Mutagenesis of the gene encoding cytochrome c550 of Paracoccus denitrificans and analysis of the resultant physiological effects

Spanning, Rob J.M. van; Wansell, C. W.; Harms, N.; Oltmann, L. F.; Stouthamer, A. H.

doi:10.1128/jb.172.6.3534.1990

Cited by 34 publications

(70 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Timkovich and Dickerson [9] determined an X-ray structure at 0.25-nm resolution for cytochrome c550 from strain LMD 22.21, which was based, in part, on an incorrect amino acid sequence and contained only backbone atoms for the C-terminal 13 residues. The sequence was corrected by Ambler et al [10] and confirmed by the sequence of the gene [11]. Benning et al [12] presented a higher resolution X-ray structure based on the correct sequence, but lacking residues 1Ϫ2 and 123Ϫ135, which were missing perhaps as a result of proteolysis during preparation.…”

Section: Resultsmentioning

confidence: 99%

The surface‐charge asymmetry and dimerisation of cytochrome c550 from Paracoccus denitrificans @MM implications for the interaction with cytochrome c peroxidase

Pettigrew

Gilmour

Goodhew

et al. 1998

European Journal of Biochemistry

View full text Add to dashboard Cite

The implications of the dimeric state of cytochrome c550 for its binding to Paracoccus cytochrome c peroxidase and its delivery of the two electrons required to restore the active enzyme during catalysis have been investigated. The amino acid sequence of cytochrome c550 of Paracoccus denitrificans strain LMD 52.44 was determined and showed 21 differences from that of strain LMD 22.21. Based on the X-ray structure of the latter, a structure for the cytochrome c550 monomer from strain 52.44 is proposed and a dipole moment of 945 debye was calculated with an orientation close to the exposed haem edge. The behaviour of the cytochrome on molecular-exclusion chromatography is indicative of an ionic strength-dependent monomer (15 kDa)/dimer (30 kDa) equilibrium that can also be detected by 1 H-NMR spectroscopy. The apparent mass of 50 kDa observed at very low ionic strength was consistent with the presence of a strongly asymmetric dimer. This was confirmed by cross-linking studies, which showed that a cross-linked species of mass 30 kDa on SDS behaved with an apparent mass of 50 kDa on molecular-exclusion chromatography. A programme which carried out and evaluated molecular docking of two monomers to give a dimer generated a most probable dimer in which the monomer dipoles lay almost antiparallel to each other. The resultant dipole moment of the dimer is therefore small. Although this finding calls into question the possibility of preorientation of a strongly asymmetrically charged cytochrome as it collides with a redox partner, the stoichiometry of complex formation with cytochrome c peroxidase as studied by 1 H-NMR spectroscopy shows that it is the monomer that binds.Keywords : cytochrome; peroxidase ; electron transfer; electrostatic ; dimerisation.Paracoccus denitrificans cytochrome c550 is the electron donor for Paracoccus cytochrome c peroxidase (CCP). We have used these two redox proteins as a model system, with which to investigate and understand electron transfer between proteins. They represent a useful parallel system to the well-studied yeast cytochrome c and yeast CCP [1,2] in that the cytochrome c donors are structurally similar but the peroxidases are quite distinct.Like the yeast peroxidase, the Paracoccus enzyme requires two electrons to restore the active form after oxidation by hydrogen peroxide. In the yeast case, these are probably delivered singly by two ferrocytochrome c molecules sequentially encountering the peroxidase surface. The Paracoccus enzyme contains two haems rather than one, and in separate domains of the protein [3]. One haem is believed to perform an electrontransferring function and the other, a peroxidatic function. With two domains, each carrying an oxidising centre in the intermedi-

show abstract

Section: Resultsmentioning

confidence: 99%

The surface‐charge asymmetry and dimerisation of cytochrome c550 from Paracoccus denitrificans @MM implications for the interaction with cytochrome c peroxidase

Pettigrew

Gilmour

Goodhew

et al. 1998

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…A 4.6-kb NotI fragment of this plasmid, which contains the mutated cycM gene, was cloned in suicide vector pRVS2, yielding vector pRTd28.22. This plasmid was used in the gene exchange technique described earlier (51) , which all have marked mutations in their chromosomal cycM genes. The correctnesses of the mutations were confirmed by Southern analyses of their chromosomal DNA.…”

Section: Methodsmentioning

confidence: 99%

“…Much of our knowledge on how electrons are transferred from the dehydrogenases to the terminal oxidoreductases stems from the analyses of mutants with mutations in genes encoding electron carriers that make up the respiratory network (9,11,50,51,52,53). However, it has thus far been difficult to understand the exact role of the different types of cytochrome c in situ since mutations in their corresponding genes did not give clear phenotypes if at all.…”

mentioning

confidence: 99%

Cytochromes c ₅₅₀ , c ₅₅₂ , and c ₁ in the Electron Transport Network of Paracoccus denitrificans : Redundant or Subtly Different in Function?

