Formate dehydrogenase from methylosinus trichosporium Ob3b: Purification and spectroscopic characterization of the cofactors

David R. Jollie, John D Lipscomb

Research output: Contribution to journalArticle

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Abstract

NAD+-coupled formate dehydrogenase has been purified to near-homogeneity from the obligate methanotroph Methylosinus trichosporium OB3b. The inclusion of stabilizing reagents in the purification buffers has resulted in a 3-fold increase in specific activity (98 μM/min/mg; turnover number 600 s-1) and as much as a 25-fold increase in yield over previously reported purification protocols. The enzyme, (molecular weight 400,000 ± 20,000) is composed of four subunit types (α, 98,000; β, 56,000; γ, 20,000; δ, 11,500) apparently associated as 2 αβγδ protomers. The holoenzyme contains flavin (1.8 ± 0.2), iron (46 ± 6), inorganic sulfide (38 ± 4), and molybdenum (1.5 ± 0.1). The flavin is optically similar to the common flavin cofactors, but it is chromatographically distinct. Anaerobic incubation of the enzyme with formate, NADH, or sodium dithionite, resulted in ∼50% reduction of the iron and elicited an electron paramagnetic resonance (EPR) spectrum (∼2.5 spins/protomer) from which the spectra of five distinct EPR-active centers could be resolved in the g = 1.94 region. Four of these spectra were characteristic of [Fe-S]x clusters. The fifth (gave = 1.99; ∼0.1 spins/protomer) was similar to that observed for the molybdenum cofactor of xanthine oxidase, and it exhibited the expected hyperfine splitting when the enzyme was enriched with 95Mo (I = 5/2). Mössbauer spectroscopy showed that all of the iron in the enzyme became reduced upon the addition of a redox mediator, proflavin, to the dithionite reduced enzyme at pH 8.0. Nevertheless, a decrease in the EPR-active spin concentration in the g = 1.94 region of the spectrum occurred and was attributed to the reduction of the molybdenum center to the EPR-silent Mo(IV) state (S = 1). The fully reduced enzyme also exhibited a new species with an S = 3/2 ground state (1-2 spins/protomer). Addition of 50% ethylene glycol to the fully reduced enzyme revealed no new species, but caused an increase in the EPR-detectable spin quantitation to 5-6 spins/protomer. This suggests that cluster spin-spin interactions may occur in both the partially and fully reduced native enzyme.

Original languageEnglish (US)
Pages (from-to)21853-21863
Number of pages11
JournalJournal of Biological Chemistry
Volume266
Issue number32
StatePublished - Dec 1 1991

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Methylosinus trichosporium
Formate Dehydrogenases
Purification
Electron Spin Resonance Spectroscopy
Protein Subunits
Paramagnetic resonance
Enzymes
Dithionite
formic acid
Molybdenum
Iron
NAD
Proflavine
Holoenzymes
Ethylene Glycol
Xanthine Oxidase
Sulfides
Ground state
Oxidation-Reduction
Spectrum Analysis

Cite this

Formate dehydrogenase from methylosinus trichosporium Ob3b : Purification and spectroscopic characterization of the cofactors. / Jollie, David R.; Lipscomb, John D.

In: Journal of Biological Chemistry, Vol. 266, No. 32, 01.12.1991, p. 21853-21863.

