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.
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)
Department of Chemistry, University of New Hampshire, Durham 03824.
N D
ND
Chasteen
J K
JK
Grady
K I
KI
Skorey
K J
KJ
Neden
D
D
Riendeau
M D
MD
Percival
D001094:Arachidonate 5-Lipoxygenase / Q000737:chemistry*; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D005290:Ferric Compounds / Q000737:chemistry; D005296:Ferrous Compounds / Q000737:chemistry; D006801:Humans; D008667:Metalloproteins / Q000737:chemistry*; D019159:Nonheme Iron Proteins; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D005290:Ferric Compounds; D005296:Ferrous Compounds; D008667:Metalloproteins; D019159:Nonheme Iron Proteins; D001094:Arachidonate 5-Lipoxygenase