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 apo-miniLOX (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.