Studies On The Iron-Molybdenum Nitrogenase From The NifZ Deleted Strain DJ194 Of Azotobacter Vinelandii

Through the anaerobic chromatography on the columns of DEAE 52, Q-Sepharose and Sephacryl S-200, the purer ΔnifZ MoFe protein (ΔnifZ Av1) preparation was obtained from the crude extract of a nifZ-deletion mutant of Azotobacter vinelandii (DJ194). The comparison of anoxic native electrophoresis and SDS-PAGE of the ΔnifZ Av1 preparation previously obtained revealed that the previous ΔnifZ Av1 preparation was mainly contaminated by three proteins, which were chaperon GroEL (member of Hsp60 family), 6-phosphate glucose isomerase (PGI, a multifunctional enzyme of Embden-Meyerhof glycolytic pathway) and bacterioferritin (Bfr). The primary identifications of the three protein in the ΔnifZ Av1 preparation showed that GroEL was a 14-mers of 55kD subunit, and PGI was a 10-mers of 62 kD subunit, and Bfr was a 24-mers of 20kD subunit. PGI in the presence of 10-mers was firstly reported. Though the electrophoretic mobilities of GroEL and PGI are both lower than that of ΔnifZ Av1 at anoxic native electrophoresis, their subunits have the similar mobilities to the subunits of ΔnifZ Av1 at SDS-PAGE, leading to overlapping each other. Although the subunit of Bfr could not contaminate the subunits of ΔnifZ Av1 at SDS-PAGE, the Bfr was easier to be crystallized from the crystallization of ΔnifZ Av1 preparation, disturbing the study on the growth and analysis of ΔnifZ Av1 crystal (Zhao et al., 2004). A ΔnifZ Av1 preparation with high purity of >90% was obtained by the more fine collection on the Sephacryl S-200 and NaCl gradient elution on the Q-Sepharose. The native electrophoresis, Western blotting and SDS-PAGE of this preparation showed that ΔnifZ Av1 was similar to wild type MoFe protein (OP Av1) in the electrophoretic mobility, molecular weight and subunit composition. This indicated that nifZ-deletion did not change the α2β2 composition of ΔnifZ Av1.And ΔnifZ Av1 was also similar to OP Av1 in the molybdenum content, EPR signal (g≈4.3, 3.65 and 2.01), and the molar extinction coefficient (Δε) of circular dichroism (CD) at 660 nm region. All of these indicated that the ΔnifZ Av1 contained equal amount of reductive FeMoco in the spin state of S=3/2 to that of 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 Δε at around 450 nm, which reflects P-cluster in Av1, was obviously lower than that of OP Av1. In addition, the EPR signal of ΔnifZ Av1 at g≈2.01 region was very different with that of His-tagged ΔnifZ Av1 (Hu et al., 2004) that was thought to contain a P-cluster precursor of a pair of [4Fe-4S], It was possible to suggest that the difference between ΔnifZ Av1 and OP Av1 was neither the structure, content and redox state of FeMoco or the structure and redox state of P-cluster, and was only the about 50% decrease of P-cluster content of ΔnifZ Av1. 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. As indicated by Lee et al.(1998), the products of nifZ (NifZ) and nifW (NifW) could interact with each other to make a multimeric complex, and both proteins might exert their regulation on the nitrogenase by a common pathway, a higher order complex of NifW and NifZ (expressed by [NifWx-NifZy]). So a possible mechanism of NifZ is supposed here. The complex of [NifWx-NifZy] would be needed during either the insertion of the precursor of P-cluster (such as [4Fe-4S] cluster) or the maturity of P-cluster, and NifZ of this complex might have greater effect on the P-cluster formation in one αβ subunit pair than on in the other pair; some mutants of nifZ would not affect the formation of [NifWx-NifZy] complex, but these [NifWx-NifZy] with abnormal NifZ would have different effects on the P-cluster formation of the αβ subunit pair more affected by NifZ. The crystallization of this ΔnifZ Av1 preparation with >90% purity showed that the big crystals would be obtained after the incubation with the 25%PEG 6K/MgCl2 precipitant in Tris buffer system for a relative short period. Under some conditions, the two different pH (7.5 and 8.3) of the precipitant solutions have not obviously different effects on the number or size of crystals. The concentrations of PEG 6K and MgCl2 have the similar effects on the crystal size. The bigger crystals of ΔnifZ Av1were easier to be obtained when both the concentrations of PEG 6K and MgCl2 were relatively lower or higher. Though the purity of this ΔnifZ Av1 preparation was more than 90% and Bfr was not determined by the iron-stained experiment, a kind of Bfr crystals with a deep red-brown color rather than the reported brick-red was identified by the SDS-PAGE. The different color of Bfr crystals perhaps related with the redox state of Fe in Bfr. In all the 5 tubes containing crystals, Bfr and the ΔnifZ Av1 were simultaneously crystallized in only one tube, indicating that the crystallization probability of Bfr would be greatly decreased with the increase of the purity of ΔnifZ Av1 preparation and the optimization of the crystallization condition.