The overall goals of the proposed work are to obtain data that will allow descriptions at the molecular level how the terminal two heme synthetic pathway enzymes, protoporphyrinogen oxidase (PRO) and ferrochelatase, function. The goals of the current proposal are to define catalytic and structural features of human PRO and ferrochelatase and bacterial oxygen-independent PRO. Potential protein-protein interactions involving these enzymes as well as cytochrome c and frataxin will be examined. The data obtained will describe the catalytic consequences of naturally occurring mutations in humans that lead to the inherited diseases known as porphyrias, and this should be of value in patient treatment and counseling. The data will also identify and characterize any key differences that exist between the human and microbial enzymes. These results may set the stage for development of new classes of antimicrobial agents.
Specific aims of this proposal are to: 1.) define structure-function characteristics of human PRO through the use of kinetics, site-directed mutagenesis, and structure-based studies, determine if cytochrome c is a bona fide electron acceptor for PPO and characterize this interaction, and isolate and characterize bacterial oxygen-independent protoporphyrinogen oxidase. 2.) define structure-function characteristics of ferrochelatase through the use of kinetics, site-directed mutagenesis, and structure-based studies, characterize the ferrochelatase:frataxin interaction and determine its relevance to ERR patients with ringed sideroblasts, and characterize the [2Fe-2S] cluster in animal and bacterial ferrochelatases through site-directed mutagenesis and physical examination. 3.) identify and characterize protein-protein interactions between ferrochelatase and PPO.
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