Otten

Oost²,

Reijnders³

et al. 2001

J Bacteriol

View full text Add to dashboard Cite

Paracoccus denitrificans strains with mutations in the genes encoding the cytochrome c 550 , c 552 , or c 1 and in combinations of these genes were constructed, and their growth characteristics were determined. Each mutant was able to grow heterotrophically with succinate as the carbon and free-energy source, although their specific growth rates and maximum cell numbers fell variably behind those of the wild type. Maximum cell numbers and rates of growth were also reduced when these strains were grown with methylamine as the sole free-energy source, with the triple cytochrome c mutant failing to grow on this substrate. Under anaerobic conditions in the presence of nitrate, none of the mutant strains lacking the cytochrome bc 1 complex reduced nitrite, which is cytotoxic and accumulated in the medium. The cytochrome c 550 -deficient mutant did denitrify provided copper was present. The cytochrome c 552 mutation had no apparent effect on the denitrifying potential of the mutant cells. The studies show that the cytochromes c have multiple tasks in electron transfer. The cytochrome bc 1 complex is the electron acceptor of the Q-pool and of amicyanin. It is also the electron donor to cytochromes c 550 and c 552 and to the cbb 3 -type oxidase. Cytochrome c 552 is an electron acceptor both of the cytochrome bc 1 complex and of amicyanin, as well as a dedicated electron donor to the aa 3 -type oxidase. Cytochrome c 550 can accept electrons from the cytochrome bc 1 complex and from amicyanin, whereas it is also the electron donor to both cytochrome c oxidases and to at least the nitrite reductase during denitrification. Deletion of the c-type cytochromes also affected the concentrations of remaining cytochromes c, suggesting that the organism is plastic in that it adjusts its infrastructure in response to signals derived from changed electron transfer routes.

show abstract

“…For rapid screening, plasmid DNA was isolated by the alkaline lysis method (15). Chromosomal DNA of P. denitrificans was isolated as described earlier (28). DNA restriction fragments were purified from agarose gels by using GeneClean (Bio 101, Inc., San Diego, Calif.).…”

Section: Methodsmentioning

confidence: 99%

Integration of heterologous DNA into the genome of Paracoccus denitrificans is mediated by a family of IS1248-related elements and a second type of integrative recombination event

1995

Self Cite

View full text Add to dashboard Cite

All members of the IS1248 family residing in the genome of Paracoccus denitrificans have been isolated by using a set of insertion sequence entrapment vectors. The family consists of five closely related members that integrate the entrapment vectors at distinct sites. One of these, IS1248b, was sequenced and, except for a single base change, shown to be identical to the previously isolated IS1248a. Southern analysis of genomic DNA with labeled IS1248 revealed different hybridization patterns for different isolates of P. denitrificans and Thiosphaera pantotropha. No hybridization was observed with DNA from Thiobacillus versutus and more distantly related species. From a comparison of the fingerprints it was shown that one of the members of the IS1248 family found in P. denitrificans DSM413 is absent in strain NCIB8944, although they are catalogued in international strain catalogues as identical strains. Furthermore, strains Pd1222 and Pd1235, both derivatives of P. denitrificans DSM413, were shown to have different patterns of IS1248 hybridizing restriction fragments. In 14 of 18 strains, the entrapment vectors used in this study were incorporated into the genome via IS1248-mediated cointegrate formation. In the other four strains, the entrapment vectors were shown to be integrated through a different mechanism not involving IS1248.

show abstract

Mutagenesis of the gene encoding cytochrome c550 of Paracoccus denitrificans and analysis of the resultant physiological effects

Abstract: Volume 172, no. 2, p. 990, Fig. 2: The amino acids under nucleotides 388 to 423 were transposed with those under nucleotides 424 to 474; the correct amino acid sequence of cytochrome c550 is shown below.

Cited by 34 publications

References 0 publications

The surface‐charge asymmetry and dimerisation of cytochrome c550 from Paracoccus denitrificans @MM implications for the interaction with cytochrome c peroxidase

The surface‐charge asymmetry and dimerisation of cytochrome c550 from Paracoccus denitrificans @MM implications for the interaction with cytochrome c peroxidase

Cytochromes c ₅₅₀ , c ₅₅₂ , and c ₁ in the Electron Transport Network of Paracoccus denitrificans : Redundant or Subtly Different in Function?

Integration of heterologous DNA into the genome of Paracoccus denitrificans is mediated by a family of IS1248-related elements and a second type of integrative recombination event

Contact Info

Product

Resources

About

Mutagenesis of the gene encoding cytochrome c550 of Paracoccus denitrificans and analysis of the resultant physiological effects

Abstract: Volume 172, no. 2, p. 990, Fig. 2: The amino acids under nucleotides 388 to 423 were transposed with those under nucleotides 424 to 474; the correct amino acid sequence of cytochrome c550 is shown below.

Cited by 34 publications

References 0 publications

The surface‐charge asymmetry and dimerisation of cytochrome c550 from Paracoccus denitrificans @MM implications for the interaction with cytochrome c peroxidase

The surface‐charge asymmetry and dimerisation of cytochrome c550 from Paracoccus denitrificans @MM implications for the interaction with cytochrome c peroxidase

Cytochromes c 550 , c 552 , and c 1 in the Electron Transport Network of Paracoccus denitrificans : Redundant or Subtly Different in Function?

Integration of heterologous DNA into the genome of Paracoccus denitrificans is mediated by a family of IS1248-related elements and a second type of integrative recombination event

Contact Info

Product

Resources

About

Cytochromes c ₅₅₀ , c ₅₅₂ , and c ₁ in the Electron Transport Network of Paracoccus denitrificans : Redundant or Subtly Different in Function?