[Study of the nitrogenase from Azotobacter vinelandii by the method of spin labels].
The interaction of nitrogenase with spin labels of four types have been studied. Conclusion about the presence of two SH-groups in the nitrogenase active site (one in Mo-Fe-protein and one in the Fe-protein) have been drawn from the correlation between the degree of inhibition of nitrogenfixing activity by the labels derived from p-Cl-Hg-benzoate and degree of binding of these labels to the nitrogenase molecule. Anaysis of EPR spectra of spin-labeled nitrogenase at 77 degrees K and at room temperature have shown that the labels bind to the free SH-groups and interact with iron containing center (ICC) of nitrogenase through the exchange mechanism. Distance between SH-group and ICC have been found to be 12 A. Spin labels derived from isocyanide have been bound directly to ICC in amount of 6--10 labels per one nitrogenase molecule. Due to the exchange interaction between these labels they give the singlet ESR spectra both at 77 degrees and at room temperature which is characteristic for the closely disposed labels. From this fact a conclusion have been drawn about the cluster structure of ICC. The labels derived from iodoacetamide ana maleimide bind SH- and NH2-groups of nitrogenase molecules. Analysis of temperature dependence of the effective rotational frequency of this labels have revealed a conformational transition in nitrogenase molecule at 19 degrees C, that has made it possible to explain the break in the Arrenius plots of enzyme activity.
D001395:Azotobacter / Q000201:enzymology*; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008433:Mathematics; D009591:Nitrogenase / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013113:Spin Labels; D013438:Sulfhydryl Compounds / Q000032:analysis; D013696:Temperature; D013816:Thermodynamics
D013113:Spin Labels; D013438:Sulfhydryl Compounds; D009591:Nitrogenase
Issledovanie nitrogenazy Asotobacter vinelandii metodom spinovykh metok