Spectral studies of tert-butyl isothiocyanate-inactivated P450 2E1

Inactivation of cytochrome P450 2E1 by tert-butyl isothiocyanate (tBITC) re sulted in a loss in the spectrally detectable P450-reduced CO complex. The heme prosthetic group does not appear to become modified, since little loss of the heme was observed in the absolute spectra or the pyridine hemochrom e spectra, or in the amount of heme recovered from HPLC analysis of the tBI TC-inactivated samples. Prolonged incubations of the inactivated P450 2E1 w ith dithionite and CO resulted in a recovery of both the CO complex and the enzymatic activity. Inactivated samples that were first reduced with dithi onite for 1 h prior to CO exposure recovered their CO spectrum to the same extent as samples not pretreated with dithionite, suggesting that the major defect was an inability of the inactivated sample to bind CO. Spectral bin ding studies with 4-methylpyrazole indicated that the inactivated P450 2E1 had an impaired ability to bind the substrate. Enzymatic activity could not be restored with iodosobenzene as the alternate oxidant, EPR analysis indi cated that approximately 24% of the tBITC-inactivated P450 2E1 was EPR-sile nt. Of the remaining tBITC-inactivated P450 2E1, approximately 45% exhibite d an unusual low-spin EPR signal that was attributed to the displacement of a, water molecule at the sixth position of the heme by a tBITC modificatio n to the apoprotein. ESI-LC-MS analysis of the inactivated P450 2E1 showed an increase in the mass of the apoprotein of 115 Da. In combination, the da ta suggest that tBITC inactivated P450 2E1 by binding to a critical active site amino acid residue(s). This modified amino acid(s) presumably acts as the sixth ligand to the heme, thereby interfering with oxygen binding and s ubstrate binding.