Mathé Christelle, Weill Claire O, Mattioli Tony A, Berthomieu Catherine, Houée-Levin Chantal, Tremey Emilie, Nivière Vincent
Laboratoire de Chimie et Biologie des Métaux, iRTSV-CEA Grenoble/CNRS/Université Joseph Fourier, 17 Avenue des Martyrs, 38054 Grenoble Cedex 9, France.
J Biol Chem. 2007 Jul 27;282(30):22207-16. doi: 10.1074/jbc.M700279200. Epub 2007 Jun 1.
Superoxide reductase is a novel class of non-heme iron proteins that catalyzes the one-electron reduction of O(2)(.) to H(2)O(2), providing an antioxidant defense in some bacteria. Its active site consists of an unusual non-heme Fe(2+) center in a [His(4) Cys(1)] square pyramidal pentacoordination. In this class of enzyme, the cysteine axial ligand has been hypothesized to be an essential feature in the reactivity of the enzyme. Previous Fourier transform infrared spectroscopy studies on the enzyme from Desulfoarculus baarsii revealed that a protonated carboxylate group, proposed to be the side chain of Glu(114), is in interaction with the cysteine ligand. In this work, using pulse radiolysis, Fourier transform infrared, and resonance Raman spectroscopies, we have investigated to what extent the presence of this Glu(114) carboxylic lateral chain affects the strength of the S-Fe bond and the reaction of the iron active site with superoxide. The E114A mutant shows significantly modified pulse radiolysis kinetics for the protonation process of the first reaction intermediate. Resonance Raman spectroscopy demonstrates that the E114A mutation results in both a strengthening of the S-Fe bond and an increase in the extent of freeze-trapping of a Fe-peroxo species after treatment with H(2)O(2) by a specific strengthening of the Fe-O bond. A fine tuning of the strength of the S-Fe bond by the presence of Glu(114) appears to be an essential factor for both the strength of the Fe-O bond and the pK(a) value of the Fe(3+)-peroxo intermediate species to form the reaction product H(2)O(2).
超氧化物还原酶是一类新型的非血红素铁蛋白，可催化将超氧阴离子（O₂⁻）单电子还原为过氧化氢（H₂O₂），在一些细菌中提供抗氧化防御。其活性位点由一个处于[His₄Cys₁]方锥五配位结构中的异常非血红素Fe²⁺中心组成。在这类酶中，半胱氨酸轴向配体被认为是酶反应活性的一个关键特征。先前对巴氏脱硫弧菌（Desulfoarculus baarsii）来源的该酶进行的傅里叶变换红外光谱研究表明，一个质子化的羧基基团（推测为Glu114的侧链）与半胱氨酸配体相互作用。在这项工作中，我们使用脉冲辐解、傅里叶变换红外光谱和共振拉曼光谱，研究了Glu114羧基侧链的存在在多大程度上影响S-Fe键的强度以及铁活性位点与超氧化物的反应。E114A突变体在第一个反应中间体的质子化过程中表现出显著改变的脉冲辐解动力学。共振拉曼光谱表明，E114A突变导致S-Fe键增强，并且在用H₂O₂处理后，通过特异性增强Fe-O键，Fe-过氧物种的冻结捕获程度增加。Glu114的存在对S-Fe键强度的微调似乎是Fe-O键强度以及Fe³⁺-过氧中间物种形成反应产物H₂O₂的pKa值的一个关键因素。
