Periplasmic Proteins ◽  
Bona Fide

The CXXCH motif is usually recognized in the bacterial periplasm as a haem attachment site in apocytochromes c. There is evidence that the Escherichia coli Ccm (cytochrome c maturation) system recognizes little more than the CXXCH sequence. A limited number of periplasmic proteins have this motif and yet are not c-type cytochromes. To explore how unwanted haem attachment to CXXCH might be avoided, and to determine whether haem attachment to the surface of a non-cytochrome protein would be possible, we converted the active-site CXXCK motif of a thioredoxin-like protein into CXXCH, the C-terminal domain of the transmembrane oxidoreductase DsbD (cDsbD). The E. coli Ccm system was found to catalyse haem attachment to a very small percentage of the resultant protein (∼0.2%). We argue that cDsbD folds sufficiently rapidly that only a small fraction fails to avoid the Ccm system, in contrast with bona fide c-type cytochromes that only adopt their tertiary structure following haem attachment. We also demonstrate covalent haem attachment at a low level in vivo to the periplasmic disulfide isomerase DsbC, which contains a native CXXCH motif. These observations provide insight into substrate recognition by the Ccm system and expand our understanding of the requirements for covalent haem attachment to proteins. The possible evolutionary relationship between thioredoxins and c-type cytochromes is discussed.

scholarly journals Control of DegP-Dependent Degradation of c-Type Cytochromes by Heme and the Cytochrome c Maturation System in Escherichia coli

2007 ◽  
Vol 189 (17) ◽  
pp. 6253-6259 ◽  
Author(s):  
Tao Gao ◽  
Mark R. O'Brian
Keyword(s):  
Escherichia Coli ◽  
Binding Motif ◽  
Dependent Manner ◽  
Heme Binding ◽  
Prosthetic Group ◽  
Cytochrome C Maturation ◽  
E Coli ◽  
Heme Prosthetic Group ◽  
Low Levels ◽  
Covalently Bound

ABSTRACT c-type cytochromes are located partially or completely in the periplasm of gram-negative bacteria, and the heme prosthetic group is covalently bound to the protein. The cytochrome c maturation (Ccm) multiprotein system is required for transport of heme to the periplasm and its covalent linkage to the peptide. Other cytochromes and hemoglobins contain a noncovalently bound heme and do not require accessory proteins for assembly. Here we show that Bradyrhizobium japonicum cytochrome c 550 polypeptide accumulation in Escherichia coli was heme dependent, with very low levels found in heme-deficient cells. However, apoproteins of the periplasmic E. coli cytochrome b 562 or the cytosolic Vitreoscilla hemoglobin (Vhb) accumulated independently of the heme status. Mutation of the heme-binding cysteines of cytochrome c 550 or the absence of Ccm also resulted in a low apoprotein level. These levels were restored in a degP mutant strain, showing that apocytochrome c 550 is degraded by the periplasmic protease DegP. Introduction of the cytochrome c heme-binding motif CXXCH into cytochrome b 562 (c-b 562) resulted in a c-type cytochrome covalently bound to heme in a Ccm-dependent manner. This variant polypeptide was stable in heme-deficient cells but was degraded by DegP in the absence of Ccm. Furthermore, a Vhb variant containing a periplasmic signal peptide and a CXXCH motif did not form a c-type cytochrome, but accumulation was Ccm dependent nonetheless. The data show that the cytochrome c heme-binding motif is an instability element and that stabilization by Ccm does not require ligation of the heme moiety to the protein.

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