Kinetics of Bis-Allylic Hydroperoxide Synthesis in the Iron-Containing Lipoxygenase 2 from Cyanothece and the Effects of Manganese Substitution.

Lipoxygenases (LOX) catalyze the regio- and stereospecific insertion of dioxygen into polyunsaturated fatty acids. While the catalytic metal of LOX is typically a non-heme iron, some fungal LOX contain manganese as catalytic metal (MnLOX). In general, LOX insert dioxygen at C9 or C13 of linoleic acid leading to the formation of conjugated hydroperoxides. MnLOX (EC 1.13.11.45), however, catalyze the oxygen insertion also at C11, resulting in bis-allylic hydroperoxides. Interestingly, the iron-containing CspLOX2 (EC 1.13.11.B6) from Cyanothece PCC8801 also produces bis-allylic hydroperoxides. What role the catalytic metal plays and how this unusual reaction is catalyzed by either MnLOX or CspLOX2 is not understood. Our findings suggest that only iron is the catalytically active metal in CspLOX2. The enzyme loses its catalytic activity almost completely when iron is substituted with manganese, suggesting that the catalytic metal is not interchangeable. Using kinetic and spectroscopic approaches, we further found that first a mixture of bis-allylic and conjugated hydroperoxy products is formed. This is followed by the isomerization of the bis-allylic product to conjugated products at a slower rate. These results suggest that MnLOX and CspLOX2 share a very similar reaction mechanism and that LOX with a Fe or Mn cofactor have the potential to form bis-allylic products. Therefore, steric factors are probably responsible for this unusual specificity. As CspLOX2 is the LOX with the highest proportion of the bis-allylic product known so far, it will be an ideal candidate for further structural analysis to understand the molecular basis of the formation of bis-allylic hydroperoxides.