The key reaction for carbon dioxide fixation in reductive TCA cycle

为了阐明还原性TCA循环在嗜热氢化细菌——嗜热氢化菌中是有效的，我们演示了以下内容：1。铁氧还蛋白的纯化与表征依次采用无细胞提取液纯化铁氧还蛋白。Q-Sepharose， DEAE-5PW和Superdex 75。对蛋白进行了表征。丙酮酸合成酶的纯化和表征丙酮酸合成酶的纯化是通过将无细胞提取物依次应用于DEAE-Sepharose CL-6B、PA-QA、Hydroxyapatite和Superdex 200进行。并对该蛋白进行了表征。铁氧还蛋白对2-氧葡萄糖酸合酶或丙酮酸合酶的反应性第一步，沿CO_2演化方向进行反应；两种酶都与铁氧还蛋白反应。在下一步中，还原的铁氧还蛋白被用作还原剂来显示羧化反应。然而，由于辅酶a化合物的不稳定性，只发生了少量的羧化反应。最后，通过酶促反应（ATP：柠檬酸裂解酶和琥珀酰辅酶a合成酶）产生辅酶a化合物。通过该系统检测了羧基化反应，并确定了预期产物。

In order to clarify that reductive TCA cycle is operative in a thermophilic hydrogenxidizing bacterium, Hydrogenobater thermophilus, the followings were demonstrated.1.Purification and characterization of ferredoxinPurification of ferredoxin was performed by applying cell-free extract successively. to Q-Sepharose, DEAE-5PW,and Superdex 75. Characterization of the prorein was also perfprmed.2.Purification and characterization of pyruvate synthasePurification of pyruvate synthase was performed by applying cell-free extract successively to DEAE-Sepharose CL-6B,PA-QA,Hydroxyapatite, and Superdex 200. Characterization of the protein was also performed.3.Reactivity of ferredoxin toward 2-oxoglutarate synthase or pyruvate synthaseIn the first step, reaction was performed toward CO_2 evolution direction ; both enzymes reacted with ferredoxin. In the next step, reduced ferredoxin was used as a reductant to show the carboxylation reaction. However, only a little carboxylation reaction occurred probably due to the instability of CoA-compounds. Finally, CoA-compounds were produced through enzymatic reactions ( ATP : citrate lyase and succinyl-CoA synthetase ) . By this system carboxylation reaction was detected and also the expected products were identified.