Radicals in anaerobic aromatic metabolism

自由基在酶催化中的作用将用细菌Thauera aromatica中的两种厌氧芳香代谢酶进行研究。(1)苯甲酰辅酶A还原酶通过偶联电子转移和ATP水解还原芳香环。该反应被认为类似于Birch还原。该模型意味着交替的单电子转移和质子化步骤，其中第一电子转移的E 'o为-1.9 V。将通过改变底物的电子特性、通过使用抑制剂和同位素以及通过检测动力学同位素效应来探测“Birch”假设。将通过EPF-、ENDOR-和FT-IR光谱技术检测中间底物和/或酶自由基。为了比较，将研究来自厌氧细菌的环还原酶，其被认为在不与ATP水解偶联的情况下转移电子。(2)磷酸苯酯羧化酶催化磷酸苯酯对位羧化为4-羟基苯甲酸酯和14 CO2/羧酸盐同位素交换。将纯化对氧和自由基捕获剂敏感的酶，并通过与苯甲酰-CoA还原酶所述类似的方法研究潜在的自由基反应机制。

The role of radicals in enzyme catalysis will be studied with two enzymes of anaerobic aromatic metabolism in the bacterium Thauera aromatica. (1) Benzoyl-CoA reductase reduces the aromatic ring by coupling electron transfer to ATP-hydrolysis. This reaction is thought to be similar to a Birch reduction. This model implies alternate one-electron transfer and protonation steps, with E'o of the first electron transfer of -1.9 V. The 'Birch' hypothesis will be probed by altering the electronic characteristics of the substrate, by the use of of inhibitors and isotopes and by the detection of kinetic isotope effects. Intermediate substrates and/or enzyme radicals will be detected by EPF-, ENDOR- and FT-IR spectroscopic techniques. For comparison, ring reducing enzymes will be studied from anaerobic bacteria which are thought to transfer electrons without coupling to ATP hydrolysis. (2) Phenylphosphate carboxylase catalyzes phenylphosphate para-carboxylation to 4-hydroxybenzoate and 14CO2/carboxylate isotope exchange. The enzyme which is sensitive towards oxygen and radical trapping agents will be purified and the potential radical reaction mechanism will be investigated by similar methods as described for benzoyl-CoA reductase.