The Molar Light Absorption of Pyridine Ferroprotoporphrin (Pyridine Haemochromogen).

Coral allene oxide synthase (AOS), a hemoprotein with weak sequence homology to catalase, is the N-terminal domain of a naturally occurring fusion protein with an 8R-lipoxygenase. AOS converts 8R-hydroperoxyeicosatetraenoic acid to the corresponding allene oxide. The UV-visible absorption and magnetic circular dichroism spectra of ferric AOS and of its cyanide and azide complexes, and the electron paramagnetic resonance spectra of native AOS (high-spin, g ) 6.56, 5.22, 2.00) and of its cyanide adduct (low-spin, g ) 2.86, 2.24, 1.60) closely resemble the corresponding spectra of bovine liver catalase (BLC). These results provide strong evidence for tyrosinate ligation to the heme iron of AOS as has been established for catalases. On the other hand, the positive circular dichroism bands in the Soret region for all three derivatives of ferric AOS are almost the mirror image of those in catalase. In addition, the cyanide affinity of native AOS (K d ) 10 mM at pH 7) is about 3 orders of magnitude lower than that of BLC. Thus, while these results conclusively support a common tyrosinate-ligated heme in AOS as in catalase, significant differences exist in the interaction between their respective heme prosthetic groups and protein environments, and in the access of small molecules to the heme iron.Interest in the enzymology underlying the high prostaglandin content of the Caribbean sea whip coral, Plexaura homomalla, has been the impetus for several biochemical investigations. Arachidonic acid is metabolized in extracts of P. homomalla and related corals by a lipoxygenase (LOX) 1 pathway that appeared initially to relate to the production of cyclopentenone prostaglandins (1, 2). An 8R-lipoxygenase converts arachidonic acid to 8R-hydroperoxyeicosatetraenoic acid (8R-HPETE), and the fatty acid hydroperoxide is then enzymatically dehydrated to an allene oxide, an epoxide with a propensity to cyclize to cyclopentenone derivatives. Brash et al., in 1997, isolated the cDNA of an 8R-lipoxygenase from P. homomalla and found it to occur with an extra sequence in the open reading frame that encodes the allene oxide synthase (AOS) (3). This natural fusion protein in coral consists of a LOX C-terminal domain (79 kDa) and an AOS N-terminal domain (43 kDa). The truncated (AOS) construct catalyzes an identical reaction to the AOS domain in the fusion protein, converting 8R-HPETE solely to the allene oxide. The truncated construct exhibits very high turnover number (1400/s) (4), although, due to the weaker expression of the fusion protein in E. coli, the comparable values for the fusion protein remain to be determined. 2 Although allene oxide has not proven to be an intermediate in the prostaglandin synthesis pathway (5), it may be linked to prostanoid type products such as clavulones and punaglandins of other corals (2,6,7). Allene oxides are very unstable epoxides; they readily hydrolyze into ketols, cyclopentenones, and other rearrangement products (8-10).The production of allene oxides from fatty acid hydroperoxides also ...

show abstract

“…The heme content of the coral AOS was determined using the pyridine hemochrome assay (29). For 2.2 nmol of protein, 1.8 nmol of heme was found.…”

Section: Heme Content and The Molar Extinction Coefficient Of Aosmentioning

confidence: 99%

Characterization of the Coral Allene Oxide Synthase Active Site with UV−Visible Absorption, Magnetic Circular Dichroism, and Electron Paramagnetic Resonance Spectroscopy: Evidence for Tyrosinate Ligation to the Ferric Enzyme Heme Iron

et al. 2001

View full text Add to dashboard Cite

show abstract

“…Deuterated reagents were obtained from Cambridge Isotope Laboratories. The concentration of protoheme in samples was determined by the pyridine hemochromogen method [14].…”

Section: Methodsmentioning

confidence: 99%

Identification of the axial heme ligands of cytochrome b₅₅₆ in succinate: Ubiquinone oxidoreductase from Escherichia coli

