Physical characterisation of Dcytb

Summary: Hemochromatosis is the most common hereditary disorder among Caucasians. In this disease, the regulation of iron homeostasis as an interplay between iron uptake from nutrition and iron excretion via the feces and urine is disturbed. This leads to an increased iron uptake despite full body iron stores. Radical formation as a consequence of increased iron deposits in the organs may lead to organ dysfunction and failure, especially in the liver, heart and pancreas. In molecular terms, the iron transporter divalent metal transporter-1 (DMT-1) and its associated reductase duodenal cytochrome b (Dcytb) are mainly responsible for iron uptake from nutrition in the enterocyte. Dcytb reduces ferric iron that predominates in the diet to ferrous iron which can be used as substrate for DMT-1. Both protein levels are higher in hemochromatotic than in healthy individuals leading to the increased iron absorption. Aim of the study: As phlebotomy is the only therapy available for hemochromatotics medicamentous treatment that limits iron uptake from the diet and increases iron excretion would be beneficial. Therefore, I want to characterize the reductase Dcytb by physical and electrochemical measurements for further elucidation of the reaction mechanism, the kinetic and the dependence of intra- and extracellular factors. These investigations should finally allow to develop of antagonists of Dcytb. Methods: Dcytb will be purified from prokaryotic and eukaryotic expression systems. The reduced and oxidized forms of Dcytb will be first characterized in dependence of the pH value and temperature with electronic, electroparamagnetic(EPR) and circular dichroism(CD) spectroscopy. The electron transfer properties of Dcytb will be studied by photochemical titration, stopped-flow spectroscopy and flash photolysis. Generation of mutants should provide insight into the reaction mechanism of Dcytb with iron and ascorbic acid.