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Abstract

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 (E78^II) in the Paracoccus denitrificans cytochrome c oxidase do not affect its catalytic activity at all (E78^IIQ) or reduce it to about 50% (E78^IIA); 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 E78^II 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 approximately 1760 to 1710 cm^-1 excludes the protonation of E78^II 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 E78^II in the Paracoccus enzyme.