The key reaction for carbon dioxide fixation in reductive TCA cycle

为了阐明还原TCA循环在一株嗜热性氢氧化菌--嗜热氢杆菌中的作用，进行了以下研究：1.铁氧还蛋白的纯化和表征。Q-琼脂糖凝胶、DEAE-5PW和Superdex 75。2.丙酮酸合成酶的纯化和鉴定依次用DEAE-SepharoseCL-6B、PA-QA、羟基磷灰石和Superdex 200的无细胞提取液进行丙酮酸合成酶的纯化。3.铁氧还蛋白与2-氧戊二酸合成酶或丙酮酸合成酶的反应在第一步中，反应朝着CO_2析出方向进行，两种酶都与铁氧还蛋白反应。下一步，用还原的铁还蛋白作为还原剂来显示羧化反应。然而，由于CoA-化合物的不稳定性，可能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酸辅酶A合成酶)合成辅酶A-化合物。利用该体系对羧化反应进行了检测，并对预期产物进行了鉴定。

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.