| Cytochrome c, a heme protein involved in electron transfer and energy production, is also an important protein in the initiation of apoptosis, making it an attractive therapeutic target. When a cell receives an apoptotic signal, cytochrome c is released from mitochondria into the cytosol, and the cell dies. Cytochrome c is synthesized in the cytosol as a precursor protein, imported into mitochondria, and the mature protein is formed by the enzyme-catalyzed covalent attachment of heme to the apoprotein. It was recently shown that holocytochrome c can be formed from apocytochrome c in vitro without the assistance of an enzyme over the course of 24 hours. Inspired by these recent results, we set out to design a heme-analog that when administered to cancer cells would rapidly attach to apocytochrome c in the cytosol, cause formation of cytochrome c, and induce apoptosis.; Two heme analogs were prepared by the modification of both heme vinyl groups to ethynyl groups (iron diethynyl porphyrin, FeEP) or formyl groups (iron diformyl porphyrin, FeFP). FeEP covalently attached to apocytochrome c on a similar time scale as attachment of heme to apocytochrome c. FeFP attached to apocytochrome c within 30 minutes, more than 40 times faster than attachment of heme to apocytochrome c. The FeFP-cytochrome c adduct was confirmed by electrophoresis and mass spectrometry.; Leukemia and breast cancer cells were incubated with FeFP, but contrary to our hypothesis, FeFP did not induce apoptosis and was instead cytoprotective, a property that could be utilized as a therapy for a different set of disease states in which too much apoptosis occurs. We investigated several possible properties of FeFP and the FeFP-cytochrome c adduct that could explain the cytoprotection, specifically peroxidase and peroxynitrite decomposition activities. FeFP had very weak peroxidase activity, and FeFP-cytochrome c had negligible peroxidase activity. However, both FeFP and FeFP-cytochrome c had moderate peroxynitrite decomposition activity, with FeFP having activity 3.6 times higher than FeFP-cytochrome c. Peroxynitrite decomposition catalysts are important as they can treat a number of diseases including diabetes and Alzheimer's disease or prevent damage after a heart attack or stroke. |
