Probing the access of protons to the K pathway in the Paracoccus denitrificans cytochrome c oxidase

• Published in: The FEBS journal 2005, Vol.272 (2), p.404-412
• Main Authors: Richter, Oliver-M.H, Dürr, Katharina L, Kannt, Aimo, Ludwig, Bernd, Scandurra, Francesca M, Giuffrè, Alessandro, Sarti, Paolo, Hellwig, Petra
• Format: Article
• Language: English
• Subjects: cytochrome c oxidase; electron transfer; Electron Transport Complex IV - chemistry; FTIR; Hydrogen-Ion Concentration; Mutagenesis, Site-Directed; Paracoccus denitrificans; Paracoccus denitrificans - enzymology; proton channel; Protons; site‐directed mutagenesis; Spectroscopy, Fourier Transform Infrared; Static Electricity
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2004.04480.x

In recent studies on heme-copper oxidases a particular glutamate residue in subunit II has been suggested to constitute the entry point of the so-called K pathway. In contrast, mutations of this residue (E78II) in the Paracoccus denitrificans cytochrome c oxidase do not affect its catalytic activity at all (E78IIQ) or reduce it to about 50% (E78IIA); in the latter case, the mutation causes no drastic decrease in heme a₃ reduction kinetics under anaerobic conditions, when compared to typical K pathway mutants. Moreover, both mutant enzymes retain full proton-pumping competence. While oxidized-minus-reduced Fourier-transform infrared difference spectroscopy demonstrates that E78II is indeed addressed by the redox state of the enzyme, absence of variations in the spectral range characteristic for protonated aspartic and glutamic acids at ≈ 1760 to 1710 cm⁻¹ excludes the protonation of E78II in the course of the redox reaction in the studied pH range, although shifts of vibrational modes at 1570 and 1400 cm⁻¹ reflect the reorganization of its deprotonated side chain at pH values greater than 4.8. We therefore conclude that protons do not enter the K channel via E78II in the Paracoccus enzyme.