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Resonance Raman Characterization of the Ammonia-Generated Oxo Intermediate of Cytochrome c Oxidase from Paracoccus denitrificans

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von der Hocht,  Iris
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Hilbers,  Florian
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Michel,  Hartmut
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Citation

Kozuch, J., von der Hocht, I., Hilbers, F., Michel, H., & Weidinger, I. M. (2013). Resonance Raman Characterization of the Ammonia-Generated Oxo Intermediate of Cytochrome c Oxidase from Paracoccus denitrificans. Biochemistry, 52(36), 6197-6202. doi:10.1021/bi400535m.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0024-D4A2-4
Abstract
A novel oxo state of cytochrome c oxidase from Paracoccus denitrif icans 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 −1 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 −1. 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 PN where ammonia binds to CuB once the oxo intermediate F state has been formed.