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.

The Escherichia coli cytochrome c maturation (Ccm) apparatus can mature cytochromes with an extra cysteine within or adjacent to the CXXCH motif

2006 ◽  
Vol 34 (1) ◽  
pp. 91-93 ◽  
Author(s):  
J.W.A. Allen ◽  
S.J. Ferguson
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Nitrogen Cycle ◽  
Covalent Attachment ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Cytochrome C Maturation ◽  
Free Thiol ◽  
Haem Iron

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 Cys-Xaa-Xaa-Cys-His 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. The Ccm system can correctly mature c-type cytochromes with CCXXCH, CCXCH, CXCCH and CXXCHC motifs, even though these are not found naturally and the extra cysteine might, in principle, disrupt the biogenesis proteins. The non-occurrence of these motifs probably relates to the destructive chemistry that can occur if a free thiol reacts with haem iron to generate a radical.

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.

The role of ResA in type II cytochrome c maturation

2005 ◽  
Vol 33 (1) ◽  
pp. 149-151 ◽  
Author(s):  
A. Crow ◽  
N.E. Le Brun ◽  
A. Oubrie
Keyword(s):  
Bacillus Subtilis ◽  
Cytochrome C ◽  
Crystal Structures ◽  
Nitrogen Cycle ◽  
Covalent Attachment ◽  
Type Ii ◽  
Cytochrome C Maturation ◽  
Bacterial Proteins

Numerous bacterial proteins involved in the nitrogen cycle, and other processes, require c-type haem as a cofactor. c-type cytochromes are formed by covalent attachment of haem to the conserved CXXCH motif. Here, we briefly review what is presently known about cytochrome c maturation in Bacillus subtilis with particular emphasis on the crystal structures of ResA.

Multiple haem lyase genes indicate substrate specificity in cytochrome c biogenesis

2006 ◽  
Vol 34 (1) ◽  
pp. 146-149 ◽  
Author(s):  
S. Hartshorne ◽  
D.J. Richardson ◽  
J. Simon
Keyword(s):  
Cytochrome C ◽  
Model Organism ◽  
Vital Role ◽  
Covalent Attachment ◽  
Binding Motif ◽  
Binding Motifs ◽  
Cytochrome C Biogenesis ◽  
Multiple Copies ◽  
Energy Conserving ◽  
Eukaryotic Organisms

c-Type cytochromes are a widespread class of proteins that play a vital role in the energy-conserving metabolism of prokaryotic and eukaryotic organisms. The key event in cytochrome c biogenesis is the covalent attachment of the haem cofactor to two (or rarely one) cysteine residues arranged in a haem c-binding motif such as CX2–4CH, CXXCK or X3CH. This reaction is catalysed by the membrane-bound enzyme CCHL (cytochrome c haem lyase). Different CCHLs have been described and some of them are dedicated to distinct haem c-binding motifs of cytochromes that are encoded in the vicinity of the respective CCHL gene. Various bacterial genomes contain multiple copies of CCHL-encoding genes, suggesting the presence of non-conventional or even as yet unrecognized haem c-binding motifs. This assumption is exemplified in the present study using the proteobacterium Wolinella succinogenes as a model organism whose genome encodes three CCHL isoenzymes. The discovery of a novel conserved multihaem cytochrome c (MccA) is described.

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.

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.

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.

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