ATR-FTIR Spectroscopy of the P sub(M) and F Intermediates of Bovine and Paracoccus denitrificans Cytochrome c Oxidase

• Published in: Biochemistry (Easton) Vol. 42; no. 29; pp. 8809 - 8817
• Main Authors: Iwaki, Masayo, Puustinen, A, Wikstroem, M, Rich, PR
• Format: Journal Article
• Language: English
• Published: 29.07.2003
• Subjects: Paracoccus denitrificans
• ISSN: 0006-2960
• DOI: 10.1021/bi034522d

The structures of P sub(M) and F intermediates of bovine and Paracoccus denitrificans cytochrome c oxidase were investigated by perfusion-induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. Transitions from the "fast" oxidized state to the P sub(M) or F states were initiated by perfusion with buffer containing either CO/oxygen or H sub(2)O sub(2). Intermediates were quantitated by simultaneous monitoring of visible absorption changes in the protein film. For both bovine and P. denitrificans oxidase, the major features of the IR difference spectrum of P sub(M) were similar when produced by CO/oxygen or by H sub(2)O sub(2) treatments. These IR difference spectra were distinctly different from the IR difference spectrum of F that formed with extended treatment with H sub(2)O sub(2). Some IR bands could be assigned tentatively to perturbations of heme a sub(3) ring modes and substituents, and these perturbations were greater in P sub(M) than in F. Other bands could be assigned to surrounding protein changes. Strong perturbation of the environment of a carboxylic acid, most likely E-242 (bovine numbering), occurred in P sub(M) and relaxed back in F. A second redox-sensitive carboxylic acid was also perturbed in the bovine P sub(M) intermediate. Further consistent signatures of P sub(M) in both oxidases that were absent in F were strong negative bands at 1547 and 1313 cm super(-1) in bovine oxidase (1542 and 1314 cm super(-1) in P. denitrificans) and a positive band at approximately 1519 cm super(-1). From comparison with available IR data on model compounds, it is suggested that these reflect changes in the covalent tyrosine-histidine ligand to Cu sub(B). These findings are discussed in relation to the oxidase catalytic cycle.