Spectroscopic characterization of soybean lipoxygenase-1 mutants: the role of second coordination sphere residues in the regulation of enzyme activity.

Lipoxygenases are non-heme iron enzymes, which catalyze the stereo- and regiospecific hydroperoxidation of unsaturated fatty acids. Spectroscopic studies on soybean lipoxygenase have shown that the ferrous form of the enzyme is a mixture of five- and six-coordinate species (40 and 60%, respectively). Addition of substrate leads to a purely six-coordinate form. A series of mutations in the second coordination sphere (Q697E, Q697N, Q495A, and Q495E) were generated, and the structures of the mutants were solved by crystallography [Tomchick et al. (2001) Biochemistry 40, 7509-7517]. While this study clearly showed the contribution of H-bond interactions between the first and the second coordination spheres in catalysis, no correlation with the coordination environment of the Fe(II) was observed. A recent study using density-functional theory [Lehnert and Solomon (2002) J. Biol. Inorg. Chem. 8, 294-305] indicated that coordination flexibility, involving the Asn694 ligand, is regulated via H-bond interactions. In this paper, we investigate the solution structures of the second coordination sphere mutants using CD and MCD spectroscopy since these techniques are more sensitive indicators of the first coordination sphere ligation of Fe(II) systems. Our data demonstrate that the iron coordination environment directly relates to activity, with the mutations that have the ability to form a five-coordinate/six-coordinate mixture being more active. We propose that the H-bond between the weak Asn694 ligand and the Gln697 plays a key role in the modulation of the coordination flexibility of Asn694, and thus, is crucial for the regulation of enzyme reactivity.