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2002
Stoichiometric production of hydrogen peroxide and parallel formation of ferric multimers through decay of the diferric-peroxo complex, the first detectable intermediate in ferritin mineralization, Jameson, GNL, Jin W., Krebs C., Perreira AS, Tavares P., Liu XF, Theil EC, and Huynh B. H. , Biochemistry, Volume {41}, Number {45}, p.{13435-13443}, (2002) Abstract

The catalytic step that initiates formation of the ferric oxy-hydroxide mineral core in the central cavity of H-type ferritin involves rapid oxidation of ferrous ion by molecular oxygen (ferroxidase reaction) at a binuclear site (ferroxidase site) found in each of the 24 subunits. Previous investigators have shown that the first detectable reaction intermediate of the ferroxidase reaction is a diferric-peroxo intermediate, F-peroxo, formed within 25 ms, which then leads to the release of H2O2 and formation of ferric mineral precursors. The stoichiometric relationship between F-peroxo, H2O2, and ferric mineral precursors, crucial to defining the reaction pathway and mechanism, has now been determined. To this end, a horseradish peroxidase-catalyzed spectrophotometric method was used as an assay for H2O2. By rapidly mixing apo M ferritin from frog, Fe2+, and O-2 and allowing the reaction to proceed for 70 ms when F-peroxo has reached its maximum accumulation, followed by spraying the reaction mixture into the H2O2 assay solution, we were able to quantitatively determine the amount of H2O2 produced during the decay of F-peroxo. The correlation between the amount of H2O2 released with the amount of F-peroxo accumulated at 70 ms determined by Mossbauer spectroscopy showed that F-peroxo decays into H2O2 with a stoichiometry of 1 F-peroxo:H2O2. When the decay of F-peroxo was monitored by rapid freeze-quench Mossbauer spectroscopy, multiple diferric mu-oxo/mu-hydroxo complexes and small polynuclear ferric clusters were found to form at rate constants identical to the decay rate of F-peroxo. This observed parallel formation of multiple products (H2O2, diferric complexes, and small polynuclear clusters) from the decay of a single precursor (F-peroxo) provides useful mechanistic insights into ferritin mineralization and demonstrates a flexible ferroxidase site.

2001
Structure-function studies of cytochrome c peroxidase from ps. nautica, Alves, T., Besson S., Pereira A. S., Pettigrew G. W., Moura J. J. G., and Moura I. , Journal Of Inorganic Biochemistry, Aug, Volume {86}, Number {1}, 655 AVENUE OF THE AMERICAS, NEW YORK, NY 10010 USA, p.{122}, (2001) Abstract
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Substitution of murine ferrochelatase glutamate-287 with glutamine or alanine leads to porphyrin substrate-bound variants, Franco, R., Pereira A. S., Tavares P., Mangravita A., Barber M. J., Moura I., and Ferreira G. C. , BIOCHEMICAL JOURNAL, Volume {356}, Number {1}, p.{217-222}, (2001) Abstract

Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring. Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity. Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme. In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX. Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx. 80% of the total porphyrin corresponds to protoporphyrin IX. Significantly, rapid stopped-flow experiments of the E287A and E287Q Variants demonstrate that reaction with Zn2+ results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate. Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product. Thus E287 in murine ferrochelatase appears to be critical For the catalytic process by controlling the release of the product.

1999
Simple and complex iron-sulfur proteins in sulfate reducing bacteria, Moura, I., Pereira A. S., Tavares P., and Moura J. J. G. , Volume {47}, p.{361-419}, (1999) Abstract
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Spectroscopic characterization of porphyrin binding to ferrochelatase, the last enzyme in the heme biosynthetic pathway, Franco, R., Ma J. G., Lu Y., Pereira A., Tavares P., Moura I., Shelnutt J. A., and Ferreira G. C. , Journal Of Inorganic Biochemistry, Volume {74}, Number {1-4}, p.{130}, (1999) Abstract
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