Functional necessity and physicochemical characteristics of the [2Fe-2S] cluster in mammalian ferrochelatase

Steven G. Lloyd, Ricardo Franco, José J. G. Moura, Isabel Moura, Glória C. Ferreira, Boi Hanh Huynh

Research output: Contribution to journalArticle

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Abstract

The recently discovered [2Fe-2S] cluster in mouse liver ferrochelatase has been characterized using UV-vis, EPR, and Mossbauer spectroscopic techniques. Studies are reported here for the recombinant protein purified from an overproducing transformed Escherichia coli strain. A positive correlation is observed between the presence of the [2Fe-2S] cluster and the enzymatic specific activity and demonstrates the necessity of this cofactor. Chemical analysis revealed that the preparations contained up to 1.3 Fe/molecule and indicated a 1:1 stoichiometry between Fe and acid-labile sulfide. The [2Fe-2S] cluster in the as-isolated ferrochelatase exhibits a UV-vis spectrum indicative of a [2Fe-2S]2+ cluster and is EPR-silent. The 8 T Mossbauer spectrum of the 57Fe-enriched as-isolated protein is well simulated by parameters ΔE(Q) = 0.69 ± 0.03 mm/s and δ = 0.28 ± 0.02 mm/s and confirms the presence of a diamagnetic ground state. Upon reduction with sodium dithionite, ferrochelatase shows a near-axial EPR spectrum with g-values of 2.00, 1.93, and 1.91, consistent with a S = 1/4 mixed valent Fe3+-Fe2+ cluster. The Orbach temperature dependence of the EPR line widths was used to provide an estimate of the exchange coupling J, which was determined to be in the order of 500-650 cm-1 (+JS1.S2 model). Redox titrations monitored by UV-vis and EPR spectroscopy revealed midpoint potentials of -390 ± 10 and -405 ± 10 mV, respectively. Mossbauer spectra of the sodium dithionite-reduced 57Fe-enriched ferrochelatase collected at 4.2 K in the presence of magnetic fields of 60 mT and 8 T strengths were analyzed in the mixed-valent S = 1/4 ground state. Parameters for the ferric site are ΔE(Q) = 1.2 ± 0.2 mm/s and δ = 0.28 ± 0.03 mm/s, with somewhat anisotropic hyperfine splittings; for the ferrous site, ΔE(Q) = 3.3 ± 0.1 mm/s and δ = 0.67 ± 0.04 mm/s with anisotropic hyperfine splittings characteristic of high-spin ferrous ion. The similarities and differences with other characterized [2Fe-2S]+ cluster-containing proteins are discussed.

Original languageEnglish
Pages (from-to)9892-9900
Number of pages9
JournalJournal of the American Chemical Society
Volume118
Issue number41
DOIs
Publication statusPublished - 16 Oct 1996

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Ferrochelatase
Paramagnetic resonance
Dithionite
Ground state
Sodium
Proteins
Sulfides
Magnetic Fields
Recombinant proteins
Exchange coupling
Recombinant Proteins
Oxidation-Reduction
Spectrum Analysis
Titration
Stoichiometry
Linewidth
Liver
Escherichia coli
Ions
Temperature

Cite this

@article{27612c022cc44252a776637b20a377e2,
title = "Functional necessity and physicochemical characteristics of the [2Fe-2S] cluster in mammalian ferrochelatase",
abstract = "The recently discovered [2Fe-2S] cluster in mouse liver ferrochelatase has been characterized using UV-vis, EPR, and Mossbauer spectroscopic techniques. Studies are reported here for the recombinant protein purified from an overproducing transformed Escherichia coli strain. A positive correlation is observed between the presence of the [2Fe-2S] cluster and the enzymatic specific activity and demonstrates the necessity of this cofactor. Chemical analysis revealed that the preparations contained up to 1.3 Fe/molecule and indicated a 1:1 stoichiometry between Fe and acid-labile sulfide. The [2Fe-2S] cluster in the as-isolated ferrochelatase exhibits a UV-vis spectrum indicative of a [2Fe-2S]2+ cluster and is EPR-silent. The 8 T Mossbauer spectrum of the 57Fe-enriched as-isolated protein is well simulated by parameters ΔE(Q) = 0.69 ± 0.03 mm/s and δ = 0.28 ± 0.02 mm/s and confirms the presence of a diamagnetic ground state. Upon reduction with sodium dithionite, ferrochelatase shows a near-axial EPR spectrum with g-values of 2.00, 1.93, and 1.91, consistent with a S = 1/4 mixed valent Fe3+-Fe2+ cluster. The Orbach temperature dependence of the EPR line widths was used to provide an estimate of the exchange coupling J, which was determined to be in the order of 500-650 cm-1 (+JS1.S2 model). Redox titrations monitored by UV-vis and EPR spectroscopy revealed midpoint potentials of -390 ± 10 and -405 ± 10 mV, respectively. Mossbauer spectra of the sodium dithionite-reduced 57Fe-enriched ferrochelatase collected at 4.2 K in the presence of magnetic fields of 60 mT and 8 T strengths were analyzed in the mixed-valent S = 1/4 ground state. Parameters for the ferric site are ΔE(Q) = 1.2 ± 0.2 mm/s and δ = 0.28 ± 0.03 mm/s, with somewhat anisotropic hyperfine splittings; for the ferrous site, ΔE(Q) = 3.3 ± 0.1 mm/s and δ = 0.67 ± 0.04 mm/s with anisotropic hyperfine splittings characteristic of high-spin ferrous ion. The similarities and differences with other characterized [2Fe-2S]+ cluster-containing proteins are discussed.",
author = "Lloyd, {Steven G.} and Ricardo Franco and Moura, {Jos{\'e} J. G.} and Isabel Moura and Ferreira, {Gl{\'o}ria C.} and Huynh, {Boi Hanh}",
year = "1996",
month = "10",
day = "16",
doi = "10.1021/ja954000o",
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}

Functional necessity and physicochemical characteristics of the [2Fe-2S] cluster in mammalian ferrochelatase. / Lloyd, Steven G.; Franco, Ricardo; Moura, José J. G.; Moura, Isabel; Ferreira, Glória C.; Huynh, Boi Hanh.

