Incorporation of the prosthetic heme group into cytoplasmatic and membrane proteins

含有血红素的蛋白质，如酶、传感器和电子传递细胞色素，对几乎所有生物体的新陈代谢都是必不可少的。在细菌中，血红素的转运和插入到细胞质和膜目标中所知甚少。在上一个资助期内，在乳酸乳球菌中鉴定出血红素结合蛋白HemW。重组厌氧纯化的大肠杆菌和乳杆菌HemW含有一个[4Fe-4S]簇和s -腺苷蛋氨酸（SAM）。在血红素结合后，蛋白质形成二聚体，其中电子从[4Fe-4S]簇转移到血红素上。二聚体的目标是膜提供血红素在nadh依赖的反应膜定位的电子转移蛋白。接下来，我们打算使用多种光谱方法（EPR，拉曼共振，Mössbauer， MCD， ITC）来确定共价血红素结合的性质，轴向血红素配体以及与[4Fe-4S]簇和SAM的相互作用。HemW的靶酶和nadh依赖性转移的潜在附加组分将被识别和表征。目的是测定HemW晶体结构。将研究枯草芽孢杆菌和人体内的血红蛋白相关蛋白。了解硝酸还原酶等模式酶中的合作血红素和Moco插入是一个长期的目标。

Heme containing proteins, like enzymes, sensors and electron transporting cytochromes are essential for the metabolism of almost all organisms. Little is known about the transport and insertion of hemes into their cytoplasmic and membrane targets in bacteria. During the last funding period the heme binding protein HemW was identified in Lactococcus lactis. Recombinant anaerobically purified, monomeric Escherichia coli and L. lactis HemW contained a [4Fe-4S] cluster and S-adenosylmethionine (SAM). Upon heme binding the protein forms a dimer in which electrons are transferred from the [4Fe-4S] cluster to heme. The dimer is targeted to the membrane to supply heme in a NADH-dependent reaction to membrane localized electron transfer proteins. Next, we intend to determine the nature of covalent heme binding, the axial heme ligands and the interplay with the [4Fe-4S] cluster and SAM using multiple spectroscopic methods (EPR, Raman resonance, Mössbauer, MCD, ITC). Target enzymes of HemW and potential additional components of the NADHdependent transfer will be identified and characterized. HemW crystal structure determination is intended. HemW related proteins from Bacillus subtilis and human will be investigated. The understanding of the cooperative heme and Moco insertion in model enzymes like nitrate reductase is a long time goal.