The composition and distribution of metal clusters in the MoFe protein from a nifZ deletion strain (DJ 194) of Azotobacter vinelandii

Abstract

Through the anaerobic chromatography on the columns of DEAE 52, Q-Sepharose and Sephacryl S-200, a nitrogenase MoFe protein (ΔnifZ Av1) was obtained from a nifZ deleted mutant of Azotobacter vinelandii (stain DJ194). The results of Western blotting after anoxic native electrophoresis and SDS-PAGE showed that ΔnifZ Av1 was similar to wild type MoFe protein (OP Av1) at the electrophoretic mobility, molecular weight and subunit composition. Furthermore, ΔnifZ Av1 was also similar to OP Av1 at the molybdenum content, EPR signal (g ≈ 4.3, 3.65 and 2.01), and the molar extinction coefficient (Δe) of circular dichroism (CD) at 660 nm region. All of these indicated that, besides having the same α2β2 composition as OP Av1, the ΔnifZ Av1 also contained equal amount of reductive FeMoco in the spin state of S=3/2 to OP Av1. However, the iron content and substrate (C2H2, H+ and N2)-reduction activity of ΔnifZ Av1 were 74% and 46%–50% of those of OP Av1, respectively. Furthermore, the Δe at around 450 nm, which reflects P-cluster in Av1, was obviously lower than that of OP Av1. It suggested that the difference between ΔnifZ Av1 and OP Av1 resulted from P-cluster rather than FeMoco, and from the half number of P-cluster in ΔnifZ Av1, but the composition or redox state of P-cluster in ΔnifZ Av1 were not changed. Thus it could propose that ΔnifZ 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-containing pair. Since the deletion of nifZ gene leads to the deficiency 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.