Haem staining in gels, a useful tool in the study of bacterial c-type cytochromes

Goodhew, Celia F.; Brown, Kevin R.; Pettigrew, Graham W.

doi:10.1016/0005-2728(86)90234-3

Cited by 174 publications

(128 citation statements)

References 15 publications

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“…Lane 1, untreated cytochrome c550, 0 M NaCl; lanes 2Ϫ7, cytochrome c550 reacted with 2.5 mM EDC in 0, 0.05, 0.1, 0.2, 0.5, 1.0 M NaCl, respectively; lanes 8Ϫ10, horse cytochrome c reacted with 2.5 mM EDC in 0, 0.1, 1.0 M NaCl, respectively. After electrophoresis, cytochrome bands were stained for the presence of haem [27]. The standard proteins Ϫ horse cytochrome c (M r 12 000), myoglobin (M r 17 000), carbonic anhydrase (M r 29 000) and ovalbumin (M r 45 000) were run in a separate channel and stained with Coomassie blue.…”

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

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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-

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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

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“…Gels were stained to indicate the presence of c-type cytochromes (28) or with Coomassie Brilliant Blue R-250. The following buffer was used for sample preparation irrespective of the method of protein visualization: 6 M urea, 5% (w/v) SDS, 0.1% (w/v) glycerol, and 0.05% (w/v) bromphenol blue.…”

Section: Methodsmentioning

confidence: 99%

“…faster) migration of the very hydrophobic CcoN subunit on the gels. Analysis of the holoenzyme on SDS-polyacrylamide gels that were subsequently stained for 3,3Ј,5,5Ј-tetramethylbenzidine-mediated heme peroxidase activity (28) revealed two strong bands with apparent molecular masses of 32 and 23 kDa. These correspond to the c-type heme containing subunits CcoP and CcoO.…”

Section: Purification and Characterization Of Cytochrome Cbbmentioning

confidence: 99%

Molecular and Spectroscopic Analysis of the Cytochromecbb3 Oxidase from Pseudomonas stutzeri

Pitcher¹,

Cheesman²,

Watmough³

2002

Journal of Biological Chemistry

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Cytochrome cbb 3 oxidase, a member of the heme-copper oxidase superfamily, is characterized by its high affinity for oxygen while retaining the ability to pump protons. These attributes are central to its proposed role in the microaerobic metabolism of proteobacteria. We have completed the first detailed spectroscopic characterization of a cytochrome cbb 3 oxidase, the enzyme purified from Pseudomonas stutzeri. A combination of UVvisible and magnetic CD spectroscopies clearly identified four low-spin hemes and the high-spin heme of the active site. This heme complement is in good agreement with our analysis of the primary sequence of the ccoNOPQ operon and biochemical analysis of the complex. Near-IR magnetic CD spectroscopy revealed the unexpected presence of a low-spin bishistidine-coordinated c-type heme in the complex. This was shown to be one of two c-type hemes in the CcoP subunit by separately expressing the subunit in Escherichia coli. Separate expression of CcoP also allowed us to unambiguously assign each of the signals associated with low-spin ferric hemes present in the X-band EPR spectrum of the oxidized enzyme. This work both underpins future mechanistic studies on this distinctive class of bacterial oxidases and raises questions concerning the role of CcoP in electron delivery to the catalytic subunit.

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“…The absorption maxima of the purified OxdRG enzyme were at 273, 359, 421, and 549 nm, indicating that the enzyme contains heme. Furthermore, the enzyme was positively stained by heme staining on native-PAGE gel (26). The pyridine hemochrome was prepared by mixing the enzyme with alkaline-pyridine for examining the properties of the heme in the enzyme.…”

