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A novel role for iron in modulating the activity and membrane‐binding ability of a trimmed soybean lipoxygenase‐1
Author(s) -
Dainese Enrico,
Angelucci Clotilde B.,
Sabatucci Annalaura,
De Filippis Vincenzo,
Mei Giampiero,
Maccarrone And Mauro
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.09-141390
Subject(s) - chemistry , circular dichroism , lipoxygenase , enzyme , membrane , biochemistry , glycine , fluorescence , metal , ferrous , biophysics , amino acid , biology , physics , organic chemistry , quantum mechanics
Lipoxygenases (LOXs) are iron‐containing enzymes that play critical roles in plants and animals. As yet, metal atom extraction, reconstitution, and substitution have not been successfully applied to soybean LOX‐1 [ Glycine max (L.) Merrill], a prototype member of the LOX family that is widely used in structural and kinetic studies. Here, tryptic digestion of native LOX‐1, used as a control, allowed us to isolate the 60‐kDa C‐terminal region (termed miniLOX), that retains the catalytically active iron in a more accessible position. Then, iron was removed to obtain an unprecedented apo‐miniLOX, which was reconstituted and substituted with different metal ions. These forms of miniLOX were characterized vs. native LOX‐1 by kinetic analysis, near UV circular dichroism, steady‐state fluorescence, and fluorescence resonance energy transfer. MiniLOX showed a 2‐fold increase in the membrane‐binding affinity compared with native LOX‐1 and a remarkable 4‐fold increase compared with apominiLOX ( K d =9.2±1.0, 17.9±2.0, and 45.4±4.3 μM, respectively). Furthermore, miniLOX reconstituted with Fe(II) or Fe(III) partially recovered its membrane‐binding ability ( K d =21.4±2.4 and 18.9±5.5 μM, respectively), overall supporting a novel noncatalytic role for iron in the LOX family.—Dainese, E., Angelucci, C. B., Sabatucci, A., De Filippis, V., Mei, M., Maccarrone, M. A novel role for iron in modulating the activity and membrane‐binding ability of a trimmed soybean lipoxygenase‐1. FASEB J. 24, 1725–1736 (2010). www.fasebj.org