Department of Kinetics of Chemical and Biological Processes, Institute of Problems of Chemical Physics, Russian Academy of Sciences, Street Academician Semenov, 1., Chernogolovka, 142432, Moscow Region, Russia.
World J Microbiol Biotechnol. 2018 Nov 28;34(12):184. doi: 10.1007/s11274-018-2564-y.
Nitrogen fixation is one of the major biogeochemical contributions carried out by diazotrophic microorganisms. The goal of this research is study of posttranslational modification of dinitrogenase reductase (Fe protein), the involvement of malate and pyruvate in generation of reductant in Rhodospirillum rubrum. A procedure for the isolation of the Fe protein from cell extracts was developed and used to monitor the modification of the Fe protein in vivo. The subunit pattern of the isolated the Fe protein after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was assayed by Western blot analysis. Whole-cell nitrogenase activity was also monitored during the Fe protein modification by gas chromatograpy, using the acetylene reduction assay. It has been shown, that the addition of fluoroacetate, ammonia and darkness resulted in the loss of whole-cell nitrogenase activity and the in vivo modification of the Fe protein. For fluoroacetate, ammonia and darkness, the rate of loss of nitrogenase activity was similar to that for the Fe protein modification. The addition of NADH and reillumination of a culture incubated in the dark resulted in the rapid restoration of nitrogenase activity and the demodification of the Fe protein. Fluoroacetate inhibited the nitrogenase activity of R. rubrum and resulted in the modification of the Fe protein in cells, grown on pyruvate or malate as the endogeneous electron source. The nitrogenase activity in draTG mutant (lacking DRAT/DRAG system) decreased after the addition of fluoroacetate, but the Fe protein remained completely unmodified. The results showed that the reduced state of cell, posttranslational modifications of the Fe protein and the DRAT/DRAG system are important for nitrogenase activity and the regulation of nitrogen fixation.
固氮作用是由固氮微生物进行的主要生物地球化学贡献之一。本研究的目的是研究二氮酶还原酶（Fe 蛋白）的翻译后修饰，以及苹果酸和丙酮酸在红螺菌（Rhodospirillum rubrum）中产生还原剂的参与。开发了一种从细胞提取物中分离 Fe 蛋白的程序，并用于监测 Fe 蛋白在体内的修饰。通过 Western blot 分析测定了分离的 Fe 蛋白在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳后的亚基模式。通过使用乙炔还原测定法，通过气相色谱法监测整个细胞氮酶活性，同时监测 Fe 蛋白修饰。已经表明，氟乙酸盐、氨和黑暗的添加导致整个细胞氮酶活性的丧失和 Fe 蛋白的体内修饰。对于氟乙酸盐、氨和黑暗，氮酶活性的丧失速率与 Fe 蛋白修饰的速率相似。添加 NADH 和重新光照在黑暗中培养的培养物导致氮酶活性的快速恢复和 Fe 蛋白的去修饰。氟乙酸盐抑制红螺菌的氮酶活性，并导致在以丙酮酸或苹果酸作为内源电子源生长的细胞中 Fe 蛋白的修饰。在添加氟乙酸盐后，缺乏 DRAT/DRAG 系统的 draTG 突变体（）的氮酶活性降低，但 Fe 蛋白保持完全未修饰。结果表明，细胞的还原状态、Fe 蛋白的翻译后修饰和 DRAT/DRAG 系统对于氮酶活性和氮固定的调节很重要。
