Through the anaerobic chromatography on the columns of DEAE 52, Q-Sepharose and Sephacryl S-200, a nitrogenase MoFe protein (?nifZ Av1) was obtained from a nifZ deleted mutant of Azotobacter vinelandii (stain DJ194). ...Through the anaerobic chromatography on the columns of DEAE 52, Q-Sepharose and Sephacryl S-200, a nitrogenase MoFe protein (?nifZ Av1) was obtained from a nifZ deleted mutant of Azotobacter vinelandii (stain DJ194). The results of Western blotting after anoxic native electro-phoresis and SDS-PAGE showed that ?nifZ Av1 was similar to wild type MoFe protein (OP Av1) at the electrophoretic mobility, molecular weight and subunit composition. Fur-thermore, ?nifZ Av1 was also similar to OP Av1 at the mo-lybdenum content, EPR signal (g≈4.3, 3.65 and 2.01), and the molar extinction coefficient (?ε) of circular dichroism (CD) at 660 nm region. All of these indicated that, besides having the same α2β2 composition as OP Av1, the ?nifZ Av1 also contained equal amount of reductive FeMoco in the spin state of S=3/2 to OP Av1. However, the iron content and sub-strate (C2H2, H+ and N2)-reduction activity of ?nifZ Av1 were 74% and 46%―50% of those of OP Av1, respectively. Fur-thermore, the ?ε at around 450 nm, which reflects P-cluster in Av1, was obviously lower than that of OP Av1. It suggested that the difference between ?nifZ Av1 and OP Av1 resulted from P-cluster rather than FeMoco, and from the half num-ber of P-cluster in ?nifZ Av1, but the composition or redox state of P-cluster in ?nifZ Av1 were not changed. Thus it could propose that ?nifZ Av1 is composed of two different αβ subunit pairs. One is a FeMoco- and P-cluster-containing pair, and the other is a P-cluster-deficient but FeMoco-con- taining pair. Since the deletion of nifZ gene leads to the defi-ciency of only one of two P-clusters in a α2β2 tetramer, the assembly of P-cluster may not simply depend on one gene product, and so a possible mechanism of NifZ is supposed here.展开更多
基金supported by the State Key Basic Research and Developmental Plan of China(Grant No.001CB1089-06)the National Natural Science Foundation of China(Grant No.30270296)the National Science Foundation for distinguished Young Scholars of China(Grant No.20403024).
文摘Through the anaerobic chromatography on the columns of DEAE 52, Q-Sepharose and Sephacryl S-200, a nitrogenase MoFe protein (?nifZ Av1) was obtained from a nifZ deleted mutant of Azotobacter vinelandii (stain DJ194). The results of Western blotting after anoxic native electro-phoresis and SDS-PAGE showed that ?nifZ Av1 was similar to wild type MoFe protein (OP Av1) at the electrophoretic mobility, molecular weight and subunit composition. Fur-thermore, ?nifZ Av1 was also similar to OP Av1 at the mo-lybdenum content, EPR signal (g≈4.3, 3.65 and 2.01), and the molar extinction coefficient (?ε) of circular dichroism (CD) at 660 nm region. All of these indicated that, besides having the same α2β2 composition as OP Av1, the ?nifZ Av1 also contained equal amount of reductive FeMoco in the spin state of S=3/2 to OP Av1. However, the iron content and sub-strate (C2H2, H+ and N2)-reduction activity of ?nifZ Av1 were 74% and 46%―50% of those of OP Av1, respectively. Fur-thermore, the ?ε at around 450 nm, which reflects P-cluster in Av1, was obviously lower than that of OP Av1. It suggested that the difference between ?nifZ Av1 and OP Av1 resulted from P-cluster rather than FeMoco, and from the half num-ber of P-cluster in ?nifZ Av1, but the composition or redox state of P-cluster in ?nifZ Av1 were not changed. Thus it could propose that ?nifZ Av1 is composed of two different αβ subunit pairs. One is a FeMoco- and P-cluster-containing pair, and the other is a P-cluster-deficient but FeMoco-con- taining pair. Since the deletion of nifZ gene leads to the defi-ciency of only one of two P-clusters in a α2β2 tetramer, the assembly of P-cluster may not simply depend on one gene product, and so a possible mechanism of NifZ is supposed here.
