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首页> 外文期刊>Biochemistry >Determination of the Midpoint Potential of the FAD and FMN Flavin Cofactors and of the 3Fe-4S Cluster of Glutamate Synthase
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Determination of the Midpoint Potential of the FAD and FMN Flavin Cofactors and of the 3Fe-4S Cluster of Glutamate Synthase

机译：FAD和FMN黄素辅因子和谷氨酸合酶的3Fe-4S簇中点电位的测定

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摘要

Glutamate synthase is a complex iron—sulfur flavoprotein that catalyzes the reductive transfer of the L-glutamine amide group to C(2) of 2-oxoglutarate, forming two molecules of L-glutamate. The bacterial enzyme is an c43 protomer, which contains one FAD (on the ~3 subunit, “-‘50 kDa), one FMN (on the a subunit, ~—‘ 150 kDa), and three different Fe—S clusters (one 3Fe—4S center on the a subunit and two 4Fe—4S clusters at an unknown location). To address the problem of the intramolecular electron pathway, we have measured the midpoint potential values of the flavin cofactors and of the 3Fe—4S cluster of glutamate synthase in the isolated cc and j3 subunits and in the cc/3 holoenzyme. No detectable amounts of flavin semiquinones were observed during reductive titrations of the enzyme, indicating that the midpoint potential value of each flavinox/flavinsq couple is, in all cases, significantly more negative than that of the corresponding flavinsq/flavinhq couple. Association of the two subunits to form the cc/3 protomer does not alter significantly the midpoint potential value of the FMN cofactor and of the 3Fe—4S cluster (approximately —240 and —270 mV, respectively), but it makes that of FAD some 40 mV less negative (approximately —340 mV for the /3 subunit and —300 mY for FAD bound to the holoenzyme). Binding of the nonreducible NADP~ analogue, 3-aminopyridine adenine dinucleotide phosphate, made the measured midpoint potential value of the FAD cofactor approximately 30—40 mV less negative in the isolated /3 subunit, but had no effect on the redox properties of the cc/3 holoenzyme. This result correlates with the formation of a stable charge-transfer complex between the reduced flavin and the oxidized pyridine nucleotide in the isolated /3 subunit, but not in the cc/3 holoenzyme. Binding of L-methiOnine sulfone, a glutamine analogue, had no significant effect on the redox properties of the enzyme cofactors. On the contrary, 2-oxoglutarate made the measured mid-point potential value of the 3Fe—4S cluster approximately 20 mV more negative in the isolated a sub-unit, but up to 100 mV less negative in the ci/3 holoenzyme as compared to the values of the correspond-ing free enzyme forms. These findings are consistent with electron transfer from the entry site (FAD) to the exit site (FMN) through the 3Fe—4S center of the enzyme and the involvement of at least one of the two low-potential 4Fe—4S centers, which are present in the glutamate synthase holoenzyme, but not in the isolated subunits. Furthermore, the data demonstrate a specific role of 2-oxoglutarate in promoting electron transfer from FAD to the 3Fe—4S cluster of the glutamate synthase holoenzyme. The modulatory role of 2-oxo glutarate is indeed consistent with the recently determined three-dimensional structure of the glutamate synthase a subunit, in which several polypeptide stretches are suitably positioned to mediate communication between substrate binding sites and the enzyme redox centers (FMN and the 3Fe—4S cluster) to tightly control and coordinate the individual reaction steps [Binda, C., et al. (2000) Structure8, 1299—1308].
机译：谷氨酸合酶是一种复杂的铁硫黄素蛋白，它催化L-谷氨酰胺酰胺基团还原转移至2-氧代戊二酸的C（2），形成两个L-谷氨酸分子。细菌酶是c43启动子，它包含一个FAD（在〜3个亚基上，“-'50 kDa”），一个FMN（在a个亚基上，〜-'150 kDa）和三个不同的Fe-S簇（一个3Fe-4S集中在一个子单元上，两个4Fe-4S簇位于未知位置）。为了解决分子内电子途径的问题，我们测量了分离的cc和j3亚基和cc / 3全酶中的黄素辅因子和谷氨酸合酶的3Fe-4S簇的中点电势值。在酶的还原滴定过程中未观察到可检测量的黄素半醌，表明在所有情况下，每个黄素/黄素对的中点电位值均显着大于相应黄素/黄素对的中点电位。这两个亚基形成cc / 3前体的缔合并不会显着改变FMN辅因子和3Fe-4S簇的中点电势值（分别约为-240和-270 mV），但它使FAD的中点电势有些变化负离子少40 mV（对于/ 3亚基，约为-340 mV，对于与全酶结合的FAD约为-300 mY）。不可还原的NADP〜类似物3-氨基吡啶腺嘌呤二核苷酸磷酸的结合，使所测得的FAD辅因子的中点电位值在分离的/ 3亚基中的负值降低了约30-40 mV，但对cc的氧化还原特性没有影响/ 3全酶。该结果与在分离的/ 3亚基中而不在cc / 3全酶中的还原的黄素和氧化的吡啶核苷酸之间形成稳定的电荷转移复合物有关。谷氨酰胺类似物L-甲硫氨酸砜的结合对酶辅因子的氧化还原特性没有明显影响。相反，2-氧戊二酸使3Fe-4S团簇的测量中点电位值在分离出的一个亚基中负值大约高20 mV，但与ci / 3全酶相比，负值最多降低100 mV。相应的游离酶形式的值。这些发现与电子通过酶的3Fe-4S中心从进入位点（FAD）转移到出口位点（FMN）以及两个低电势4Fe-4S中心中至少一个的参与相一致。存在于谷氨酸合酶全酶中，但不存在于分离的亚基中。此外，数据表明2-氧戊二酸酯在促进电子从FAD转移到谷氨酸合酶全酶的3Fe-4S簇中具有特殊作用。谷氨酸2-氧代的调节作用确实与最近确定的谷氨酸合酶a亚基的三维结构相一致，其中几个多肽段被适当地定位以介导底物结合位点和酶氧化还原中心之间的通讯（FMN和3Fe-4S簇）来严格控制和协调各个反应步骤[Binda，C.，et al。 （2000）Structure8，1299-1308]。

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