钼铁蛋白金属原子簇的拆卸与组装的圆二色谱的研究

STUDIES ON THE CIRCULAR DICHROISM SPECTROSCOPY OF THE DISASSEMBLY AND ASSEMBLY OF METAL CLUSTERS IN MOLYBDENUM-IRON PROTEIN

用邻菲啰啉(o-phen)和氧分别或共同处理的棕色固氮菌(Azotobacter vinelandii)固氮酶钼铁蛋白的活性、圆二色谱(CD)和金属含量都发生变化。曝氧蛋白的活性随其在660nm和450nm的△ε(圆二向色性克分子消光系数)的降低而降低;而o-phen处理的蛋白活性只随其△ε_(458nm)和铁含量的降低而降低;但在有氧时,o-phen处理的蛋白活性的降低不仅与其△ε_(458nm)和铁含量降低有关,而且还与其△ε_(660nm)和钼含量降低有关。然而,当这些处理蛋白与由Na_2MoO_4、Na_2S、柠檬酸铁和二硫苏糖醇组成的重组溶液保温后,重组蛋白的活性随它们的CD谱和金属含量的显著恢复而得到明显恢复。结果表明:(1)不仅△ε_(458nm)可反映钼铁蛋白中P-cluster的状态和数目,而且△ε_(660nm)也许也可反映蛋白中FeMoco的状态和数目;(2)o-phen不仅可螯合还原蛋白中P-cluster的铁原子,而且也可螯合曝氧蛋白中FeMoco和P-cluster的铁原子;(3)钼铁蛋白中的这两种金属原子簇都可通过缺失原子簇的钼铁蛋白与重组溶液的保温而得到组装;(4)这两种原子簇都是钼铁蛋白还原底物所必需的重要部分。

After the treatment of nitrogenase MoFe protein from Azotobacter vinelandii respectively or simultaneously with o-phenanthroline (o-phen) and O_2, the treated proteins resulted in the changes in activity, CD spectra and metals content. The activity of O_2-exposed protein was decreased with the decrease in its △ε (molecular circular dichroic extinction coefficient) at both 660 and 450nm, and the activity of the protein treated with o-phen was decreased only with the decrease in its △ε_(450nm) and Fe content, but the decrease in the activity of MoFe protein treated with ophen under the presence of O_2 was relative to the decrease not only in its △ε_(450nm) and Fe content, but also in its △ε_(660nm) and Mo content. However, when the treated proteins were incubated with the reconstituting solution which consisted of Na_2MoO_4, Na_2S, ferric citrate and DTT, their activity were significantly restored with a significant restoration of their CD spectra and metals content. The results showed that: (1) not only △ε_(450nm) is able to reflect the state and content of Pcluster in MoFe protein but △ε_(660nm) might also reflect the state and content of FeMoco in the protein; (2) o-phen is able to chelate Fe not only from P-cluster in the reduced MoFe protein, but also from FeMoco and P-cluster in the O_2-exposed MoFe protein; (3) both P-cluster and FeMoco of the MoFe protein can be assembled by the incubation of clusters-defficient MoFe protein with the reconstituting solution; (4) these clusters of MoFe protein are the necessary and important fractions for the protein to reduce substrates.