Biochemistry of coproporphyrin ferrochelatases

Heme is essential for the survival of most bacteria. Gram-positive organisms produce heme in a way that is fundamentally different from the biosynthetic pathway used by Gram-negative organisms or even mammals. Many mechanistic questions relating to this heme biosynthetic pathway, which was only described a few years ago, are currently still open. In this project, the enzyme called "coproporphyrin ferrochelatase" is being studied in detail to elucidate structure-function relationships. Ferrochelatases incorporate ferrous iron atoms into a porphyrin ring and have been investigated in protein biochemical research for several decades. However, the studies have always been carried out with a substrate that, as has just been shown, is not physiologically relevant, namely protoporphyrin IX. The correct substrate, however, is coproporphyrin III, which has two more propionate groups than protoporphyrin IX and therefore has different binding properties. Coproporphyrin ferrochelatase from the Gram-positive pathogenic bacterium Listeria monocytogenes and variants, in which individual amino acid residues are exchanged at catalytically important sites, are produced recombinantly in Escherichia coli and purified to obtain a highly pure protein for biochemical and biophysical characterizations. Using several sophisticated spectroscopic, structural and biochemical analysis methods, the natural wild-type form of coproporphyrin ferrochelatase is studied in detail and compared with the variants that are punctually altered. From these studies essential conclusions can be drawn about the mode of action of this enzyme and can be linked to its structural properties. Knowledge of the reaction mechanism of copropophryin ferrochelatases is necessary to design further studies that will attempt to inhibit enzymatic activity. A substance that can specifically inhibit the heme biosynthesis pathway of pathogenic Gram-positive bacteria is a promising starting point for the development of urgently needed novel antibiotics.

In the course of this research project, a special enzyme of bacterial heme biosynthesis was fundamentally investigated. Ferrochelatases incorporate an iron atom into a so-called porphyrin ring to produce vital heme. We have investigated exactly how this happens. The most important questions concerned the interaction of the substrates with the protein and the structural consequences. With the mechanistic knowledge we have accumulated, a big step has been made towards understanding this enzyme-catalyzed reaction. This knowledge will help us in the search for novel antibiotics, as heme biosynthesis in Gram-positive pathogens can then be specifically inhibited, preventing the production of heme, which is essential for the bacteria. In particular, we have discovered that the porphyrin ring, in which the iron is incorporated, shows very specific deformations (e.g. like a saddle) during catalysis, which are relevant for iron incorporation. We found this out by means of detailed biochemical and biophysical, especially spectroscopic methods. On the one hand, we determined the complex structures of the enzymes with the substrates using X-ray crystallography and, on the other hand, we examined the same structures in a complementary way using resonance Raman spectroscopy. In this method, laser light of a specific wavelength is scattered and the changes in the scattered light are analyzed. The research results of this project form a good basis for further investigations. Basic research is essential for progress and further development in science, in this case in the search for urgently needed new antibacterial substances. Many Gram-positive pathogens such as Staphylococcus aureus, but also Listeria monocytogenes, show multiple resistances to current antibiotic therapies. Since the bacterial heme biosynthesis of Gram-positive organisms differs substantially from that of humans, this biosynthetic pathway is a suitable starting point for such research.