A novel oxo state of cytochrome c oxidase from Paracoccus denitrificans generated by successive addition of excess H₂O₂ and ammonia was investigated using resonance Raman (RR) spectroscopy. Addition of ammonia to the H₂O₂-generated artificial F state resulted in an upshift of the oxoferryl stretching vibration from 790 to 796 cm ^-1, indicating that ammonia influences ligation of the heme-bound oxygen in the binuclear center. Concomitantly performed RR measurements in the high-frequency region between 1300 and 1700 cm^-1 showed a high-spin to low-spin transition of heme a₃ upon generation of the F state that was not altered by addition of ammonia. Removal of H₂O₂ by addition of catalase resulted in the disappearance of the oxoferryl stretching vibration and major back transformation of heme a₃ into the high-spin state. The ratio of high-spin to low-spin states was identical for intermediates created with and without ammonia, leading to the conclusion that ammonia does not interact directly with heme a₃. Only for the ammonia-created state was a band at 612 nm observed in the UV-visible difference spectrum that was shifted to 608 nm after addition of catalase. Our results support the hypothesis by von der Hocht et al. [von der Hocht, I., et al. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 3964-3969] that addition of ammonia creates a novel oxo intermediate state called P_N where ammonia binds to Cu_B once the oxo intermediate F state has been formed.