Layer Gunhild, Grage Katrin, Teschner Thomas, Schünemann Volker, Breckau Daniela, Masoumi Ava, Jahn Martina, Heathcote Peter, Trautwein Alfred X, Jahn Dieter
Institut für Mikrobiologie, Technische Universität Braunschweig, Spielmannstrasse 7, 38106 Braunschweig, Germany.
J Biol Chem. 2005 Aug 12;280(32):29038-46. doi: 10.1074/jbc.M501275200. Epub 2005 Jun 20.
The S-adenosylmethionine (AdoMet) radical enzyme oxygen-independent coproporphyrinogen III oxidase HemN catalyzes the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX during bacterial heme biosynthesis. The recently solved crystal structure of Escherichia coli HemN revealed the presence of an unusually coordinated iron-sulfur cluster and two molecules of AdoMet. EPR spectroscopy of the reduced iron-sulfur center in anaerobically purified HemN in the absence of AdoMet has revealed a 4Fe-4S cluster in two slightly different conformations. Mössbauer spectroscopy of anaerobically purified HemN has identified a predominantly 4Fe-4S cluster in which only three iron atoms were coordinated by cysteine residues (isomer shift of delta = 0.43 (1) mm/s). The fourth non-cysteine-ligated iron exhibited a delta = 0.57 (3) mm/s, which shifted to a delta = 0.68 (3) mm/s upon addition of AdoMet. Substrate binding by HemN did not alter AdoMet coordination to the cluster. Multiple rounds of AdoMet cleavage with the formation of the reaction product methionine indicated AdoMet consumption during catalysis and identified AdoMet as a co-substrate for HemN catalysis. AdoMet cleavage was found to be dependent on the presence of the substrate coproporphyrinogen III. Two molecules of AdoMet were cleaved during one catalytic cycle for the formation of one molecule of protoporphyrinogen IX. Finally, the binding site for the unusual second, non iron-sulfur cluster coordinating AdoMet molecule (AdoMet2) was targeted using site-directed mutagenesis. All AdoMet2 binding site mutants still contained an iron-sulfur cluster and most still exhibited AdoMet cleavage, albeit reduced compared with the wild-type enzyme. However, all mutants lost their overall catalytic ability indicating a functional role for AdoMet2 in HemN catalysis. The reported significant correlation of structural and functional biophysical and biochemical data identifies HemN as a useful model system for the elucidation of general AdoMet radical enzyme features.
S-腺苷甲硫氨酸（AdoMet）自由基酶——不依赖氧气的粪卟啉原III氧化酶HemN，在细菌血红素生物合成过程中催化粪卟啉原III氧化脱羧生成原卟啉原IX。最近解析的大肠杆菌HemN晶体结构显示存在一个配位异常的铁硫簇和两个AdoMet分子。在无氧纯化的HemN中，于不存在AdoMet的情况下对还原态铁硫中心进行电子顺磁共振光谱分析，揭示了处于两种略有不同构象的4Fe-4S簇。对无氧纯化的HemN进行穆斯堡尔光谱分析，确定了一个主要为4Fe-4S的簇，其中只有三个铁原子由半胱氨酸残基配位（δ的同质异能位移为0.43(1)mm/s）。第四个非半胱氨酸配位的铁的δ值为0.57(3)mm/s，在添加AdoMet后移至0.68(3)mm/s。HemN与底物的结合并未改变AdoMet与该簇的配位。多轮AdoMet裂解并形成反应产物甲硫氨酸，表明在催化过程中AdoMet被消耗，并确定AdoMet是HemN催化的共底物。发现AdoMet裂解依赖于底物粪卟啉原III的存在。在一个催化循环中，为了形成一分子原卟啉原IX，有两个AdoMet分子被裂解。最后，使用定点诱变技术针对与异常的第二个非铁硫簇配位的AdoMet分子（AdoMet2）的结合位点进行研究。所有AdoMet2结合位点突变体仍含有一个铁硫簇，并且大多数仍表现出AdoMet裂解，尽管与野生型酶相比有所降低。然而，所有突变体都失去了整体催化能力，这表明AdoMet2在HemN催化中具有功能作用。所报道的结构与功能的生物物理和生化数据之间的显著相关性，将HemN确定为阐明一般AdoMet自由基酶特征的有用模型系统。