Peterson¹,

Vibat²,

Gennis³

1994

FEBS Letters

View full text Add to dashboard Cite

Electron paramagnetic resonance (EPR) and near-infrared magnetic circular dichroism (MCD) have been used to identify the ligands to the cytochrome b556 component of succinate: ubiquinone oxidoreductase (succinate dehydrogenase) from Escherichia coll. The 'highly axial low spin' (HALS) EPR spectrum suggests bis(histidine) ligation of the heme with the histidines in a staggered configuration. The near-infrared MCD spectrum exhibits a low energy maximum at 1600 nm which is also clearly indicative of bis(histidine) ligation of the heme iron. The data unambiguously demonstrate that the heme b556 is ligated to E. coli succinate dehydrogenase via two histidines.

show abstract

“…The heme content of erythrocruorin was determined by the pyridine hemochromogen method [5], with an absorption coefficient at 557 nm of 34.4 mM-' cm-I for the CI band of the pyridine ferroprotoporphyrin [6].…”

Section: Heme and Iron Determinationsmentioning

confidence: 99%

Molecular Architecture of Annelid Erythrocruorins. Extracellular Hemoglobin of Arenicola marina (Polychaeta)

Pionetti

Pouyet

1980

Eur J Biochem

View full text Add to dashboard Cite

The respiratory protein, erythrocruorin, of the annelid Arenicola marina was investigated. Spectral properties show many analogies with those of vertebrate hemoglobine. 144 heme groups (ferroprotoporphyrin) were found in the whole molecule, which has a relative molecular mass of 3.56 x lo6, as determined by sedimentation equilibrium, and an isoelectric point of 4.69. Protein dissociation patterns were analysed by electrophoresis after denaturation in the presence of dodecylsulfate, with and without 2-mercaptoethanol. A tentative model associating molecular mass of the native molecule, heme content, molecular mass of the polypeptide chains and functional properties is proposed. A twelfth subunit of A . marina erythrocruorin would contain twelve heme groups arranged in three functional units made up of four protomers, half of these being covalently bound to non-heme chains; two structural chains would be spatially arranged as bonds between the subunits.

show abstract

The Molar Light Absorption of Pyridine Ferroprotoporphrin (Pyridine Haemochromogen).

Cited by 559 publications

References 0 publications

Characterization of the Coral Allene Oxide Synthase Active Site with UV−Visible Absorption, Magnetic Circular Dichroism, and Electron Paramagnetic Resonance Spectroscopy: Evidence for Tyrosinate Ligation to the Ferric Enzyme Heme Iron

Characterization of the Coral Allene Oxide Synthase Active Site with UV−Visible Absorption, Magnetic Circular Dichroism, and Electron Paramagnetic Resonance Spectroscopy: Evidence for Tyrosinate Ligation to the Ferric Enzyme Heme Iron

Identification of the axial heme ligands of cytochrome b₅₅₆ in succinate: Ubiquinone oxidoreductase from Escherichia coli

Molecular Architecture of Annelid Erythrocruorins. Extracellular Hemoglobin of Arenicola marina (Polychaeta)

Contact Info

Product

Resources

About

The Molar Light Absorption of Pyridine Ferroprotoporphrin (Pyridine Haemochromogen).

Cited by 559 publications

References 0 publications

Characterization of the Coral Allene Oxide Synthase Active Site with UV−Visible Absorption, Magnetic Circular Dichroism, and Electron Paramagnetic Resonance Spectroscopy: Evidence for Tyrosinate Ligation to the Ferric Enzyme Heme Iron

Characterization of the Coral Allene Oxide Synthase Active Site with UV−Visible Absorption, Magnetic Circular Dichroism, and Electron Paramagnetic Resonance Spectroscopy: Evidence for Tyrosinate Ligation to the Ferric Enzyme Heme Iron

Identification of the axial heme ligands of cytochrome b556 in succinate: Ubiquinone oxidoreductase from Escherichia coli

Molecular Architecture of Annelid Erythrocruorins. Extracellular Hemoglobin of Arenicola marina (Polychaeta)

Contact Info

Product

Resources

About

Identification of the axial heme ligands of cytochrome b₅₅₆ in succinate: Ubiquinone oxidoreductase from Escherichia coli