Kinetic, spectroscopic, and structural investigations of the soybean lipoxygenase-1 first-coordination sphere mutant, Asn694Gly.

ABSTRACT : In wild-type soybean LO-1 (WT sLO-1), Asn694 is a weak sixth ligand that is thought to becritical for enzymatic catalysis. In this investigation, N694G sLO-1 was studied to probe its contributionat this sixth ligand position to the kinetic and spectroscopic properties. The k cat value of N694G is ˘230times lower than that of WT sLO-1 at 25°C, which can be partially explained by a lowered reductionpotential of the iron as seen as a shift in the visible ligand-to-metal charge-transfer band ( i max ) 410 nmfor N694G and i max ) 425 nm for WT sLO-1). This conclusion was supported by a faster rate of oxidationof N694G by the product than that of WT sLO-1 (k 2 ) 606 s -1 for N694G and k 2 ) 349 s -1 for WTsLO-1). These results suggest a stronger ligand at the active site iron than the native Asn694, which isconfirmed to be a water bound to the Fe(II) in the crystal structure. This produces a six-coordinate circulardichroism/magnetic circular dichroism (CD/MCD) spectra for ferrous N694G and an intermediate rhombicelectron paramagnetic resonance (EPR) signal for ferric N694G. The EPR spectrum and its pH dependencesuggest that the coordination environment of ferric N694G contains one hydroxide and one water. On thebasis of both kinetic and structural factors, we propose that the Asn694 water-derived ligand would likelybe a hydroxide and the active site, water-derived ligand a water in the ferric state, hence lowering thereaction rate of N694G more than would be expected from the lowered reduction potential alone.Lipoxygenase (LO) is a mononuclear, non-heme irondioxygenase that catalyzes the incorporation of molecularoxygen into a 1,4-cis,cis-pentadiene fatty acid to formhydroperoxide products. In plants, soybean LO isoform-1(sLO-1)