Incorporation of the prosthetic heme group into cytoplasmic and membrane proteins

Abstract

Tetrapyrroles are macromolecules involved in nearly all fundamental biologically processes and further represent essential components of many organisms metabolism. They play a central role in electron transfer-dependent, energy-generating processes like photosynthesis and respiration. Heme, an iron containing tetrapyrrole acts as a prosthetic group for e. g. hemoglobin, which mediates oxygen transport in the blood. It was investigated how heme arrives at its functionally destination in cytoplasmic or membrane-bound proteins. The aim of this study was the identification of heme-binding or transporting proteins in vivo by a Pseudomonas aeruginosa Bacterial Adenylate Cyclase Two-Hybrid System. By means of these screenings a possible heme-transporter was isolated among many hydrophobic so far not characterized proteins. Further studies revealed this protein to be an iron-transporter, potentially responsible for iron transport to ferrochelatase. In a second project the E. coli protein HemW was to be biochemically and biophysically characterized. A Lactococcus lactis hemW mutant was known to accumulate free heme in the cell however not able to respire on supplementation with heme. An E. coli hemW mutant was successfully complemented with the hemW gene of L. lactis. The E. coli protein HemW was isolated under anaerobic conditions and chromatographically purified HemW contains a [4Fe-4S] cluster, like its L. lactis relative. Furthermore HemW dimerizes and binds heme specifically and covalently. During this binding-process an electron transfer from the [Fe-S] cluster to an as of yet to be identified bound heme takes place.