Kinetics of manganese lipoxygenase with a catalytic mononuclear redox center

Manganese lipoxygenase was isolated from the take-all fungus, Gaeumannomyce s graminis, and the oxygenation mechanism was investigated, A kinetic isoto pe effect, k(H)/k(D) = 21-24, was observed with [U-H-2]linoleic acid as a s ubstrate, The relative biosynthesis of (13R)-hydroperoxylinoleate (11S-HPOD E) and (13R)-hydroperoxylinoleate (13R-HPODE) was pH-dependent and changed by [U-H-2]linoleic acid. Stopped-flow kinetic traces of linoleic and alpha- linolenic acids indicated catalytic lag times of similar to 45 ms, which we re followed by bursts of enzyme activity for similar to 60 ms and then by s teady state (k(cat) similar to 26 and similar to 47 s(-1), respectively). 1 1S-HPODE was isomerized by manganese lipoxygenase to 13R-HPODE and formed f rom linoleic acid at the same rates (k(cat) 7-9 s(-1)). Catalysis was accom panied by collisional quenching of the long wavelength fluorescence (640-68 5 nm) by fatty acid substrates and 13R-HPODE, Electron paramagnetic resonan ce (EPR) of native manganese lipoxygenase showed weak 6-fold hyperfine spli tting superimposed on a broad resonance indicating two populations of Mn-II bound to protein. The addition of linoleic acid decreased both components, and denaturation of the lipoxygenase liberated similar to 0.8 Mn2+ atoms/l ipoxygenase molecule. These observations are consistent with a mononuclear Mn-II center in the native state, which is converted during catalysis to an EPR silent Mn-III state. We propose that manganese lipoxygenase has kineti c and redox properties similar to iron lipoxygenases.