Direct spectroscopic and kinetic evidence for the involvement of a peroxodiferric intermediate during the ferroxidase reaction in fast ferritin mineralization

Alice S. Pereira, William Small, Carsten Krebs, Pedro Tavares, Dale E. Edmondson, Elizabeth C. Theil, Boi Hanh Huynh

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Abstract

Rapid freeze-quench (RFQ) Mossbauer and stopped-flow absorption spectroscopy were used to monitor the ferritin ferroxidase reaction using recombinant (apo) frog M ferritin; the initial transient ferric species could be trapped by the RFQ method using low iron loading (36 Fe2+/ferritin molecule). Biphasic kinetics of ferroxidation were observed and measured directly by the Mossbauer method; a majority (85%) of the ferrous ions was oxidized at a fast rate of ~80 s-1 and the remainder at a much slower rate of ~ 1.7 s-1. In parallel with the fast phase oxidation of the Fe2+ ions, a single transient iron species is formed which exhibits magnetic properties (diamagnetic ground state) and Mossbauer parameters (ΔE(Q) = 1.08 ± 0.03 mm/s and δ = 0.62 ± 0.02 mm/s) indicative of an antiferromagnetically coupled peroxodiferric complex. The formation and decay rates of this transient diiron species measured by the RFQ Mossbauer method match those of a transient blue species (λ(max) = 650 nm) determined by the stopped-flow absorbance measurement. Thus, the transient colored species is assigned to the same peroxodiferric intermediate. Similar transient colored species have been detected by other investigators in several other fast ferritins (H and M subunit types), such as the human H ferritin and the Escherichia coli ferritin, suggesting a similar mechanism for the ferritin ferroxidase step in all fast ferritins. Peroxodiferric complexes are also formed as early intermediates in the reaction of O2 with the catalytic diiron centers in the hydroxylase component of soluble methane monooxygenase (MMOH) and in the D84E mutant of the R2 subunit of E. coli ribonucleotide reductase. The proposal that a single protein site, with a structure homologous to the diiron centers in MMOH and R2, is involved in the ferritin ferroxidation step is confirmed by the observed kinetics, spectroscopic properties, and purity of the initial peroxodiferric species formed in the frog M ferritin.

Original languageEnglish (US)
Pages (from-to)9871-9876
Number of pages6
JournalBiochemistry
Volume37
Issue number28
DOIs
StatePublished - Jul 14 1998

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Ceruloplasmin
Ferritins
Kinetics
Apoferritins
methane monooxygenase
Anura
Escherichia coli
Iron
Ions
Ribonucleotide Reductases
Flow measurement
Mixed Function Oxygenases
Absorption spectroscopy
Ground state
Spectrum Analysis
Magnetic properties
Research Personnel
Oxidation
Molecules

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Pereira, Alice S. ; Small, William ; Krebs, Carsten ; Tavares, Pedro ; Edmondson, Dale E. ; Theil, Elizabeth C. ; Huynh, Boi Hanh. / Direct spectroscopic and kinetic evidence for the involvement of a peroxodiferric intermediate during the ferroxidase reaction in fast ferritin mineralization. In: Biochemistry. 1998 ; Vol. 37, No. 28. pp. 9871-9876.
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abstract = "Rapid freeze-quench (RFQ) Mossbauer and stopped-flow absorption spectroscopy were used to monitor the ferritin ferroxidase reaction using recombinant (apo) frog M ferritin; the initial transient ferric species could be trapped by the RFQ method using low iron loading (36 Fe2+/ferritin molecule). Biphasic kinetics of ferroxidation were observed and measured directly by the Mossbauer method; a majority (85{\%}) of the ferrous ions was oxidized at a fast rate of ~80 s-1 and the remainder at a much slower rate of ~ 1.7 s-1. In parallel with the fast phase oxidation of the Fe2+ ions, a single transient iron species is formed which exhibits magnetic properties (diamagnetic ground state) and Mossbauer parameters (ΔE(Q) = 1.08 ± 0.03 mm/s and δ = 0.62 ± 0.02 mm/s) indicative of an antiferromagnetically coupled peroxodiferric complex. The formation and decay rates of this transient diiron species measured by the RFQ Mossbauer method match those of a transient blue species (λ(max) = 650 nm) determined by the stopped-flow absorbance measurement. Thus, the transient colored species is assigned to the same peroxodiferric intermediate. Similar transient colored species have been detected by other investigators in several other fast ferritins (H and M subunit types), such as the human H ferritin and the Escherichia coli ferritin, suggesting a similar mechanism for the ferritin ferroxidase step in all fast ferritins. Peroxodiferric complexes are also formed as early intermediates in the reaction of O2 with the catalytic diiron centers in the hydroxylase component of soluble methane monooxygenase (MMOH) and in the D84E mutant of the R2 subunit of E. coli ribonucleotide reductase. The proposal that a single protein site, with a structure homologous to the diiron centers in MMOH and R2, is involved in the ferritin ferroxidation step is confirmed by the observed kinetics, spectroscopic properties, and purity of the initial peroxodiferric species formed in the frog M ferritin.",
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Direct spectroscopic and kinetic evidence for the involvement of a peroxodiferric intermediate during the ferroxidase reaction in fast ferritin mineralization. / Pereira, Alice S.; Small, William; Krebs, Carsten; Tavares, Pedro; Edmondson, Dale E.; Theil, Elizabeth C.; Huynh, Boi Hanh.