In: Journal of the American Chemical Society, Vol. 118, No. 41, 16.10.1996, p. 9892-9900.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Functional necessity and physicochemical characteristics of the [2Fe-2S] cluster in mammalian ferrochelatase

AU - Lloyd, Steven G.

AU - Franco, Ricardo

AU - Moura, José J. G.

AU - Moura, Isabel

AU - Ferreira, Glória C.

AU - Huynh, Boi Hanh

PY - 1996/10/16

Y1 - 1996/10/16

N2 - The recently discovered [2Fe-2S] cluster in mouse liver ferrochelatase has been characterized using UV-vis, EPR, and Mossbauer spectroscopic techniques. Studies are reported here for the recombinant protein purified from an overproducing transformed Escherichia coli strain. A positive correlation is observed between the presence of the [2Fe-2S] cluster and the enzymatic specific activity and demonstrates the necessity of this cofactor. Chemical analysis revealed that the preparations contained up to 1.3 Fe/molecule and indicated a 1:1 stoichiometry between Fe and acid-labile sulfide. The [2Fe-2S] cluster in the as-isolated ferrochelatase exhibits a UV-vis spectrum indicative of a [2Fe-2S]2+ cluster and is EPR-silent. The 8 T Mossbauer spectrum of the 57Fe-enriched as-isolated protein is well simulated by parameters ΔE(Q) = 0.69 ± 0.03 mm/s and δ = 0.28 ± 0.02 mm/s and confirms the presence of a diamagnetic ground state. Upon reduction with sodium dithionite, ferrochelatase shows a near-axial EPR spectrum with g-values of 2.00, 1.93, and 1.91, consistent with a S = 1/4 mixed valent Fe3+-Fe2+ cluster. The Orbach temperature dependence of the EPR line widths was used to provide an estimate of the exchange coupling J, which was determined to be in the order of 500-650 cm-1 (+JS1.S2 model). Redox titrations monitored by UV-vis and EPR spectroscopy revealed midpoint potentials of -390 ± 10 and -405 ± 10 mV, respectively. Mossbauer spectra of the sodium dithionite-reduced 57Fe-enriched ferrochelatase collected at 4.2 K in the presence of magnetic fields of 60 mT and 8 T strengths were analyzed in the mixed-valent S = 1/4 ground state. Parameters for the ferric site are ΔE(Q) = 1.2 ± 0.2 mm/s and δ = 0.28 ± 0.03 mm/s, with somewhat anisotropic hyperfine splittings; for the ferrous site, ΔE(Q) = 3.3 ± 0.1 mm/s and δ = 0.67 ± 0.04 mm/s with anisotropic hyperfine splittings characteristic of high-spin ferrous ion. The similarities and differences with other characterized [2Fe-2S]+ cluster-containing proteins are discussed.

AB - The recently discovered [2Fe-2S] cluster in mouse liver ferrochelatase has been characterized using UV-vis, EPR, and Mossbauer spectroscopic techniques. Studies are reported here for the recombinant protein purified from an overproducing transformed Escherichia coli strain. A positive correlation is observed between the presence of the [2Fe-2S] cluster and the enzymatic specific activity and demonstrates the necessity of this cofactor. Chemical analysis revealed that the preparations contained up to 1.3 Fe/molecule and indicated a 1:1 stoichiometry between Fe and acid-labile sulfide. The [2Fe-2S] cluster in the as-isolated ferrochelatase exhibits a UV-vis spectrum indicative of a [2Fe-2S]2+ cluster and is EPR-silent. The 8 T Mossbauer spectrum of the 57Fe-enriched as-isolated protein is well simulated by parameters ΔE(Q) = 0.69 ± 0.03 mm/s and δ = 0.28 ± 0.02 mm/s and confirms the presence of a diamagnetic ground state. Upon reduction with sodium dithionite, ferrochelatase shows a near-axial EPR spectrum with g-values of 2.00, 1.93, and 1.91, consistent with a S = 1/4 mixed valent Fe3+-Fe2+ cluster. The Orbach temperature dependence of the EPR line widths was used to provide an estimate of the exchange coupling J, which was determined to be in the order of 500-650 cm-1 (+JS1.S2 model). Redox titrations monitored by UV-vis and EPR spectroscopy revealed midpoint potentials of -390 ± 10 and -405 ± 10 mV, respectively. Mossbauer spectra of the sodium dithionite-reduced 57Fe-enriched ferrochelatase collected at 4.2 K in the presence of magnetic fields of 60 mT and 8 T strengths were analyzed in the mixed-valent S = 1/4 ground state. Parameters for the ferric site are ΔE(Q) = 1.2 ± 0.2 mm/s and δ = 0.28 ± 0.03 mm/s, with somewhat anisotropic hyperfine splittings; for the ferrous site, ΔE(Q) = 3.3 ± 0.1 mm/s and δ = 0.67 ± 0.04 mm/s with anisotropic hyperfine splittings characteristic of high-spin ferrous ion. The similarities and differences with other characterized [2Fe-2S]+ cluster-containing proteins are discussed.

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U2 - 10.1021/ja954000o

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JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

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