9513047 Erman The long-term goal of this research is to elucidate the structural features which control heme protein reactivity and determine heme protein function. The research will compare proteins from two different functional classes: (1) the heme enzymes and (2) the oxygen transport/storage proteins. Of particular interest is the chemical reactivity of the Fe(III) state of heme proteins. The properties of CcP will be compared with those of the Fe(III) state of two oxygen transport/storage proteins, mammalian metmyoglobin (MetMb) and Glycera dibranchiata methemoglobin (metGHb) in an effort to understand the structural features which are responsible for the reactivity differences in these two classes of heme proteins. The first aspect of Fe(III) heme protein reactivity to be investigated is the reaction which characterizes the peroxidases, the reaction between the Fe(III) protein and H2O2 to form an oxidized enzyme intermediate called Compound I. Initial studies, using site-directed mutagenesis to alter protein structure, indicate that two amino acid residues in CcP are critical in the formation of Compound I, the distal histidine His-52 and a distal arginine (Arg-48). Arg-48 facilitates oxygen-oxygen bond cleavage while His-52 promotes peroxide binding to the heme iron. Elucidation of the differences in the properties of the distal histidine in the peroxidases and the oxygen transport/storage heme proteins is one of the specific objectives of this proposal. A key question concerning the reactivity of the Fe(III) state of heme proteins is access of small reactants and ligands to the distal heme pocket. Active-site access will be probed by investigating the rates of binding of small ligands such as fluoride, cyanide, and nitric oxide to the iron in CcP, metMb, metGHb and their mutants. Discrimination between the neutral and charged forms of the ligands will be determined. Steric constraints, acid-base behavior, and the polarity of the heme pocket will be investigated through site-directed mutagenesis. The studies outlined in this proposal will provide a more sophisticated level of understanding of heme protein reactivity and function. It will provide a basis for rational protein design. The proposal includes plans to convert metGHb into an efficient peroxidase and more importantly, to convert CcP into a versatile hydroxylase, utilizing H2O2 rather than O2. %%% Heme proteins perform many important functions in living organisms including the transport of oxygen to its site of utilization and the protection of the organism from reactive oxygen species. (Reactive oxygen species such as superoxide, hydrogen peroxide, and the hydroxyl radical are thought to accelerate the aging process.) The goal of the research proposed in this project is to determine how the structure of various heme proteins govern their biological function. Two heme proteins, with different functions, will be extensively investigated by systematic manipulation of their structures. Observation of the biological reactivity of the altered proteins will allow correlations to be made concerning structure and function. The studies will provide a sophisticated level of understanding of heme protein reactivity and function. These studies will also provide a basis for the rational design of heme proteins to carry out processes which may have important commercial applications. ***
