Davis Mindy I, Orville Allen M, Neese Frank, Zaleski Jeffrey M, Lipscomb John D, Solomon Edward I
Department of Chemistry, Stanford University, Stanford, California 94305-5080, USA.
J Am Chem Soc. 2002 Jan 30;124(4):602-14. doi: 10.1021/ja011945z.
The geometric and electronic structure of the high-spin ferric active site of protocatechuate 3,4-dioxygenase (3,4-PCD) has been examined by absorption (Abs), circular dichroism (CD), magnetic CD (MCD), and variable-temperature-variable-field (VTVH) MCD spectroscopies. Density functional (DFT) and INDO/S-CI molecular orbital calculations provide complementary insight into the electronic structure of 3,4-PCD and allow an experimentally calibrated bonding scheme to be developed. Abs, CD, and MCD indicate that there are at least seven transitions below 35 000 cm(-1) which arise from tyrosinate ligand-to-metal-charge transfer (LMCT) transitions. VTVH MCD spectroscopy gives the polarizations of these LMCT bands in the principal axis system of the D-tensor, which is oriented relative to the molecular structure from the INDO/S-CI calculations. Three transitions are associated with the equatorial tyrosinate and four with the axial tyrosinate. This large number of transitions per tyrosinate is due to the pi and importantly the sigma overlap of the two tyrosinate valence orbitals with the metal d orbitals and is governed by the Fe-O-C angle and the Fe-O-C-C dihedral angles. The previously reported crystal structure indicates that the Fe-O-C angles are 133 degrees and 148 degrees for the equatorial and axial tyrosinate, respectively. Each tyrosinate has transitions at different energies with different intensities, which correlate with differences in geometry that reflect pseudo-sigma bonding to the Fe(III) and relate to reactivity. These factors reflect the metal-ligand bond strength and indicate that the axial tyrosinate-Fe(III) bond is weaker than the equatorial tyrosinate-Fe(III) bond. Furthermore, it is found that the differences in geometry, and hence electronic structure, are imposed by the protein. The consequences to catalysis are significant because the axial tyrosinate has been shown to dissociate upon substrate binding and the equatorial tyrosinate in the enzyme-substrate complex is thought to influence asymmetric binding of the chelated substrate moiety via a strong trans influence which activates the substrate for reaction with O2.
通过吸收光谱（Abs）、圆二色光谱（CD）、磁圆二色光谱（MCD）以及变温变场（VTVH）MCD光谱，对原儿茶酸3,4-双加氧酶（3,4-PCD）的高自旋铁活性位点的几何结构和电子结构进行了研究。密度泛函（DFT）和INDO/S-CI分子轨道计算为3,4-PCD的电子结构提供了互补的见解，并有助于制定经过实验校准的成键方案。Abs、CD和MCD表明，在35000 cm⁻¹以下至少有七个跃迁，这些跃迁源自酪氨酸盐配体到金属的电荷转移（LMCT）跃迁。VTVH MCD光谱给出了这些LMCT带在D张量主轴系统中的极化情况，该主轴系统相对于INDO/S-CI计算得出分子结构定向。三个跃迁与赤道面酪氨酸盐相关，四个与轴向酪氨酸盐相关。每个酪氨酸盐的跃迁数量众多，这是由于两个酪氨酸盐价轨道与金属d轨道的π重叠，更重要的是σ重叠，并且受Fe-O-C角和Fe-O-C-C二面角的控制。先前报道的晶体结构表明，赤道面和轴向酪氨酸盐的Fe-O-C角分别为133°和148°。每个酪氨酸盐在不同能量下具有不同强度的跃迁，这与反映与Fe(III)的假σ键合以及与反应性相关的几何差异相关。这些因素反映了金属-配体键强度，并表明轴向酪氨酸盐-Fe(III)键比赤道面酪氨酸盐-Fe(III)键弱。此外，发现几何结构以及因此的电子结构差异是由蛋白质造成的。对催化作用的影响很大，因为已表明轴向酪氨酸盐在底物结合时会解离，并且酶-底物复合物中的赤道面酪氨酸盐被认为通过强烈的反位影响来影响螯合底物部分的不对称结合，从而激活底物与O₂反应。
