Spectral Studies of tert-Butyl Isothiocyanate-Inactivated P450 2E1

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• 作者：Ute M. Kent ; Elizabeth S. Roberts-Kirchhoff ; Namdoo Moon ; William R. Dunham ; and Paul F. Hollenberg
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：June 19, 2001
• 年：2001
• 卷：40
• 期：24
• 页码：7253 - 7261
• 全文大小：149K
• 年卷期：v.40,no.24(June 19, 2001)
• ISSN：1520-4995

文摘

Inactivation of cytochrome P450 2E1 by tert-butyl isothiocyanate (tBITC) resulted in a lossin the spectrally detectable P450-reduced CO complex. The heme prosthetic group does not appear tobecome modified, since little loss of the heme was observed in the absolute spectra or the pyridinehemochrome spectra, or in the amount of heme recovered from HPLC analysis of the tBITC-inactivatedsamples. Prolonged incubations of the inactivated P450 2E1 with dithionite and CO resulted in a recoveryof both the CO complex and the enzymatic activity. Inactivated samples that were first reduced withdithionite for 1 h prior to CO exposure recovered their CO spectrum to the same extent as samples notpretreated with dithionite, suggesting that the major defect was an inability of the inactivated sample tobind CO. Spectral binding studies with 4-methylpyrazole indicated that the inactivated P450 2E1 had animpaired ability to bind the substrate. Enzymatic activity could not be restored with iodosobenzene as thealternate oxidant. EPR analysis indicated that approximately 24% of the tBITC-inactivated P450 2E1was EPR-silent. Of the remaining tBITC-inactivated P450 2E1, approximately 45% exhibited an unusuallow-spin EPR signal that was attributed to the displacement of a water molecule at the sixth position ofthe heme by a tBITC modification to the apoprotein. ESI-LC-MS analysis of the inactivated P450 2E1showed an increase in the mass of the apoprotein of 115 Da. In combination, the data suggest that tBITCinactivated P450 2E1 by binding to a critical active site amino acid residue(s). This modified aminoacid(s) presumably acts as the sixth ligand to the heme, thereby interfering with oxygen binding andsubstrate binding.