Incorporation of the prosthetic heme group into cytoplasmatic and membrane proteins

点击翻译按钮获取中文摘要

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.