N_2和NH_4^+培养下粪产碱菌固氮酶钼铁蛋白的合成及其特性

Synthesis and Properties of MoFe Protein of Nitrogenase from N_2-grown and NH_4^+-grown Alcaligenes faecalis

应用柱层析和制备电泳分别将N_2和 NH_4^+培养的粪产碱菌固氮酶钼铁蛋白(Af 1和Af 1)分离并纯化,两者理化性质十分相似。分子量分别为226 kD和 222 kD;α亚基和β亚基分子量分别为57 kD和60 kD;氨基酸种类相同,总残基数分别为1790和1750;每分子Af 1和Af 1均含有2个原子Mo和32个原子 Fe;金属原子簇的氧化还原当量数为6。Af 1的比活性为 1477 nmolC_2H_4 mg^(-1)protein min^(-1),Af 1无活性;分子结构两者有差异。

It is well known that NH_4^+ inhibits nitroge-nase activity in diazotrophic bacteria. It alsobas been reported that the biosynthesis of nitro-genase is inhibited by NH_4^+. However, we havefound that the nitrogenase might be synthesisedin the NH_4^+-grown cells from Alcaligenes faecalisstrain A-15 by using serological method and ro-ket, crossed and line immunoelectrophoresis.But these techniques could not be able to identi-fy the nitrogenase biosynthesis and its propertiessufficiently. The purpose of this paper is to elu-cidate the synthesis of MoFe protein of nitrogenasefrom N_2-grown and NH_4^+-grown cells and to com-pare their differences. A. faecalis A--15 isolated from rice root wasused and were grown in N-free and N-combined(45～30 mmo1/L of NH_4^+ ) culture medium respec-tively. Thc MoFe protein from N_2-grown cells(Afl) and NH_4^+-grown cells (Af* 1) were purifiedby passing the cell-free crude extract through theDEAE-cellulose column and Sephacryl S-300columm for gel filtration and then through thepreparative electrophoresis. The specific activi-ty of Afl is 1477 nmol C_2H_4 mg^(-1) protein min^(-1).No activity was detected from Af*1. Both Afland Af*1 were homogeneous as shown by gel elec-trophoretogram. The amino acid composition,metal content (Mo and Fe), molecular weight,UV-visible spectrum, Fluorescence and Circu-lar dichroism spectrum were measured by rou-tine methods. The redox equivalents were titra-ted with thionine and measured by fluorescencespectrophotometer and CD spectrograph. Both Afl and Af*1 contained 2 Mo and 32 Feper molecule. The molecular weight for Aflwas 226 kD and for Af*1 was 222 kD. The ami-no acid residues composition was 1790 for Afland 1750 for Af*1. And for both of them the aci-dic residues were twice as many as the basic ones.Both MoFe protein were composed of 2 kinds ofsubunits. which were 57 kD and 60 kD respec-tively. Estimating with their molecular weight.both MoFe protein wcre α_2β_2 tetramer. BothMoFe proteins had a maximum absorption at277 nm and a broad absorption band from 350--700 nm. A small shoulder appeared at 420nm when the proteins were exposed to oxygen for20 min (Fig. 3). By aid of fluorescence and circular dichroism(CD) spectrum, thionine was used as an oxidantto titrate both Afl and Af*1. It was shown thatboth MoFe protein could be oxidized by thioninewith 6 redox equivalents. And the oxidationprocesses were divided in two steps which were 4and 2 equivalents respectively (Fig. 7, 8). It issuggested that both of them contained 2 kindsof metal clusters: 4 P-clusters and 2 M-clusters. Afl and Af*1 differed from each other onlyat the CD spectra (Fig. 4,5), suggesting their mo-lecular structure might be different.