PURIFICATION OF A 2ND ALTERNATIVE NITROGENASE FROM A NIFHDK DELETION STRAIN OF AZOTOBACTER-VINELANDII
PURIFICATION OF A 2ND ALTERNATIVE NITROGENASE FROM A NIFHDK DELETION STRAIN OF AZOTOBACTER-VINELANDII
复制标题
DOI：
10.1128/jb.170.1.27-33.1988
复制
发表时间：
1988-01-01
影响因子：
3.2
通讯作者：
BISHOP, PE
中科院分区：
文献类型：
--
作者：
CHISNELL, JR;PREMAKUMAR, R;BISHOP, PE
从一株缺失nifHDK的棕色固氮菌中分离纯化了第二个可供选择的固氮酶复合体(固氮酶3)。该活性复合体由两个组分组成，即二氮原酶3和二氮原酶还原酶3。二氮原酶3含有两个蛋白质亚基(α，MR 58000和β，MR 50,000)，它们至少组装成活性构型：α2β2(二氮酶3S)和α1β2(二氮酶3F)。每个MR 216,000个二固氮酶3S含有24个Fe和18个酸不稳定的S2-离子；每个MR 158,000个固氮酶3F含有11个Fe和9个酸不稳定的S2-离子。二氮原酶还原酶3由两个相同的Mr(32,500)蛋白质亚基组成，每个Mr 65,000含有4个Fe和4个酸不稳定的S2-离子。在二维凝胶上，固氮酶3复合体的蛋白质亚单位与四种最初被指定为N2ase B的可抑制Mo-、V-和NH4+的蛋白质共迁移：固氮酶被假设存在于1980年首次描述的替代的氮气固定系统中(P.E.Bishop，D.M.L.Jarlenski和D.R.Hetherington，Proc.娜塔莉。阿卡德。SCI。美国77：7342-7346,1980)。中子活化分析表明，固氮酶3缺乏大量的Mo、V、Cr、Re和W，但在固氮酶3S和固氮酶3F中发现了一些锌。底物还原效率模式为H+>；n2>；C2H2。氮气还原的最大比活力为每毫克蛋白质(脱氮酶3S)每分钟38nmoNH3。固氮酶3对O2极为敏感，在冷冻和解冻过程中不能重复地保持活性。
A second alternative nitrogenase complex (nitrogenase 3) was purified from a nifHDK deletion strain of Azotobacter vinelandii. The active complex is made up of two components, dinitrogenase 3 and dinitrogenase reductase 3. Dinitrogenase 3 contains two protein subunits (.alpha., Mr 58,000, and .beta., Mr 50,000) which assemble into at least to active configurations: .alpha.2.beta.2 (dinitrogenase 3S) and .alpha.1.beta.2 (dinitrogenase 3F). Dinitrogenase 3S contains 24 Fe and 18 acid-labile S2- ions per Mr 216,000, and dinitrogenase 3F contains 11 Fe and 9 acid-labile S2- ions per Mr 158,000. Dinitrogenase reductase 3 is composed of two protein subunits of identical Mr (32,500) and contains four Fe and four acid-labile S2- ions per Mr 65,000. On two-dimensional gels, the protein subunits of the nitrogenase 3 complex comigrated with the four Mo-, V-, and NH4+-repressible proteins originally designated as N2ase B: the nitrogenase hypothesized to exist in the alternative N2 fixation system first described in 1980 (P.E. Bishop, D.M.L. Jarlenski, and D.R. Hetherington, Proc. Natl. Acad. Sci. USA 77:7342-7346, 1980). Neutron activation analysis indicated that the nitrogenase 3 lacked significant amounts of Mo, V, Cr, Re, and W. Some Zn, however, was found in the dinitrogenase 3S and dinitrogenase 3F preparations. The pattern of substrate reduction efficiency was H+ > N2 > C2H2. The maximum specific activity found for N2 reduction was 38 nmol of NH3 per min per mg of protein (dinitrogenase 3S). Nitrogenase 3 was found to be extremely sensitive to O2, and activities could not be reproducibly maintained during freezing and thawing.