Department of Chemistry, Division of Biochemistry, BOKU, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.
J Inorg Biochem. 2010 Jun;104(6):648-56. doi: 10.1016/j.jinorgbio.2010.02.006. Epub 2010 Mar 3.
Catalase-peroxidases are the only heme peroxidases with substantial hydrogen peroxide dismutation activity. In order to understand the role of the redox chemistry in their bifunctional activity, catalatically-active and inactive mutant proteins have been probed in spectroelectrochemical experiments. In detail, wild-type KatG from Synechocystis has been compared with variants with (i) disrupted KatG-typical adduct (Trp122-Tyr249-Met275), (ii) mutation of the catalytic distal His123-Arg119 pair, and (iii) altered accessibility to the heme cavity (Asp152, Ser335) and modified charge at the substrate channel entrance (Glu253). A valuable insight into the mechanism of reduction potential (E degrees ') modulation in KatG has been obtained from the parameterization of the corresponding enthalpic and entropic components, determined from the analysis of the temperature dependence of E degrees '. Moreover, model structures of ferric and ferrous Synechocystis KatG have been computed and used as reference to analyze and discuss the experimental data. The results, discussed by reference to published resonance Raman data on the strength of the proximal iron-imidazole bond and catalytic properties, demonstrate that E degrees ' of the Fe(III)/Fe(II) couple is not strongly correlated with the bifunctional activity. Besides the importance of an intact Trp-Tyr-Met adduct, it is the architecture of the long and constricted main channel that distinguishes KatGs from monofunctional peroxidases. An ordered matrix of oriented water dipoles is important for H(2)O(2) oxidation. Its disruption results in modification of enthalpic and entropic contributions to E degrees ' that reflect reduction-induced changes in polarity, electrostatics, continuity and accessibility of solvent to the metal center as well as alterations in solvent reorganization.
过氧化氢酶过氧化物酶是唯一具有大量过氧化氢歧化活性的血红素过氧化物酶。为了了解氧化还原化学在其双功能活性中的作用，已经在光谱电化学实验中探测了催化活性和非活性突变体蛋白。详细地说，已经将来自集胞藻的野生型 KatG 与具有以下特征的变体进行了比较：（i）破坏了典型的 KatG 加合物（Trp122-Tyr249-Met275）；（ii）催化远端 His123-Arg119 对的突变；以及（iii）改变血红素腔的可及性（Asp152、Ser335）和修饰底物通道入口处的电荷（Glu253）。通过参数化相应的焓和熵分量，从 E°的温度依赖性分析中获得了对 KatG 还原电位（E°）调制机制的有价值的见解。此外，还计算了 Ferric 和 Ferrous Synechocystis KatG 的模型结构，并将其用作参考，以分析和讨论实验数据。讨论参考了发表的关于近端铁-咪唑键强度和催化性质的共振拉曼数据的结果表明，Fe（III）/Fe（II）对的 E°与双功能活性没有很强的相关性。除了完整的 Trp-Tyr-Met 加合物的重要性之外，长而狭窄的主通道的结构将 KatG 与单功能过氧化物酶区分开来。有序排列的定向水分子偶极子矩阵对于 H2O2 氧化很重要。其破坏导致 E°的焓和熵贡献的修饰，反映了还原诱导的极性、静电、溶剂连续性和对金属中心的可及性以及溶剂重组的变化。
