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 FeII 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 FeII 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.脂氧合酶（Lipoxygenases）是一类非血红素铁酶，可催化不饱和脂肪酸发生立体专一且区域专一的氢过氧化反应。针对大豆脂氧合酶的光谱学研究显示，该酶的亚铁形式为五配位与六配位物种的混合物，占比分别为40%与60%。底物的加入会使酶完全转变为六配位形式。研究人员构建了一系列第二配位层突变体（Q697E、Q697N、Q495A及Q495E），并通过晶体学解析了这些突变体的结构[Tomchick等(2001) 《Biochemistry》40, 7509−7517]。尽管该研究明确揭示了第一与第二配位层间的氢键相互作用在催化过程中的贡献，但未发现其与FeII配位环境的相关性。近期一项基于密度泛函理论（density-functional theory）的研究[Lehnert与Solomon(2002) 《J. Biol. Inorg. Chem.》8, 294−305]表明，涉及Asn694配体的配位柔性可通过氢键相互作用进行调控。本文中，我们采用圆二色光谱（CD spectroscopy）与磁圆二色光谱（MCD spectroscopy）对第二配位层突变体的溶液结构展开研究，因为这两种技术能够更灵敏地反映FeII体系的第一配位层配位状态。我们的实验数据表明，铁的配位环境与酶活性直接相关：能够形成五配位/六配位混合物的突变体活性更高。我们提出，弱结合配体Asn694与Gln697之间的氢键在调控Asn694的配位柔性过程中发挥关键作用，因此对酶反应活性的调控至关重要。