Flavodoxin 1 of Azotobacter vinelandii: Characterization and role in electron donation to purified assimilatory nitrate reductase
Flavodoxin 1 of Azotobacter vinelandii: Characterization and role in electron donation to purified assimilatory nitrate reductase
复制标题
DOI：
10.1042/bj3170103
复制
发表时间：
1996-07-01
影响因子：
4.1
通讯作者：
Eady, RR
中科院分区：
文献类型：
--
作者：
Gangeswaran, R;Eady, RR
以硝酸根为氮源，利用单Q柱FPLC分离得到黄伏氧毒素1 （AvFld 1）和黄伏氧毒素2 （AvFld 2）。两种蛋白在SDS/PAGE上呈单条带迁移。avfield 1大约是。在未解析黄氧还毒素混合物中，AvFld 2的含量是AvFld 2的5倍。n端氨基酸分析表明，AvFld 2序列对应于氮酶电子供体nif F基因产物。这些序列还表明，这些物种对应于从密切相关的细菌固氮菌（Azotobacter choococcum）的n -2培养物中分离出的黄毒毒素Fld A和Fld B [Bagby， Barker, Hill， Eady和Thorneley (1991) Biochem]。[j].科学通报，1999,19(2):1 - 4。电喷雾质谱法分别测定了19430+/-3和19533+/-5多肽的M(r)值。p -31核磁共振测量表明，除了与FMN相关的磷酸盐（δ = -136.3 p.p.m.和-135.48 p.p.m.）外，AvFld 1在δ = -142.1 p.p.m.和AvFld 2在δ = -138.59 p.p.m.处存在亚化学计量量的FMN信号。这些似乎来自不稳定的物种，因为它们在进一步的蛋白质操作中很容易丢失。AvFld 1和AvFld 2的半醌-对苯二酚氧化还原对的中点电位分别为-330 mV和-493 mV，但只有AvFld 1能够给纯化的葡萄树吸收性硝酸盐还原酶提供电子，催化将硝酸盐还原为亚硝酸盐。从NH4+培养的细胞中分离的黄氧还蛋白（Fld 3）作为电子供体的速率也只有AvFld 1的一半，而从A. chroococum中分离的铁氧还蛋白1则没有。
Flavodoxins synthesized by Azotobacter vinelandii strain UW 136 during growth on nitrate as nitrogen source were separated by FPLC on a Mono Q column into two species, flavodoxin 1 (AvFld 1) and flavodoxin 2 (AvFld 2). Both proteins migrated as single bands on SDS/PAGE. AvFld 1 was approx. 5-fold more abundant than AvFld 2 in the unresolved flavodoxin mixture. N-terminal amino acid analysis showed the sequence of AvFld 2 to correspond to the nif F gene product, an electron donor to nitrogenase. The sequences also show that these species corresponded to the flavodoxins Fld A and Fld B isolated from N-2-grown cultures of the closely related organism Azotobacter chroococcum [Bagby, Barker, Hill, Eady and Thorneley (1991) Biochem. J. 277, 313-319]. Electrospray mass spectrometry gave M(r) values for the polypeptides of 19430+/-3 and 19533+/-5 respectively. P-31-NMR measurements showed that in addition to the phosphate associated with the FMN (delta = -136.3 p.p.m. and -135.48 p.p.m.), AvFld 1 had a signal at delta = -142.1 p.p.m. and AvFld 2 at delta = -138.59 p.p.m. present in substoichiometric amounts with FMN. These appeared to arise from unstable species since they were readily lost on further manipulation of the proteins. The mid-point potentials of the semiquinone-hydroquinone redox couples were -330 mV and -493 mV for AvFld 1 and AvFld 2 respectively, but only AvFld 1 was competent in donating electrons to the purified assimilatory nitrate reductase of A. vinelandii to catalyse the reduction of nitrate to nitrite. Flavodoxin isolated from NH4+-grown cells (Fld 3) also functioned as electron donor at half the rate of AvFld 1, but ferredoxin 1 from A. chroococcum did not.