Section: Screening For Aldoxime-degrading Microorganismsmentioning

confidence: 99%

A Gene Cluster Responsible for Alkylaldoxime Metabolism Coexisting with Nitrile Hydratase and Amidase in Rhodococcus globerulus A-4^,

et al. 2003

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An enzyme "alkylaldoxime dehydratase (OxdRG)" was purified and characterized from Rhodococcus globerulus A-4, in which nitrile hydratase (NHase) and amidase coexisted with the enzyme. The enzyme contains heme b as a prosthetic group, requires reducing reagents for the reaction, and is most active at a neutral pH and at around 30°C, similar to the phenylacetaldoxime dehydratase from Bacillus sp. OxB-1 (OxdB). However, some differences were seen in subunit structure, substrate specificity, and effects of activators and inhibitors. The corresponding gene, oxd, encoding a 1059-base pair ORF consisting of 353 codons, was cloned, sequenced, and overexpressed in Escherichia coli. The predicted polypeptide showed 30.3% identity to OxdB. The gene is mapped just upstream of the gene cluster encoding the enzymes involved in the metabolism of aliphatic nitriles, i.e., NHase and amidase, and their regulatory and activator proteins. We report here the existence of an aldoxime dehydratase genetically linked with NHase and amidase, and responsible for the metabolism of alkylaldoxime in R. globerulus.Aldoximes derived from amino acids are considered to be intermediates in the biosynthesis of cyanogenic glucosides and glucosinolates in plants (1). However, information on aldoxime metabolism is quite limited and the genetics and enzymology have not been well characterized. One oximemetabolizing enzyme (cytochrome P450 CYP71E1) has been reported to catalyze the conversion of aldoxime to R-hydroxynitrile in the pathway for biosynthesis of cyanogenic glucoside dhurrin in Sorghum bicolor (2-4). Other cytochrome P450s, namely, CYP83 homologues (A1 and B1), have also been identified as oxime-metabolizing enzymes, which catalyze the conversion of indoleacetaldoxime to the corresponding aci-nitro compound, the first step in the biosynthesis of indole glucosinolates in Arabidopsis thaliana. However, the level of activity is quite low, and the mechanisms involved have not been studied. Indoleacetaldoxime is known to be a metabolic branch point between the production of indoleacetic acid and indole glucosinolates in A. thaliana (5-7), but the enzymes responsible for the metabolism have yet to be purified and characterized.Asano et al. have isolated various nitrile-degrading microorganisms, e.g., Rhodococcus rhodochrous (formerly Arthrobacter sp.) strains J-1 and I-9 (K22, AKU 629) (8) and Pseudomonas chlororaphis B23 (9). They first purified, characterized, and named nitrile hydratase (NHase, EC. 4.2.1.84) from R. rhodochrous J-1 (10-12). They also found that P. chlororaphis B23 accumulates large quantities of amides from nitriles and is suitable for the industrial production of acrylamide from acrylonitrile (9, 12). Moreover, nicotinamide and 5-cyanovaleramide are also industrially produced by NHase (13,14). Despite its important uses, the physiological function of NHase in nature remains unclear.We have studied the metabolism of aldoximes from a physiological as well as an applicative perspective, and have isolated Bacillus sp. O...

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Haem staining in gels, a useful tool in the study of bacterial c-type cytochromes

Cited by 174 publications

References 15 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

Molecular and Spectroscopic Analysis of the Cytochromecbb3 Oxidase from Pseudomonas stutzeri

A Gene Cluster Responsible for Alkylaldoxime Metabolism Coexisting with Nitrile Hydratase and Amidase in Rhodococcus globerulus A-4^,

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Haem staining in gels, a useful tool in the study of bacterial c-type cytochromes

Cited by 174 publications

References 15 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

Molecular and Spectroscopic Analysis of the Cytochromecbb3 Oxidase from Pseudomonas stutzeri

A Gene Cluster Responsible for Alkylaldoxime Metabolism Coexisting with Nitrile Hydratase and Amidase in Rhodococcus globerulus A-4,

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A Gene Cluster Responsible for Alkylaldoxime Metabolism Coexisting with Nitrile Hydratase and Amidase in Rhodococcus globerulus A-4^,