The reaction of peroxynitrite (PN) with purified human cytochrome P450 3A4 (CYP3A4) resulted in the loss of the reduced-CO difference spectrum, but the absolute absorption spectrum of the heme was not significantly altered. The loss of 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC) O-debenzylation activity of CYP3A4 was concentration-dependent with respect to PN, and the loss of BFC activity supported by NADPH-cytochrome P450 reductase (CPR) was much greater than that supported by tert-butyl hydroperoxide. Moreover, the PN-treated CYP3A4 exhibited a reduced-CO spectrum when reduced by CPR that was much smaller than when it was reduced by dithionite. These results suggest that modification of CYP3A4 by PN may impair its interaction with CPR, leading to the loss of catalytic activity. Tyrosine nitration, as measured by an increase in mass of 45 Da due to the addition of a nitro group, was used as a biomarker for protein modification by PN. PN-treated CYP3A4 was digested by trypsin and endoproteinase Glu C, and nitrotyrosine formation was then determined by using electrospray ionization-liquid chromatography-tandem mass spectrometry. Tyr residues 99, 307, 347, 430, and 432 were found to be nitrated. Using the GRAMM-X docking program, the structure for the CYP3A4-CPR complex shows that Tyr99, Tyr347, and Tyr430 are on the proximal side of CYP3A4 and are in close contact with three acidic residues in the FMN domain of CPR, suggesting that modification of one or more of these tyrosine residues by PN may influence CPR binding or the transfer of electrons to CYP3A4. Mutagenesis of Tyr430 to Phe or Val revealed that both the aromatic and the hydroxyl groups of Tyr are required for CPR-dependent catalytic activity and thus support the idea that the proximal side Tyr participates in the 3A4-CPR interaction. In conclusion, modification of tyrosine residues by PN and their subsequent identification can be used to enhance our knowledge of the structure/function relationships of the P450s with respect to the electron transfer steps, which are critical for P450 activity.