Under a suitable condition of crystallization, dark brown short rhombohedron crystals could be obtained from nitrogenase MnFe protein purified from a mutant UW3 of Azotobacter vinelandii Lipmann grown in Mn-containing but Mo- and NH3-free medium. The possibility of crystallization, and number, size and quality of crystals were obviously dependent on concentrations of NaCl, MgCl2, PEG 8000,Tris and Hepes buffer and on methods for crystallization. PEG concentration affected on the shape of the crystals. The optimal, concentrations of the chemicals for crystallization of MnFe protein were slightly different from those for crystallization of Delta nifZ MoFe protein from a nifZ deleted strain of Azotobacter vinelandii. SDS-PAGE showed that the protein from the dissolved crystals was almost the same as MnFe protein before crystallization, indicating that the crystal was formed from MnFe protein.
Under a given condition of crystallization, dark brown short rhombohedron crystals could be obtained from Δ nifZ MoFe protein purified from a nifZ deleted mutant strain of Azotobacter vinelandii Lipmann. Systematic studies on the effect of concentrations of PEG 8000,MgCl 2, NaCl,Tris and buffer pH on the crystallization and crystal growth of the protein showed that the protein could not be crystallized in lower concentrations of the chemicals and lower buffer pH. A large amount of smaller crystals of the protein appeared in a week with gradual increasing in the chemical concentrations and pH≥8.0. When the chemical concentrations were further increased, the time for crystallization was increased and a few high grade crystals of larger size were formed. If the concentrations of the chemicals were continuously increased, many crystals with smaller size, and, sometimes of poor quality appeared again and eventually ceased to produce any crystals. The optimal concentration for each of the above mentioned chemicals varies with other variable factors. Only one bigger crystal (both of the longest two sides: 0.16 mm) could be obtained in a hanging drop of protein sample when the concentrations of PEG 8000, MgCl 2, NaCl,Tris and protein were kept at 1.86%, 300 mmol/L, 400 mmol/L, 53 mmol/L and 4.64 g/L , respectively, with Tris buffer pH 8.2.
A mutant UW 3, which is unable to fix N 2 in the presence of Mo (Nif -) but undergo phenotypic reversal to Nif + under Mo deficiency, was able to grow in Mo- and NH 3-deficient medium containing Mn, and the growth was accelerated by Mn at low concentration. A partly purified nitrogenase component Ⅰ protein separated from UW 3 grown in the Mn-containing medium was shown to contain Fe and Mn atoms (ratio of Fe/Mo/Mn: 10.41/0.19/1.00) with C 2H 2- and H +-reducing activity which almost equal to half of that of MoFe protein purified from wild-type mutant of Azotobacter vinelandii Lipmann. This protein was obviously different from MoFe protein in both absorption spectrum and circular dichroism, and the molecular weight of subunits in Mn-containing protein was close to that of α subunit in MoFe protein. The preliminary results indicated that the protein containing Mn might be a nitrogenase component Ⅰ protein.
In order to meet the requirement for crystalline growth of O-2-susceptible proteins in space, crystallization conditions on the earth was optimized for the proteins using a simple and suitable device for anaerobic addition of the protein samples. Nitrogenase is susceptible to O-2. Delta nifZ MoFe protein from a nifZ deleted strain and MnFe protein from mutant strain UW3 grown on a medium containing Mn were crystallized at the first time in the world using an anaerobic device equipped with plastic bags or using a small simplified box, as a replacement for the cumbersome dry box. And the proteins could be also crystallized far from laboratory by sitting-drop method using a much lighter device. It was equipped with a smaller plastic food bag and a first-aid bag filled with Ar, as a substitute for the cumbersome dry box and the Ar cylinder, respectively. The results showed that the device could meet the requirement for studies on crystal growth of the above anaerobic proteins in space.
