Studies of Biological Iron Oxidation

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0000989StahlTransition metals play an important role in cellular redox reactions. Iron, the most abundant transition metal ion in biology, poses a two-fold challenge: the reduced state can be toxic and the oxidized state is very insoluble under physiological conditions. Consequently, biological systems must maintain fine control over the uptake, transport, storage, and use of iron. This research project is to investigate a ferroxidase enzyme that plays a critical role in biological metabolism. Molecular biological and genetic studies of the organism Saccharomyces cerevisiae revealed the existence of a novel eukaryotic iron uptake pathway. A cell surface ferrireductase provides ferrous ions for two different membrane transport systems. One mediates iron(II) uptake under iron-replete conditions and also transports other divalent metal ions. The other consists of an oxidase-permease complex that operates under low-iron conditions and is very selective for iron(II). This latter one, Fet3p, is a 72 kDa membrane-bound ferroxidase that belongs to a class of enzymes known as multi-copper oxidases. Fet3p is a smaller protein that appears more amenable to detailed study because of the availability of a soluble variant that lacks the membrane domain. This project will explore several kinetic and structural aspects of Fet3p in order to gain mechanistic insights into the role of multicopper ferroxidases in iron homeostasis. This research will include aspects of dioxygen reduction as well as iron oxidation and will provide fundamental insights into the function of multicopper ferroxidases and their role in iron metabolism. Techniques that will be used to accomplish this task include, stopped-flow spectroscopic studies under different conditions such as pH, equilibrium dialysis, and NMR spectroscopy.l