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)
Chasteen
N D
ND
Department of Chemistry, University of New Hampshire, Durham 03824.
Grady
J K
JK
Skorey
K I
KI
Neden
K J
KJ
Riendeau
D
D
Percival
M D
MD
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