Heme peptide as a model substance for halogenomethane activation and heme modification.

Octa-heme peptide (CHP) obtained from Candida krusei cytochrome c was tested for suicidal activation of halogenomethanes. Under anaerobic conditions, CHP was kept in the reduced state in the presence of NADPH and NADPH-cytochrome P-450 reductase. Addition of CBrCl3 to the reduced CHP caused spectral changes such as rapid disappearance of alpha and beta bands and gradual decrease in the gamma-peak height, accompanied by oxidation of NADPH. Heme content of the reaction mixture, determined as pyridine hemochrome, also decreased NADPH dependently. CCl4 was less effective than CBrCl3, while CHCl3 had almost no effect. N-tert-butyl-alpha-phenylnitrone (PBN) suppressed the CBrCl3-induced heme damage, and resulted in the formation of radical adduct .PBN-CCl3 as evidenced by ESR spectroscopy. Radical formation was also observed with CCl4. The CHP damage induced by CBrCl3 was also accompanied by the release of Br- about 11-12-times molar excess of CHP, whereas the release of CHCl3 was about 20% that of Br-.FD-MS assay of the product of CHP reaction suggested that 10 trichloromethyl radicals bonded with CHP. Thus, CBrCl3 undergoes single-electron reduction in the presence of reduced CHP to trichloromethyl radicals, which covalently bind to CHP molecules. Heme peptide may be a useful tool in the study of mechanisms involved in the destruction of cytochrome P-450 by halogenomethanes.