Under a suitable condition of crystallization, dark brown rhombohedron crystals (the lengths of the longest two diagonals were 0.25 and 0.12 mm, respectively) could be obtained from nitrogenase CrFe protein purified from a mutant UW3 of Azotobacter vinelandii Lipmann grown in Cr-containing but NH3-free Medium, The possibility of crystallization, as well as the. number, size and quality of crystals obviously depended on the concentrations of PEG 8000, MgCl2, NaCl, Tris and Hepes buffer, and methods of crystallization. The optimum concentrations of the chemicals for crystallization of CrFe protein were slightly different from those for crystallization of MnFe protein from UW3 grown in Mn and DeltanifZ MoFe protein from a nifZ deleted strain of A. vinelandii. The crystal seemed to be formed from CrFe protein.
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.
By incubating the reduced MoFe protein from Azotobacter vinelandii with O phenanthroline under air and chromatographying the incubated solution on Sephadex G 25 column, inactive MoFe protein could be obtained. Its acetylene reduction activity was remarkably recovered not only by incubation with the reconstituent solution composed of KMnO 4,ferric homocitrate, Na 2S and dithiothreitol,Azotobacter vinelandii with O phenanthroline under air and chromatographying the incubated solution on Sephadex G 25 column, inactive MoFe protein could be obtained. Its acetylene reduction activity was remarkably recovered not only by incubation with the reconstituent solution composed of KMnO 4,ferric homocitrate, Na 2S and dithiothreitol, with O phenanthroline under air and chromatographying the incubated solution on Sephadex G 25 column, inactive MoFe protein could be obtained. Its acetylene reduction activity was remarkably recovered not only by incubation with the reconstituent solution composed of KMnO 4,ferric homocitrate, Na 2S and dithiothreitol, but also with a mixture of 4Fe∶4S clusters and another cluster which had two structure units of 1Mo∶3Fe∶4S bridged by three -OCH 3- at the Mo atoms. Neither the reconstituent solution nor the mixture could reactivate apo MoFe proteins from the mutants deleting nifE and nifH genes and from the mutant UW 45 , which could be reactivated by the FeMoco extracted from the MoFe protein. The results indicated that the FeMoco deficient MoFe proteins from these mutants seemed to be reconstituted only by the clusters which were probably structures only similar to FeMoco. The partially metallocluster deficient MoFe protein could be reconstituted by the clusters with a certain kind of structure and composition; and was changed into different nitrogenase proteins with the ability to fix nitrogen.
