Halbleib C M, Zhang Y, Ludden P W
Department of Biochemistry, Center for the Study of Nitrogen Fixation, University of Wisconsin, Madison, Wisconsin 53706, USA.
J Biol Chem. 2000 Feb 4;275(5):3493-500. doi: 10.1074/jbc.275.5.3493.
The nitrogenase-regulating enzymes dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductase-activating glycohydrolase (DRAG), from Rhodospirillum rubrum, were shown to be sensitive to the redox status of the Fe(4)S(4) cluster of nitrogenase Fe protein from R. rubrum or Azotobacter vinelandii. DRAG had <2% activity with oxidized R. rubrum Fe protein relative to activity with reduced Fe protein. The activity of DRAG with oxygen-denatured Fe protein or a low molecular weight substrate, N(alpha)-dansyl-N(omega)-(1,N(6)-etheno-ADP-ribosyl)-arginine methyl ester, was independent of redox potential. The redox midpoint potential of DRAG activation of Fe protein was -430 mV versus standard hydrogen electrode, coinciding with the midpoint potential of the [Fe(4)S(4)] cluster from R. rubrum Fe protein. DRAT was found to have a specificity opposite that of DRAG, exhibiting low (<20%) activity with 87% reduced R. rubrum Fe protein relative to activity with fully oxidized Fe protein. A mutant of R. rubrum in which the rate of oxidation of Fe protein was substantially decreased had a markedly slower rate of ADP-ribosylation in vivo in response to 10 mM NH(4)Cl or darkness stimulus. It is concluded that the redox state of Fe protein plays a significant role in regulation of the activities of DRAT and DRAG in vivo.
来自红螺菌的固氮酶调节酶二氮酶还原酶ADP-核糖基转移酶（DRAT）和二氮酶还原酶激活糖水解酶（DRAG），被证明对来自红螺菌或棕色固氮菌的固氮酶铁蛋白的Fe(4)S(4)簇的氧化还原状态敏感。相对于还原型铁蛋白的活性，DRAG对氧化型红螺菌铁蛋白的活性<2%。DRAG对氧变性铁蛋白或低分子量底物N(α)-丹磺酰-N(ω)-(1,N(6)-乙烯基-ADP-核糖基)-精氨酸甲酯的活性与氧化还原电位无关。铁蛋白DRAG激活的氧化还原中点电位相对于标准氢电极是-430 mV，与红螺菌铁蛋白的[Fe(4)S(4)]簇的中点电位一致。发现DRAT具有与DRAG相反的特异性，相对于完全氧化的铁蛋白的活性，DRAT对87%还原的红螺菌铁蛋白的活性较低（<20%）。红螺菌的一个突变体，其中铁蛋白的氧化速率显著降低，在体内对10 mM NH(4)Cl或黑暗刺激的ADP-核糖基化速率明显较慢。得出结论，铁蛋白的氧化还原状态在体内DRAT和DRAG活性的调节中起重要作用。
