Helton M E, Pacheco A, McMaster J, Enemark J H, Kirk M L
Department of Chemistry, The University of New Mexico, Albuquerque 87131-1096, USA.
J Inorg Biochem. 2000 Jul 1;80(3-4):227-33. doi: 10.1016/s0162-0134(00)00082-9.
Temperature-dependent magnetic circular dichroism (MCD) spectroscopy has been used for the first time to probe the electronic structure of the Mo active site in sulfite oxidase (SO). The enzyme was poised in the catalytically relevant [Mo(V):Fe(II)] state by anaerobic reduction of the enzyme with the natural substrate, sulfite, in the absence of the physiological oxidant cytochrome c. The [Mo(V):Fe(II)] state is of particular importance, as it is proposed to be a catalytic intermediate in the oxidative half reaction, where SO is reoxidized to the resting [Mo(VI):Fe(III)] state by two sequential one-electron transfers to cytochrome c. The MCD spectrum of the enzyme shows no charge transfer transitions below approximately 17000 cm(-1). This has been interpreted to result from (1) a severe reduction in ene-1,2-dithiolate sulfur in-plane and out-of-plane p orbital mixing, (2) a decrease in the dithiolate sulfur out-of-plane p-Mo d(xy) orbital overlap, and (3) an orthogonal orientation between the vertical cysteine sulfur p (perpendicular to the Mo-Scys sigma-bond) and Mo d(xy) orbitals. The spectroscopically determined cysteine sulfur p-Mo d(xy) bonding scheme in the [Mo(V):Fe(II)] state is consistent with the crystallographically determined O-Mo-Scys-C dihedral angle of approximately 90 degrees and precludes a covalent interaction between the vertical cysteine sulfur p orbital and Mo d(xy), effectively decoupling the cysteine from an effective through-bond electron transfer pathway. We have tentatively assigned a 22250 cm(-1) positive C-term feature in the MCD as the cysteine S(sigma)-->Mo d(xy) charge transfer that becomes allowed by a combination of configuration interaction and low-symmetry; however, the orbital overlap is anticipated to be quite small due to the near orthogonality of these orbitals. Therefore, we propose that the primary role of the coordinated cysteine is to decrease the effective nuclear charge on Mo by charge donation to the metal, statically poising the active site at more negative reduction potentials during electron transfer (ET) regeneration. Finally, the results of this study are consistent with the pyranopterin ene-1,2-dithiolate acting to couple the Mo site into efficient superexchange pathways for ET regeneration following oxygen atom transfer to the substrate.
温度依赖型磁圆二色性（MCD）光谱首次被用于探究亚硫酸盐氧化酶（SO）中钼活性位点的电子结构。在没有生理氧化剂细胞色素c的情况下，用天然底物亚硫酸盐对该酶进行厌氧还原，使酶处于催化相关的[Mo(V):Fe(II)]状态。[Mo(V):Fe(II)]状态尤为重要，因为它被认为是氧化半反应中的催化中间体，在此过程中，SO通过两个连续的单电子转移给细胞色素c而被重新氧化为静止的[Mo(VI):Fe(III)]状态。该酶的MCD光谱在约17000 cm⁻¹以下没有电荷转移跃迁。这被解释为是由于：（1）ene-1,2-二硫醇盐硫在平面内和平面外的p轨道混合严重减少；（2）二硫醇盐硫平面外的p-Mo d(xy)轨道重叠减少；（3）垂直的半胱氨酸硫p轨道（垂直于Mo-Scys σ键）和Mo d(xy)轨道之间呈正交取向。光谱测定的[Mo(V):Fe(II)]状态下的半胱氨酸硫p-Mo d(xy)键合方案与晶体学测定的O-Mo-Scys-C二面角约为90度一致，并且排除了垂直的半胱氨酸硫p轨道与Mo d(xy)之间的共价相互作用，有效地使半胱氨酸与有效的通过键电子转移途径解耦。我们初步将MCD中22250 cm⁻¹的正C项特征归为半胱氨酸S(σ)→Mo d(xy)电荷转移，这种电荷转移因组态相互作用和低对称性的组合而变得允许；然而，由于这些轨道近乎正交，预计轨道重叠相当小。因此，我们提出配位半胱氨酸的主要作用是通过向金属供电荷来降低钼上的有效核电荷，在电子转移（ET）再生过程中使活性位点静态地处于更负的还原电位。最后，本研究结果与吡喃蝶呤ene-1,2-二硫醇盐在氧原子转移到底物后将钼位点耦合到有效的超交换ET再生途径中的作用一致。
