EPR spectral evidence for a binuclear Mn(II) center in dinitrogenase reductase-activating glycohydrolase from Rhodospirillum rubrum
EPR spectral evidence for a binuclear Mn(II) center in dinitrogenase reductase-activating glycohydrolase from Rhodospirillum rubrum
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DOI：
10.1021/ja9818912
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发表时间：
1998-09-02
影响因子：
15
通讯作者：
Ludden, PW
中科院分区：
文献类型：
--
作者：
Antharavally, BS;Poyner, RR;Ludden, PW
细菌固氮是由二氮酶和二氮酶还原酶两种氧不稳定蛋白催化的，它们组成了氮酶复合物。1 .氮酶复合物催化大气氮还原为铵，这是一个非常耗能的反应，因为每还原一个氮分子至少要水解16个MgATP分子。因此，在能量应激或氮充足的条件下，红红螺旋菌2,3通过在二氮酶还原酶同型二聚体的单个亚基上进行Arg-101的可逆adp核糖基化，使翻译后的氮酶失活。adp -核糖片段从NAD+转移到二氮化酶还原酶是由二氮化酶还原酶adp -核糖基转移酶（DRAT）催化的。adp -核糖基蛋白链的糖水解激活是由二氮化酶还原酶激活糖水解酶（DRAG）催化的。5-7 DRAG是一种32 kd的单体酶，需要MgATP和游离二价金属才能发挥活性，adp核基化二氮酶还原酶作为底物6，因此二价金属的结合位点被认为是在DRAG蛋白上。8, 9 Mn2+被认为是生理辅助因子，尽管已经报道了Fe2+、Mg2+和Co3+激活DRAG酶。我们在此报告EPR证据，从红红螺旋菌分离的DRAG含有一个自旋偶联的Mn-(II)-Mn （II）中心。从过表达的rubrum菌株ur276中纯化了DRAG。6通过二氮化酶还原酶与二氮化酶还原乙炔的活性偶联来测量活性，11和蛋白质浓度根据公布的程序使用比辛醌酸测定。用Pall滤池将酶浓缩至12 mg/mL，在50 mM MOPS， 0.2 M NaCl，
Bacterial nitrogen fixation is catalyzed by the two oxygenlabile proteins dinitrogenase and dinitrogenase reductase, which compose the nitrogenase enzyme complex. 1 The nitrogenase enzyme complex catalyzes the reduction of atmospheric nitrogen to ammonium, which is a very energy-intensive reaction, in that at least 16 MgATP molecules are hydrolyzed per nitrogen molecule reduced. Therefore, Rhodospirillum rubrum2, 3 posttranslationally inactivates nitrogenase by reversible ADP-ribosylation of Arg-101 on a single subunit of the dinitrogenase reductase homodimer under the conditions of energy stress or nitrogen sufficiency. The transfer of the ADP-ribose moiety from NAD+ onto dinitrogenase reductase is catalyzed by dinitrogenase reductase ADP-ribosyltransferase (DRAT). 4 Activation via glycohydrolysis of the ADP-ribosyl protein linkage is catalyzed by dinitrogenase reductase-activating glycohydrolase (DRAG). 5-7 DRAG is a 32-kD monomeric enzyme which requires MgATP and free divalent metal for its activity with ADP-ribosylated dinitrogenase reductase as substrate, 6 and thus the binding site for the divalent metal is thought to be on the DRAG protein. 8, 9 Mn2+ is thought to be the physiological cofactor, although activation of the DRAG enzyme by Fe2+, Mg2+, and Co3+ has been reported. 10 We report here EPR evidence that DRAG isolated from Rhodospirillum rubrum contains a spin-coupled Mn-(II)-Mn (II) center.DRAG was purified from an overexpressing R. rubrum strain UR 276 following the published procedure. 6 Activity was measured by coupling the activity of dinitrogenase reductase with the reduction of acetylene by dinitrogenase, 11 and protein concentration was determined using bicinchoninic acid following the published procedure. 12 Samples for EPR spectroscopy were prepared by concentrating the enzyme with a Pall Filtron microconcentrator to 12 mg/mL in 50 mM MOPS, 0.2 M NaCl,