A STUDY ON A NOVEL ENERGY-COUPLING PROTEIN COMPLEX THAT REDUCE FERREDOXIN BY NADH

一种通过NADH还原铁氧化还原蛋白的新型能量耦合蛋白复合物的研究

• 批准号: 09640771
• 负责人: SAEKI Kazuhiko
• 金额: $ 1.73万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09640771/
• 关键词: RHODOBACTER; FERREDOXIN; ELECTRON-TRANSPORT; NITROGEN-FIXATION; NADH; MEMBANE-PROTEIN-COMPLEX; 光合成細菌; rnf遺伝子

The rnf(rbodobacter nitrogen fixation)operon in Rhodobacter capsutatus is essential for nitrogen fixation under light. We showed that products of the rnf operon constitute a protein complex in chromatophore membrane and proposed that the complex has a chimerie construct of two subcomplexes, one transmembrane subcomplex similar to Na^+-translocating NADH quinone oxidoreductase and the other peripheral subcomptex similar to H^+-translocating NADH quinone oxidoreductase. Its function should : be energy-consuming NADH-ferredoxin oxidoreductase. 1) We revealed that the complex is also required for nitrogen fixation under dark anaerobic conditions using dimethylsulfoxide as a terminal electron acceptor this agrees that the Rnf complex functions without direct link to photosynthetic reaction center. 2) Presence of at least one Fe-S center in Rnf complex was identified by EPR analysis The cluster showed a novel signal, g=1 .84, at 10K with extremely low redox potential. Since other strong signals from succinate-quinone oxidoreductase interfered detailed analysis, we are currently seeking conditions and mutants to overcome this difficulty. 3) Escheriehia coil and four other non-diazotrophic bacteria possess homologous of the rnf operon. Over expression of the E.coil homologue in E.ccli seemed lethal. This E.coli operon did not complemented the rnf-null mutants of R.capsulatus. 4) Chemical crosslinking studies showed that RnfC subunit of the complex interacts closely with a 20K Dalton peptide, which is possibly RnfB or RnfA peptide.

冠状红杆菌中的rnf操纵子是光下固氮所必需的。我们发现RNF操纵子的产物在发色细胞膜上形成了一个蛋白质复合体，并提出该复合体具有两个亚复合体的嵌合体结构，一个类似于Na~+-转位NADH苯醌氧化还原酶的跨膜亚复合体，另一个类似于H~+-转位NADH苯醌氧化还原酶的外周亚复合体。它的功能应该是：耗能的NADH-铁氧还蛋白氧化还原酶。1)我们发现，在黑暗厌氧条件下，以二甲基亚砜为末端电子受体的RNF复合体也是固氮所必需的，这证实了RNF复合体的功能与光合作用反应中心没有直接联系。2)电子顺磁共振分析表明，RNF络合物中至少存在一个Fe-S中心，该簇在10K时出现一个新的信号，g=1.84，具有极低的氧化还原电势。由于来自琥珀酸醌氧化还原酶的其他强信号干扰了详细的分析，我们目前正在寻找条件和突变体来克服这一困难。3)大肠杆菌和其他四种非重氮菌具有Rnf操纵子的同源性。在E.ccli中过量表达E.cos同源物似乎是致命的。该操纵子并不能补充囊状杆菌rnf缺失的突变体。4)化学交联研究表明，该复合体的RnfC亚基与一个20K的Dalton多肽结合紧密，可能是RnfB或RnfA多肽。

项目成果

Saeki K: "The rnf gene products in Rhodobacter capsulatus play an essential role in nitrogen fixation under dark DMSO-dependent anaerobic respiratory conditins" Archive of Microbiology. 375 (in press). (1998)

Saeki K：“荚膜红杆菌中的 rnf 基因产物在黑暗 DMSO 依赖性厌氧呼吸条件下的固氮中发挥重要作用”微生物学档案。

K.Saeki: "The rnf gene products in Rhodobacter capsulatus play an essential role in nitrogen fixation during anaerobic DMSO-dependent growth in the dark" Archive of Microbiology. 169. 464-467 (1998)

K.Saeki：“荚膜红杆菌中的 rnf 基因产物在黑暗中厌氧 DMSO 依赖性生长过程中的固氮中发挥着重要作用”微生物学档案馆。

H.Kumagai and K.Saeki: ""Comparative and Functional Study on Rhodobacter capsulatus rnf Gene Products and Their Homologs"" in Biological Nitrogen Fixation for the 21st Century (C.Elmerich, A.Kondorosi & W.E.Newton eds.) Kluwer Academic Publishers, Dortrec

H.Kumagai 和 K.Saeki：“21 世纪生物固氮”中的“荚膜红杆菌 rnf 基因产物及其同源物的比较和功能研究”（C.Elmerich、A.Kondorosi）

C.Elmerich: "Biological Nitrogen Fixation for the 21st Century" Kluwer Academic Publishers,Dortrecht,Netherlands, 580 (1998)

C.Elmerich：“21 世纪的生物固氮”Kluwer 学术出版社，多特雷赫特，荷兰，580 (1998)

H.Kumagai, T.Fujiwara, H.Matsubara and K.Saeki: ""Membrane Localization, Topology and Mutual Stabilization of the rnfABC Gene Products in Rhodobacter capsulatus and Implications for A New Family of Energy-Coupling NADH-Oxidoreductases"" Biochemistry. 36.

H.Kumagai、T.Fujiwara、H.Matsubara 和 K.Saeki：“荚膜红杆菌中 rnfABC 基因产物的膜定位、拓扑结构和相互稳定性以及对能量耦合 NADH 氧化还原酶新家族的影响”生物化学。