Tracing the D-pathway in reconstituted site-directed mutants of cytochromec oxidase from Paracoccus denitrificans

Heme-copper terminal oxidases use the free energy of oxygen reduction to es tablish a transmembrane proton gradient. While the molecular mechanism of c oupling electron transfer to proton pumping is still under debate, recent s tructure determinations and mutagenesis studies have provided evidence for two pathways for protons within subunit I of this class of enzymes. Here, w e probe the D-pathway by mutagenesis of the cytochrome c oxidase of the bac terium Paracoccus denitrificans; amino acid replacements were selected with the rationale of interfering with the hydrophilic lining of the pathway, i n particular its assumed chain of water molecules. Proton pumping was assay ed in the reconstituted vesicle system by a stopped-flow spectroscopic appr oach, allowing a reliable assessment of proton translocation efficiency eve n at low turnover rates. Several mutations at positions above the cytoplasm ic pathway entrance (Asn 131, Asn 199) and at the periplasmic exit region ( Asp 399) led to complete inhibition of proton pumping; one of these mutants , N13 ID, exhibited an ideal decoupled phenotype, with a turnover comparabl e to that of the wild-type enzyme. Since sets of mutations in other positio ns along the presumed course of the pathway showed normal proton translocat ion stoichiometries, we conclude that the D-pathway is too wide in most are as above positions 131/199 to be disturbed by single amino acid replacement s.