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)

Helix variants of troponin C with tailored calcium affinities.

Muscle fiber contraction is regulated through calcium-induced changes in the conformation of troponin C. In this study, we explored the relationship between the stability of a specific helix in the protein and the metal ion affinity of associated binding sites. Serial replacement of the amino acid at position 130 caused the calcium affinity of the paired Ca2+/Mg2+ sites to be attenuated. In the crystal structures of chicken and turkey troponin C, position 130 is the N-cap residue of the G-helix. The ion affinities of variant proteins were shifted in the order Ile < Gly < Asp < Asn < Thr < Ser. Although differing in ion affinities, the variant proteins all exhibited high cooperativity. The results of this study point to a specific relationship between alpha-helix stability and ion affinity in troponin C and suggest that troponin C may be a paradigm for protein folding problems.