Characterization of the non-heme iron center of human 5-lipoxygenase by electron paramagnetic resonance, fluorescence, and ultraviolet-visible spectroscopy: redox cycling between ferrous and ferric states
| Autor: | N. D. Chasteen, J. K. Grady, K. J. Neden, K. I. Skorey, Denis Riendeau, M. D. Percival |
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| Rok vydání: | 1993 |
| Předmět: |
Reducing agent
Analytical chemistry Photochemistry Biochemistry Ferric Compounds Fluorescence spectroscopy Nonheme Iron Proteins law.invention Ferrous Metal Ultraviolet visible spectroscopy law Metalloproteins medicine Humans Ferrous Compounds Electron paramagnetic resonance Arachidonate 5-Lipoxygenase Binding Sites Chemistry Electron Spin Resonance Spectroscopy Fluorescence Spectrometry Fluorescence visual_art visual_art.visual_art_medium Ferric Spectrophotometry Ultraviolet Oxidation-Reduction medicine.drug |
| Zdroj: | Biochemistry. 32(37) |
| ISSN: | 0006-2960 |
| Popis: | Purified human 5-lipoxygenase, a non-heme iron containing enzyme, has been characterized by electron paramagnetic resonance, (EPR), ultraviolet (UV)-visible and fluorescence spectroscopy. As isolated, the enzyme is largely in the ferrous state and shows a weak X-band EPR signal extending from 0 to 700 G at 15 K, tentatively ascribed to integer spin Fe(II). Titration of the protein with 13-HPOD (13-hydroperoxyoctadecadienoic acid) generates a strong multicomponent EPR signal in the g' approximately 6 region, a yellow color associated with an increased absorption between 310 and 450 nm (epsilon 330nm = 2400 M-1 cm-1), and a 17% decrease in the intrinsic protein fluorescence. The multiple component nature of the g' approximately 6 signal indicates that the metal center in its oxidized state exists in more than one but related forms. The g' approximately 6 EPR signal and the yellow color reach a maximum when approximately 1 mol of 13-HPOD is added/mol of iron; the resultant EPR spectrum accounts quantitatively for all of the iron in the protein with a signal at g' = 4.3 representing less than 3% of the total iron in the majority of samples. Addition of a hydroxyurea reducing agent abolished the g' approximately 6 signal and yellow color of the protein and also reversed the decrease in fluorescence caused by the oxidant 13-HPOD. The results indicate that the g' approximately 6 EPR signal, the yellow color, and the decreased fluorescence are associated with the formation of the Fe(III) form of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS) |
| Databáze: | OpenAIRE |
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