Characterization of a FeMo cofactor-deficient MoFe protein from anifE-deleted strain (DJ35) ofAzotobacter vinelandii
- 15 Downloads
A MoFe protein (ΔnifE Avl) with a purity of ∼80% was purified from a nifE-deleted mutant ofAzotobacter vinelandii DJ35. Compared with MoFe protein purified from wild-type strain OP (OP Av1), ΔnifE Av1 had the same subunits composition, and had immune reaction with antibody to OP Av1, but its relative mobility in anaerobic native polyacrylamide gel electrophoresis (PAGE) was a little larger than that of OP Av1. Metal analysis showed that Mo and Fe contents of ΔnifE Av1 both apparently decreased. When complemented with OP Fe protein, ΔnifE Av1 had no C2H2-reduction activity, but it could bein vitro activated by FeMoco extracted from OP Av1. The circular dichroism (CD) spectrum of ΔnifE Av1 at ∼450 nm was similar to that of OP Av1, while the EPR signal at g∼3.7 was absolutely silent, and the signal intensities at g∼=4.3 and 2.0 decreased by 75% and 50%, respectively. The results indicated that ΔnifE Av1 purified from DJ35 was a FeMoco-deficient but P-cluster-containing MoFe protein.
KeywordsAzotobacter vinelandii ΔnifE Av1 FeMoco P-cluster EPR
Unable to display preview. Download preview PDF.
- 5.Tal, S., Chun, T. W., Gavini, N. et al., The ΔniB (or ΔnifE FeMo cofactor-deficient MoFe protein is different from the ΔnifH protein, J. Biol. Chem., 1991, 266: 10654–10657.Google Scholar
- 15.Zhao, J. F., Zhao, Y., Wang, Z. P. et al., Purification and activationin vitro of MoFe protein from anifE deleted mutant strain ofAzotobacter vinelandii, Acta Bot. Sin., 2003, 45: 815–819.Google Scholar
- 16.Zhao, Y., Zhao, J. F., Lü, Y. B. et al., Crystallization of nitrogenase MoFe protein from a mutantnifE deleted strain ofAzotobacter vinelandii, Acta Bot. Sin., 2003, 45: 427–431.Google Scholar
- 17.Brigle, K. E., Weiss, M. C., Newton, W. E. et al., Products of the iron-molybdenum cofactor-specific biosynthetic genes,nifE andnifN, are structurally homologous to the products of nitrogenase molybdemum-iron protein genes,nifD andnifK, J. Bacteriol., 1987, 169: 1547–1553.Google Scholar
- 19.Burgess, B. K., Jacobs, D. B., Stiefel, E. I., Large scale purification of high activityAzotobacter vinelandii nitrogenase, Biochim. Biophys. Acta, 1980, 614: 196–209.Google Scholar
- 22.Luo, A. L., Wang, J. W., Li, J. G., A simple method for protein blotting and immunoassay, Chinese Bull. Bot., 1995, 12: 63–64.Google Scholar
- 25.Robinson, A. C., Chun, T. W., Li, J. G. et al., Iron-molybdenum cofactor insertion into the apo-MoFe protein of nitrogenase involves the iron protein-MgATP complex, J. Biol. Chem., 1989, 264: 10088–10095.Google Scholar
- 30.Hawkes, T. R., Smith, B. E., Purification and characterization of the inactive MoFe protein (NifB-Kp1) of the nitrogenase fromnifB mutants ofKlebsiella pneumoniae, Biochem. J., 1983, 209: 43–50.Google Scholar
- 31.Hawkes, T. R., Smith, B. E., The inactive MoFe protein (NifB− Kpl) of the nitrogenase fromnifB mutantsof Klebsiella pneumoniae: its interaction with FeMo-cofactor and the properties of the active MoFe protein formed, Biochem. J., 1984, 223: 783–792.Google Scholar
- 34.Huang, J. F., Luo, A. L., Xie, X. M. et al., Study on the circular dichroism spectroscopy of the disassembly and assembly of metal clusters in molybdenum-iron protein, Acta Microbiol. Sin., 1991, 31: 232–239.Google Scholar