Bacillus subtilis mutant succinate dehydrogenase lacking covalently bound flavin: identification of the primary defect and studies on the iron-sulfur clusters in mutated and wild-type enzyme.
Bacillus subtilis mutant succinate dehydrogenase lacking covalently bound flavin: identification of the primary defect and studies on the iron-sulfur clusters in mutated and wild-type enzyme.
复制标题
缺乏共价结合黄素的枯草芽孢杆菌突变体琥珀酸脱氢酶：主要缺陷的鉴定以及突变型和野生型酶中铁硫簇的研究。
DOI：
10.1021/bi00366a033
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Hederstedt,L
中科院分区：
文献类型：
--
作者：
Maguire,JJ;Magnusson,K;Hederstedt,L
劳伦斯伯克利实验室膜生物能学小组，加州大学生理解剖学系，伯克利，加州94720，卡罗林斯卡学院细菌学系，S-104 01斯德哥尔摩，瑞典1986年1月31日接收;修订版手册1986年4月14日接收摘要：琥珀酸脱氢酶由两个蛋白亚基组成，含有一个FAD和三个铁硫簇。黄素与较大的Fp亚基中的组氨酸共价结合。枯草芽孢杆菌野生型膜结合酶中命名为S1、S-2和S-3的簇的还原氧化中点电位分别测定为+80、-240和-25 mV。用EPR谱研究了团簇S1和S-2以及S1和S-3之间的磁自旋相互作用。4个B的点突变。定位了具有缺陷型Fp亚基的枯草杆菌突变体。克隆了特异性缺乏共价结合黄素的突变体的基因。从DNA序列中确定突变为甘氨酸到天冬氨酸的取代，该取代位于野生型酶中结合黄素的组氨酸下游7个残基的保守位点。突变的琥珀酸脱氢酶中的铁硫簇的氧化还原中点电位和磁自旋相互作用与野生型没有区别。这表明黄素在测量的磁自旋相互作用或铁硫簇的结构和稳定性中没有作用。从序列和突变体研究中得出结论，组氨酰-FAD周围的保守氨基酸残基对于FAD结合是重要的;然而，位于Fp亚基中组氨酰下游超过100个残基的氨基酸也可以影响黄素化。
Membrane Bioenergetics Group, Lawrence Berkeley Laboratory, and Department of Physiology-Anatomy, University of California, Berkeley, California 94720, and Department of Bacteriology, Karolinska Institutet, S-104 01 Stockholm, Sweden Received January 31, 1986; Revised Manuscript Received April 14, 1986 abstract: Succinate dehydrogenase consists of two protein subunitsand contains one FAD and three iron-sulfur clusters. The flavin is covalently bound to a histidine in the larger, Fp, subunit. The reduction oxidation midpoint potentials of the clusters designated Sl, S-2, and S-3 in Bacillus subtilis wild-type membrane-bound enzyme were determined as+ 80,-240, and-25 mV, respectively. Magnetic spin interactions between clusters Sl and S-2 and between Sl and S-3 were detected by using EPR spectroscopy. The point mutations of four B. subtilis mutants with defective Fp subunits were mapped. The gene of the mutant specifically lacking covalently bound flavin inthe enzyme was cloned. The mutation was determined from the DNA sequence as a glycine to aspartate substitution at a conserved site seven residues downstream from the hisitidinethat binds the flavin in wild-type enzyme. The redox midpoint potential of the iron-sulfur clusters and the magnetic spin interactions in mutated succinate dehdyrogenases were indistinguishable from the those of the wild type. This shows that flavin has no role in the measured magnetic spin interactions or in the structure and stability of the iron-sulfur clusters. It is concluded from sequence and mutant studies that conserved amino acid residues around the histidyl-FAD are important for FAD binding; however, amino acids located more than 100 residuesdownstream from the histidyl in the Fp subunit can also effect flavinylation.