Redox State of Flavin Adenine Dinucleotide Drives Substrate Binding and Product Release in Escherichia coli Succinate Dehydrogenase

详细信息    查看全文

• 作者：Victor W. T. Cheng ; Ramanaguru Siva Piragasam ; Richard A. Rothery ; Elena Maklashina ; Gary Cecchini ; Joel H. Weiner
• 刊名：Biochemistry
• 出版年：2015
• 出版时间：February 3, 2015
• 年：2015
• 卷：54
• 期：4
• 页码：1043-1052
• 全文大小：392K
• ISSN：1520-4995

文摘

The Complex II family of enzymes, comprising respiratory succinate dehydrogenases and fumarate reductases, catalyzes reversible interconversion of succinate and fumarate. In contrast to the covalent flavin adenine dinucleotide (FAD) cofactor assembled in these enzymes, soluble fumarate reductases (e.g., those from Shewanella frigidimarina) that assemble a noncovalent FAD cannot catalyze succinate oxidation but retain the ability to reduce fumarate. In this study, an SdhA-H45A variant that eliminates the site of the 8伪-N3-histidyl covalent linkage between the protein and FAD was examined. Variants SdhA-R286A/K/Y and -H242A/Y that target residues thought to be important for substrate binding and catalysis were also studied. The variants SdhA-H45A and -R286A/K/Y resulted in the assembly of a noncovalent FAD cofactor, which led to a significant decrease (鈭?7 mV or more) in its reduction potential. The variant enzymes were studied by electron paramagnetic resonance spectroscopy following stand-alone reduction and potentiometric titrations. The 鈥渇ree鈥?and 鈥渙ccupied鈥?states of the active site were linked to the reduced and oxidized states of FAD, respectively. Our data allow for a proposed model of succinate oxidation that is consistent with tunnel diode effects observed in the succinate dehydrogenase enzyme and a preference for fumarate reduction catalysis in fumarate reductase homologues that assemble a noncovalent FAD.