Heme proteins are involved in a large number of physiologically important processes, including: delivery of oxygen to the tissues; derivation of stored energy from oxidation of nutrients; protective destruction of potentially dangerous peroxides; and activation and elimination of toxic pollutants and carcinogens. Despite this remarkable functional diversity, all of these species employ the heme group as the active component. Identification of the factors responsible for this varied function would be of obvious benefit for attaining an understanding of the behavior in normal and abnormal states. The long term objective of this research program is the elucidation of the molecular mechanisms by which these various proteins interact with the common heme group to effectively regulate its reactivity. Basically, our approach is to systematically perturb the structure of the heme group and quantitatively determine the functional consequences using appropriate methodology. The equilibria and kinetics of ligand binding to oxygen transport proteins will be investigated using stopped-flow instrumentation and a continuous oxygenation curve analyzer. Commonly accepted enzyme assays will be used to characterize the function of modified derivatives of other heme proteins to be studied. In order to elucidate the molecular basis of altered function, a number of powerful spectroscopic techniques will be used to probe key interactions between the heme and protein and to determine the effect of the structural perturbation on the electronic structure and reactivity of the active site. Spectroscopic methods to be used include: resonance Raman, nuclear magnetic resonance (NMR), infrared, circular dichroism (CD) and electronic absorption spectroscopy. This multifaceted approach, which utilizes a variety of techniques, applied to systematically modified proteins represents a realistic attempt to elucidate the important molecular control mechanisms responsible for such remarkable functional diversity.