In: Biochemistry, Vol. 37, No. 28, 14.07.1998, p. 9871-9876.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Direct spectroscopic and kinetic evidence for the involvement of a peroxodiferric intermediate during the ferroxidase reaction in fast ferritin mineralization

AU - Pereira, Alice S.

AU - Small, William

AU - Krebs, Carsten

AU - Tavares, Pedro

AU - Edmondson, Dale E.

AU - Theil, Elizabeth C.

AU - Huynh, Boi Hanh

PY - 1998/7/14

Y1 - 1998/7/14

N2 - Rapid freeze-quench (RFQ) Mossbauer and stopped-flow absorption spectroscopy were used to monitor the ferritin ferroxidase reaction using recombinant (apo) frog M ferritin; the initial transient ferric species could be trapped by the RFQ method using low iron loading (36 Fe2+/ferritin molecule). Biphasic kinetics of ferroxidation were observed and measured directly by the Mossbauer method; a majority (85%) of the ferrous ions was oxidized at a fast rate of ~80 s-1 and the remainder at a much slower rate of ~ 1.7 s-1. In parallel with the fast phase oxidation of the Fe2+ ions, a single transient iron species is formed which exhibits magnetic properties (diamagnetic ground state) and Mossbauer parameters (ΔE(Q) = 1.08 ± 0.03 mm/s and δ = 0.62 ± 0.02 mm/s) indicative of an antiferromagnetically coupled peroxodiferric complex. The formation and decay rates of this transient diiron species measured by the RFQ Mossbauer method match those of a transient blue species (λ(max) = 650 nm) determined by the stopped-flow absorbance measurement. Thus, the transient colored species is assigned to the same peroxodiferric intermediate. Similar transient colored species have been detected by other investigators in several other fast ferritins (H and M subunit types), such as the human H ferritin and the Escherichia coli ferritin, suggesting a similar mechanism for the ferritin ferroxidase step in all fast ferritins. Peroxodiferric complexes are also formed as early intermediates in the reaction of O2 with the catalytic diiron centers in the hydroxylase component of soluble methane monooxygenase (MMOH) and in the D84E mutant of the R2 subunit of E. coli ribonucleotide reductase. The proposal that a single protein site, with a structure homologous to the diiron centers in MMOH and R2, is involved in the ferritin ferroxidation step is confirmed by the observed kinetics, spectroscopic properties, and purity of the initial peroxodiferric species formed in the frog M ferritin.

AB - Rapid freeze-quench (RFQ) Mossbauer and stopped-flow absorption spectroscopy were used to monitor the ferritin ferroxidase reaction using recombinant (apo) frog M ferritin; the initial transient ferric species could be trapped by the RFQ method using low iron loading (36 Fe2+/ferritin molecule). Biphasic kinetics of ferroxidation were observed and measured directly by the Mossbauer method; a majority (85%) of the ferrous ions was oxidized at a fast rate of ~80 s-1 and the remainder at a much slower rate of ~ 1.7 s-1. In parallel with the fast phase oxidation of the Fe2+ ions, a single transient iron species is formed which exhibits magnetic properties (diamagnetic ground state) and Mossbauer parameters (ΔE(Q) = 1.08 ± 0.03 mm/s and δ = 0.62 ± 0.02 mm/s) indicative of an antiferromagnetically coupled peroxodiferric complex. The formation and decay rates of this transient diiron species measured by the RFQ Mossbauer method match those of a transient blue species (λ(max) = 650 nm) determined by the stopped-flow absorbance measurement. Thus, the transient colored species is assigned to the same peroxodiferric intermediate. Similar transient colored species have been detected by other investigators in several other fast ferritins (H and M subunit types), such as the human H ferritin and the Escherichia coli ferritin, suggesting a similar mechanism for the ferritin ferroxidase step in all fast ferritins. Peroxodiferric complexes are also formed as early intermediates in the reaction of O2 with the catalytic diiron centers in the hydroxylase component of soluble methane monooxygenase (MMOH) and in the D84E mutant of the R2 subunit of E. coli ribonucleotide reductase. The proposal that a single protein site, with a structure homologous to the diiron centers in MMOH and R2, is involved in the ferritin ferroxidation step is confirmed by the observed kinetics, spectroscopic properties, and purity of the initial peroxodiferric species formed in the frog M ferritin.

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