Cecchini G, Sices H, Schröder I, Gunsalus R P
Molecular Biology Division, Veterans Administration Medical Center, San Francisco, California 94121, USA.
J Bacteriol. 1995 Aug;177(16):4587-92. doi: 10.1128/jb.177.16.4587-4592.1995.
Fumarate reductase from Escherichia coli functions both as an anaerobic fumarate reductase and as an aerobic succinate dehydrogenase. A site-directed mutation of E. coli fumarate reductase in which FrdB Pro-159 was replaced with a glutamine or histidine residue was constructed and overexpressed in a strain of E. coli lacking a functional copy of the fumarate reductase or succinate dehydrogenase complex. The consequences of these mutations on bacterial growth, assembly of the enzyme complex, and enzymatic activity were investigated. Both mutations were found to have no effect on anaerobic bacterial growth or on the ability of the enzyme to reduce fumarate compared with the wild-type enzyme. The FrdB Pro-159-to-histidine substitution was normal in its ability to oxidize succinate. In contrast, however, the FrdB Pro-159-to-Gln substitution was found to inhibit aerobic growth of E. coli under conditions requiring a functional succinate dehydrogenase, and furthermore, the aerobic activity of the enzyme was severely inhibited upon incubation in the presence of its substrate, succinate. This inactivation could be prevented by incubating the mutant enzyme complex in an anaerobic environment, separating the catalytic subunits of the fumarate reductase complex from their membrane anchors, or blocking the transfer of electrons from the enzyme to quinones. The results of these studies suggest that the succinate-induced inactivation occurs by the production of hydroxyl radicals generated by a Fenton-type reaction following introduction of this mutation into the [3Fe-4S] binding domain. Additional evidence shows that the substrate-induced inactivation requires quinones, which are the membrane-bound electron acceptors and donors for the succinate dehydrogenase and fumarate reductase activities. These data suggest that the [3Fe-4S] cluster is intimately associated with one of the quinone binding sites found n fumarate reductase and succinate dehydrogenase.
来自大肠杆菌的延胡索酸还原酶既作为厌氧延胡索酸还原酶发挥作用，又作为需氧琥珀酸脱氢酶发挥作用。构建了大肠杆菌延胡索酸还原酶的一个定点突变体，其中将FrdB的Pro-159替换为谷氨酰胺或组氨酸残基，并在缺乏功能性延胡索酸还原酶或琥珀酸脱氢酶复合物拷贝的大肠杆菌菌株中进行过表达。研究了这些突变对细菌生长、酶复合物组装和酶活性的影响。发现与野生型酶相比，这两种突变对厌氧细菌生长或酶还原延胡索酸的能力均无影响。FrdB的Pro-159替换为组氨酸后，其氧化琥珀酸的能力正常。然而，相比之下，发现FrdB的Pro-159替换为Gln会在需要功能性琥珀酸脱氢酶的条件下抑制大肠杆菌的需氧生长，此外，在其底物琥珀酸存在下孵育时，该酶的需氧活性会受到严重抑制。通过在厌氧环境中孵育突变酶复合物、将延胡索酸还原酶复合物的催化亚基与其膜锚定物分离或阻断电子从该酶向醌的转移，可以防止这种失活。这些研究结果表明，在将此突变引入[3Fe-4S]结合结构域后，通过芬顿型反应产生的羟基自由基导致了琥珀酸诱导的失活。更多证据表明，底物诱导的失活需要醌，醌是琥珀酸脱氢酶和延胡索酸还原酶活性的膜结合电子受体和供体。这些数据表明，[3Fe-4S]簇与在延胡索酸还原酶和琥珀酸脱氢酶中发现的一个醌结合位点密切相关。
