Radicals in anaerobic aromatic metabolism

本论文将以芳香梭菌厌氧芳香族代谢的两种酶为研究对象，研究自由基在酶催化中的作用。(1)苯甲酰辅酶A还原酶通过将电子传递耦合到ATP-水解酶来还原芳环。这种反应被认为类似于白桦树的还原反应。该模型意味着交替的单电子转移和质子化步骤，第一次电子转移的E‘O为-1.9V。将通过改变底物的电子特性、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索“Birch”假说。中间底物和/或酶自由基将通过EPF-、Endor-和FT-IR光谱技术进行检测。为了进行比较，将研究厌氧细菌的环缩酶，这种细菌被认为可以传递电子，而不耦合到ATP水解。(2)苯磷酸羧基酶催化磷酸苯对位羧化为4-羟基苯甲酸酯和14CO2/羧酸同位素交换。对氧和自由基捕捉剂敏感的酶将被提纯，并将用类似于苯甲酰辅酶A还原酶的方法来研究潜在的自由基反应机理。

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.