EPR and ENDOR detection of compound I from Micrococcus lysodeikticus catalase

Benecky, Michael J.; Frew, Jane E.; Scowen, Nina; Jones, Peter; Hoffman, Brian M.

doi:10.1021/bi00095a024

Cited by 100 publications

(159 citation statements)

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“…The same derivative has also been observed with EPR spectroscopy for bacterial (Microccocus luteus) catalase (0.5 mM) following addition of peracetic acid (3 mM) (37). However, addition of a large excess amount of peracetic acid (10-25 mM) to BLC (0.01-0.5 mM) has been shown to generate a Compound II (oxo-ferryl)-type derivative as judged by UV-visible absorption spectroscopy (49); a free radical species has also been detected with EPR spectroscopy (39).…”

Section: Discussionsupporting

confidence: 60%

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

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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 ...

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Section: Discussionsupporting

confidence: 60%

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

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“…1C, the EPR spectra of the W164H (dotted line) and W164S (continuous line) variants after the addition of 1 eq of H 2 O 2 (resulting in VPI formation) are superimposed. Both VPI EPR spectra were very similar in shape, characterized by the same g iso value (2.00(1)) (with a peak to peak amplitude equal to 0.22 mT), and resembled the [Fe(IV) ϭ O Por ϩ ⅐ ] intermediate spectrum reported in other heme proteins (45)(46)(47). Unlike the organic radical signals of native VP and W164Y VPI, which were well resolved also at T ϭ 70 K, the EPR spectra of the W164H and W164S VPI showed a trend with the variation of temperature similar to that obtained for the porphyrinyl radical of horseradish peroxidase Compound I (see supplemental Fig.…”

Section: Epr Analysessupporting

confidence: 61%

Protein Radicals in Fungal Versatile Peroxidase

Ruíz-Dueñas

Pogni

Morales

et al. 2009

Journal of Biological Chemistry

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Lignin-degrading peroxidases, a group of biotechnologically interesting enzymes, oxidize high redox potential aromatics via an exposed protein radical. Low temperature EPR of Pleurotus eryngii versatile peroxidase (VP) revealed, for the first time in a fungal peroxidase, the presence of a tryptophanyl radical in both the two-electron (VPI) and the one-electron (VPII) activated forms of the enzyme. Site-directed mutagenesis was used to substitute this tryptophan (Trp-164) by tyrosine and histidine residues. No changes in the crystal structure were observed, indicating that the modified behavior was due exclusively to the mutations introduced. EPR revealed the formation of tyrosyl radicals in both VPI and VPII of the W164Y variant. However, no protein radical was detected in the W164H variant, whose VPI spectrum indicated a porphyrin radical identical to that of the inactive W164S variant. Stopped-flow spectrophotometry showed that the W164Y mutation reduced 10-fold the apparent second-order rate constant for VPI reduction (k 2app ) by veratryl alcohol (VA), when compared with over 50-fold reduction in W164S, revealing some catalytic activity of the tyrosine radical. Its first-order rate constant (k 2 ) was more affected than the dissociation constant (K D2 ). Moreover, VPII reduction by VA was impaired by the above mutations, revealing that the Trp-164 radical was involved in catalysis by both VPI and VPII. The low first-order rate constant (k 3 ) values were similar for the W164Y, W164H, and W164S variants, indicating that the tyrosyl radical in VPII was not able to oxidize VA (in contrast with that observed for VPI). VPII self-reduction was also suppressed, revealing that Trp-164 is involved in this autocatalytic process.Lignin degradation, a key step for carbon recycling in land ecosystems and a central issue for industrial use of lignocellulosic biomass (e.g. in paper pulp manufacture and bioethanol production), is initiated in nature by 1-electron oxidation of the benzenic rings of lignin by specialized high redox potential fungal peroxidases (1-5). The latter include lignin peroxidase (LiP; 6 EC 1.11.1.14), first described in Phanerochaete chrysosporium together with manganese peroxidase (EC 1.11.1.13), and versatile peroxidase (VP; EC 1.11.1.16), described in Pleurotus and Bjerkandera species as a hybrid enzyme combining the catalytic properties of LiP and manganese peroxidase (6, 7). Oxidation of high redox potential aromatics, including nonphenolic lignin models, by LiP (8, 9) and VP (10) takes place at an exposed tryptophan by long range electron transfer (LRET) to heme in the 2-electron (Compound I) and 1-electron (Compound II) activated forms of the enzyme. This basically differs from Mn 2ϩ oxidation by manganese peroxidase (11) and VP (12) and oxidation of different phenolic substrates and dyes by classical plant peroxidases (13) that is produced by direct electron transfer to the activated heme cofactor.The existence of a tryptophan radical in a peroxidase (in this case neighbor to heme) has been re...

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“…Cpd I can usually be easily recognized because the diminished aromaticity of the -cation radical of the porphyrin yields a Soret band with a significantly smaller extinction coefficient than that of Soret bands of other heme species. It has been established that the predominant form of Cpd I in horseradish peroxidase (HRP), and in many other peroxidases, is a ferryl iron (Fe IV ϭO) paired with a porphyrin -cation radical (1,(12)(13)(14). However, in cytochrome c peroxidase (CcP), the analogous species, historically referred to as Cpd ES, has a Fe IV ϭO coupled with a tryptophan radical cation (15)(16)(17)(18)(19).…”

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confidence: 99%