Proteins are targets of reactive oxygen species (ROS) that are produced in the environment and in the course of normal and xenobiotic metabolism. Protein oxidation is often detrimental, but it may be involved in normal biological processes. Hydrogen peroxide is a common ROS produced in the environment and in the cell, e.g. the mitochondrion. Hydrogen peroxide is the mildest of the ROS and is the most likely to act at sites distant from it's generation. Rhodanese, a sulfur-transfer enzyme in the mitochondrial matrix, can regulate iron-sulfur centers, and it may be controlled by oxidation. Rhodanese can be reversibly and irreversibly modified by hydrogen peroxide. Properties of modified rhodanese are those reported to be involved in damage, aging and turnover. This research program will characterize the altered forms and will determine the mechanism for conformational changes triggered by oxidation. Analyses can be pursued in unprecedented detail because of extensive kinetic and X-ray information already available for rhodanese. Our working hypotheses are: a) oxidation can be considered as a post- translational protein modification; b) oxidative damage is progressive and can be initiated by reaction at specific sites; c) oxidative effects can be propagated to distant regions of the protein; and d) oxidation and conformational changes are reciprocally linked, each facilitating the other. The specific approaches would be as follows: I. Oxidized amino acids will be located in the sequence of reversibly and irreversibly oxidized rhodanese. Oxidation states of sulfhydryl groups will be determined. II. Sequencing methods will be used to determine regions of the protein, not directly oxidized, that respond to oxidation. This is possible because oxidation reveal new antigenic determinants and exposes sites of proteolysis. III. Non-covalent conformational consequences of oxidation will be probed by physico-chemical methods. Changes in conformation, flexibility and domain interactions will be measured using: fluorescence, circular dichroism, tritium exchange, light scattering and hydrophobic chromatography. IV. Directed mutagenesis will modify regions of rhodanese suggested to either trigger or respond to oxidative changes. Site directed mutagenesis will change cys to ser and remove prolines in the interdomain tether. The N-terminal residues 1-23 will be deleted. C-terminal deletions will be produced by treating recombinant rhodanese with carboxypeptidase. These species will be studied as above.
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