Research output: Contribution to journalArticle

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title = "Formate dehydrogenase from methylosinus trichosporium Ob3b: Purification and spectroscopic characterization of the cofactors",
abstract = "NAD+-coupled formate dehydrogenase has been purified to near-homogeneity from the obligate methanotroph Methylosinus trichosporium OB3b. The inclusion of stabilizing reagents in the purification buffers has resulted in a 3-fold increase in specific activity (98 μM/min/mg; turnover number 600 s-1) and as much as a 25-fold increase in yield over previously reported purification protocols. The enzyme, (molecular weight 400,000 ± 20,000) is composed of four subunit types (α, 98,000; β, 56,000; γ, 20,000; δ, 11,500) apparently associated as 2 αβγδ protomers. The holoenzyme contains flavin (1.8 ± 0.2), iron (46 ± 6), inorganic sulfide (38 ± 4), and molybdenum (1.5 ± 0.1). The flavin is optically similar to the common flavin cofactors, but it is chromatographically distinct. Anaerobic incubation of the enzyme with formate, NADH, or sodium dithionite, resulted in ∼50{\%} reduction of the iron and elicited an electron paramagnetic resonance (EPR) spectrum (∼2.5 spins/protomer) from which the spectra of five distinct EPR-active centers could be resolved in the g = 1.94 region. Four of these spectra were characteristic of [Fe-S]x clusters. The fifth (gave = 1.99; ∼0.1 spins/protomer) was similar to that observed for the molybdenum cofactor of xanthine oxidase, and it exhibited the expected hyperfine splitting when the enzyme was enriched with 95Mo (I = 5/2). M{\"o}ssbauer spectroscopy showed that all of the iron in the enzyme became reduced upon the addition of a redox mediator, proflavin, to the dithionite reduced enzyme at pH 8.0. Nevertheless, a decrease in the EPR-active spin concentration in the g = 1.94 region of the spectrum occurred and was attributed to the reduction of the molybdenum center to the EPR-silent Mo(IV) state (S = 1). The fully reduced enzyme also exhibited a new species with an S = 3/2 ground state (1-2 spins/protomer). Addition of 50{\%} ethylene glycol to the fully reduced enzyme revealed no new species, but caused an increase in the EPR-detectable spin quantitation to 5-6 spins/protomer. This suggests that cluster spin-spin interactions may occur in both the partially and fully reduced native enzyme.",
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N2 - NAD+-coupled formate dehydrogenase has been purified to near-homogeneity from the obligate methanotroph Methylosinus trichosporium OB3b. The inclusion of stabilizing reagents in the purification buffers has resulted in a 3-fold increase in specific activity (98 μM/min/mg; turnover number 600 s-1) and as much as a 25-fold increase in yield over previously reported purification protocols. The enzyme, (molecular weight 400,000 ± 20,000) is composed of four subunit types (α, 98,000; β, 56,000; γ, 20,000; δ, 11,500) apparently associated as 2 αβγδ protomers. The holoenzyme contains flavin (1.8 ± 0.2), iron (46 ± 6), inorganic sulfide (38 ± 4), and molybdenum (1.5 ± 0.1). The flavin is optically similar to the common flavin cofactors, but it is chromatographically distinct. Anaerobic incubation of the enzyme with formate, NADH, or sodium dithionite, resulted in ∼50% reduction of the iron and elicited an electron paramagnetic resonance (EPR) spectrum (∼2.5 spins/protomer) from which the spectra of five distinct EPR-active centers could be resolved in the g = 1.94 region. Four of these spectra were characteristic of [Fe-S]x clusters. The fifth (gave = 1.99; ∼0.1 spins/protomer) was similar to that observed for the molybdenum cofactor of xanthine oxidase, and it exhibited the expected hyperfine splitting when the enzyme was enriched with 95Mo (I = 5/2). Mössbauer spectroscopy showed that all of the iron in the enzyme became reduced upon the addition of a redox mediator, proflavin, to the dithionite reduced enzyme at pH 8.0. Nevertheless, a decrease in the EPR-active spin concentration in the g = 1.94 region of the spectrum occurred and was attributed to the reduction of the molybdenum center to the EPR-silent Mo(IV) state (S = 1). The fully reduced enzyme also exhibited a new species with an S = 3/2 ground state (1-2 spins/protomer). Addition of 50% ethylene glycol to the fully reduced enzyme revealed no new species, but caused an increase in the EPR-detectable spin quantitation to 5-6 spins/protomer. This suggests that cluster spin-spin interactions may occur in both the partially and fully reduced native enzyme.

AB - NAD+-coupled formate dehydrogenase has been purified to near-homogeneity from the obligate methanotroph Methylosinus trichosporium OB3b. The inclusion of stabilizing reagents in the purification buffers has resulted in a 3-fold increase in specific activity (98 μM/min/mg; turnover number 600 s-1) and as much as a 25-fold increase in yield over previously reported purification protocols. The enzyme, (molecular weight 400,000 ± 20,000) is composed of four subunit types (α, 98,000; β, 56,000; γ, 20,000; δ, 11,500) apparently associated as 2 αβγδ protomers. The holoenzyme contains flavin (1.8 ± 0.2), iron (46 ± 6), inorganic sulfide (38 ± 4), and molybdenum (1.5 ± 0.1). The flavin is optically similar to the common flavin cofactors, but it is chromatographically distinct. Anaerobic incubation of the enzyme with formate, NADH, or sodium dithionite, resulted in ∼50% reduction of the iron and elicited an electron paramagnetic resonance (EPR) spectrum (∼2.5 spins/protomer) from which the spectra of five distinct EPR-active centers could be resolved in the g = 1.94 region. Four of these spectra were characteristic of [Fe-S]x clusters. The fifth (gave = 1.99; ∼0.1 spins/protomer) was similar to that observed for the molybdenum cofactor of xanthine oxidase, and it exhibited the expected hyperfine splitting when the enzyme was enriched with 95Mo (I = 5/2). Mössbauer spectroscopy showed that all of the iron in the enzyme became reduced upon the addition of a redox mediator, proflavin, to the dithionite reduced enzyme at pH 8.0. Nevertheless, a decrease in the EPR-active spin concentration in the g = 1.94 region of the spectrum occurred and was attributed to the reduction of the molybdenum center to the EPR-silent Mo(IV) state (S = 1). The fully reduced enzyme also exhibited a new species with an S = 3/2 ground state (1-2 spins/protomer). Addition of 50% ethylene glycol to the fully reduced enzyme revealed no new species, but caused an increase in the EPR-detectable spin quantitation to 5-6 spins/protomer. This suggests that cluster spin-spin interactions may occur in both the partially and fully reduced native enzyme.

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