Structural and Redox Properties of the Leaderless DsbE (CcmG) Protein: Both Active-Site Cysteines of the Reduced Form Are Involved in Its Function in the Escherichia coli Periplasm

2001 ◽  
Vol 382 (12) ◽  
pp. 1679-1686 ◽  
Author(s):  
Qi Li ◽  
Hong-Yu Hu ◽  
Wei-Qing Wang ◽  
Gen-Jun Xu
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Active Site ◽  
Redox Reaction ◽  
Disulfide Bonds ◽  
Biochemical Properties ◽  
Redox Properties ◽  
Reduced Form ◽  
Cytochrome C Maturation ◽  
Standard Redox Potential

AbstractThe thiol/disulfide oxidoreductases play important roles in ensuring the correct formation of disulfide bonds, of which the DsbE protein, also called CcmG, is the one implicated in electron transfer for cytochrome c maturation in the periplasm of Escherichia coli. The soluble, Nterminally truncated DsbE was overexpressed and purified to homogeneity. Here we report the structural and redox properties of the leaderless form (DsbEL ). During the redox reaction, the protein undergoes a structural transformation resulting in a more stable reduced form, but this form shows very low reactivity in thiol/ disulfide exchange of cysteine residues and low activity in accelerating the reduction of insulin. The standard redox potential (E' 0 ) for the active thiol/ disulfide was determined to be 0.186 V; only one of the two cysteines (Cys80) was suggested to be the active residue in the redox reaction. From the aspect of biochemical properties, DsbE can be regarded as a weak reductant in the Escherichia coli periplasm. This implies that the function of DsbE in cytochrome c maturation can be ascribed to its active site cysteines and the structure of the reduced form.

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 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.

Modifying Cytochrome c Maturation Can Increase the Bioelectronic Performance of Engineered Escherichia coli

2019 ◽  
Vol 9 (1) ◽  
pp. 115-124 ◽  
Author(s):  
Lin Su ◽  
Tatsuya Fukushima ◽  
Andrew Prior ◽  
Moshe Baruch ◽  
Tom J. Zajdel ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Cytochrome C Maturation ◽  
Engineered Escherichia Coli

scholarly journals High Yield of Methylophilus methylotrophus Cytochrome c″ by Coexpression with Cytochrome c Maturation Gene Cluster from Escherichia coli

2000 ◽  
Vol 20 (3) ◽  
pp. 444-450 ◽  
Author(s):  
Nicholas J. Price ◽  
Lorraine Brennan ◽  
Tiago Q. Faria ◽  
Erik Vijgenboom ◽  
Gerard W. Canters ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Gene Cluster ◽  
High Yield ◽  
Methylophilus Methylotrophus ◽  
Cytochrome C Maturation

scholarly journals Redox properties and cross-linking of the dithiol/disulphide active sites of mammalian protein disulphide-isomerase

1991 ◽  
Vol 275 (2) ◽  
pp. 341-348 ◽  
Author(s):  
H C Hawkins ◽  
M de Nardi ◽  
R B Freedman
Keyword(s):  
Redox Potential ◽  
Active Site ◽  
Active Sites ◽  
Residual Activity ◽  
Redox Properties ◽  
Redox Couple ◽  
Hill Coefficient ◽  
Cross Linking ◽  
Standard Redox Potential ◽  
Reactive Groups

1. The redox properties of the active-site dithiol/disulphide groups of PDI were determined by equilibrating the enzyme with an excess of GSH + GSSG, rapidly alkylating the dithiol form of the enzyme to inactivate it irreversibly, and determining the proportion of the disulphide form by measuring the residual activity under standard conditions. 2. The extent of reduction varied with the applied redox potential; to a first approximation, the data fitted a model in which all the enzyme dithiol/disulphide groups are independent and equivalent and the equilibrium constant between these sites and the GSH/GSSG redox couple is 42 microM at pH 7.5. 3. The standard redox potential for PDI active-site dithiol/disulphide couples was calculated from this result and found to be -0.11 V; hence PDI is a stronger oxidant and weaker reductant than GSH, nicotinamide cofactors, thioredoxin and dithiothreitol. 4. The redox equilibrium data for PDI with the GSH/GSSG redox couple showed sigmoidal deviations from linearity. The sigmoidicity could be modelled closely by assuming a Hill coefficient of 1.5. 5. This evidence of co-operative interactions between the four active sites in a PDI dimer was extended by studying the reaction between PDI and homobifunctional alkylating agents with various lengths between the reactive groups. A species whose electrophoretic mobility suggested it contained an intrachain cross-link was observed in all cases, whereas there was no evidence for cross-linking between the chains of the PDI homodimer. Most effective cross-linking was achieved with reagents containing five or more methylene spacer groups, implying a minimum distance of 1.6 nm (16 A) between the active-site reactive groups within the two thioredoxin-like domains of the PDI polypeptide.

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