| Bioinorganic chemistry is the study of metal in biological systems. While transition metals such as copper, iron and zinc are essential to all living organisms, they can also be extremely toxic. The same properties that make them necessary render them highly dangerous when not properly regulated. Redox active metal ions like Cu(I) and Fe(II) react with O2 to produce not only Cu(II) and Fe(III) but also reactive oxygen species such as O 2-, H2O2 and OH˙ that can damage biological molecules like DNA, phospholipids, and proteins. Life has evolved a complex regulatory network to maintain metal homeostasis. Intracellular copper is of particular interest because it is involved in many essential processes and pathways. Cu(I) is the biologically relevant intracellular oxidation state of copper but it is difficult to study by traditional means because it is spectroscopically silent and redox sensitive in an aerobic environment. Isothermal titration calorimetry (ITC) allows for the direct measurement of heat produce or required by chemical interactions and a single ITC experiment can determine thermodynamic parameters K, DeltaH°, DeltaG°, and DeltaS°. In this work, particular roles of copper in yeast, fungus, bacteria and human proteins are explored, primarily focusing on the thermodynamics of copper ions binding to proteins and peptides. Using ITC, it has been determined that the extracellular methionine-rich Cu(I) binding domains of Ctr1, a membrane-embedded Cu(I) transporter, have an ∼5.0 x 105 affinity for Cu(I) while human Hah1 and the fourth cytosolic domain of human Wilson's disease protein bind Cu(I) have an affinity of ∼1.6 x 1010. This is consistent with the pervading theory that intracellular Cu(I) must be tightly sequestered along its transport pathway. The binding of Cu(I) and Cu(II) to the copper enzyme galactose oxidase and copper's role in the post-translational maturation of that enzyme have been investigated. |
