Clay Michael D, Yang Tran-Chin, Jenney Francis E, Kung Irene Y, Cosper Christopher A, Krishnan Rangan, Kurtz Donald M, Adams Michael W W, Hoffman Brian M, Johnson Michael K
Department of Chemistry, University of Georgia, Athens, Georgia 30602, USA.
Biochemistry. 2006 Jan 17;45(2):427-38. doi: 10.1021/bi052034v.
We have added cyanide to oxidized 1Fe and 2Fe superoxide reductase (SOR) as a surrogate for the putative ferric-(hydro)peroxo intermediate in the reaction of the enzymes with superoxide and have used vibrational and ENDOR spectroscopies to study the properties of the active site paramagnetic iron center. Addition of cyanide changes the active site iron center in oxidized SOR from rhombic high-spin ferric (S = 5/2) to axial-like low-spin ferric (S = 1/2). Low-temperature resonance Raman and ENDOR data show that the bound cyanide adopts three distinct conformations in Fe(III)-CN SOR. On the basis of 13CN, C15N, and 13C15N isotope shifts of the Fe-CN stretching/Fe-C-N bending modes, resonance Raman studies of 1Fe-SOR indicate one near-linear conformation (Fe-C-N angle approximately 175 degrees) and two distinct bent conformations (Fe-C-N angles <140 degrees). FTIR studies of 1Fe-SOR at ambient temperatures reveals three bound C-N stretching frequencies in the oxidized (ferric) state and one in the reduced (ferrous) state, indicating that the conformational heterogeneity in cyanide binding is a characteristic of the ferric state and is not caused by freezing-in of conformational substates at low temperature. 13C-ENDOR spectra for the 13CN-bound ferric active sites in both 1Fe- and 2Fe-SORs also show three well-resolved Fe-C-N conformations. Analysis of the 13C hyperfine tensors for the three substates of the 2Fe-SOR within a simple heuristic model for the Fe-C bonding gives values for the Fe-C-N angles in the three substates of ca. 123 degrees (C3) and 133 degrees (C2), taking a reference value from vibrational studies of 175 degrees (C1 species). Resonance Raman and ENDOR studies of SOR variants, in which the conserved glutamate and lysine residues in a flexible loop above the substrate binding pocket have been individually replaced by alanine, indicate that the side chains of these two residues are not involved in direct interaction with bound cyanide. The implications of these results for understanding the mechanism of SOR are discussed.
我们已将氰化物添加到氧化态的单铁和双铁超氧化物还原酶（SOR）中，以替代该酶与超氧化物反应中假定的铁（氢）过氧中间体，并使用振动光谱和电子核双共振（ENDOR）光谱研究活性位点顺磁性铁中心的性质。添加氰化物会使氧化态SOR中的活性位点铁中心从菱形高自旋铁（S = 5/2）变为类轴向低自旋铁（S = 1/2）。低温共振拉曼光谱和ENDOR数据表明，结合的氰化物在Fe(III)-CN SOR中呈现三种不同的构象。基于Fe-CN伸缩/Fe-C-N弯曲模式的13C、15N和13C15N同位素位移，单铁SOR的共振拉曼研究表明存在一种近线性构象（Fe-C-N角约为175度）和两种不同的弯曲构象（Fe-C-N角<140度）。室温下单铁SOR的傅里叶变换红外光谱（FTIR）研究揭示，氧化态（铁态）有三种结合的C-N伸缩频率，还原态（亚铁态）有一个，这表明氰化物结合中的构象异质性是铁态的特征，并非由低温下构象亚态的冻结所致。单铁和双铁SOR中13CN结合的铁态活性位点的13C-ENDOR光谱也显示出三种分辨率良好的Fe-C-N构象。在一个简单的Fe-C键启发式模型中，对双铁SOR三种亚态的13C超精细张量进行分析，得出三种亚态中Fe-C-N角的值约为123度（C3）和133度（C2），以振动研究中的175度（C1物种）为参考值。对SOR变体进行的共振拉曼光谱和ENDOR研究表明，底物结合口袋上方柔性环中保守的谷氨酸和赖氨酸残基已分别被丙氨酸取代，这两个残基的侧链不参与与结合氰化物的直接相互作用。本文讨论了这些结果对理解SOR机制的意义。
