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  Tracing the D-Pathway in Reconstituted Site-Directed Mutants of Cytochrome c Oxidase from Paracoccus denitrificans

Pfitzner, U., Hoffmeier, K., Harrenga, A., Kannt, A., Michel, H., Bamberg, E., et al. (2000). Tracing the D-Pathway in Reconstituted Site-Directed Mutants of Cytochrome c Oxidase from Paracoccus denitrificans. Biochemistry, 39(23), 6756-6762. doi:10.1021/bi992235x.

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 Creators:
Pfitzner, Ute1, Author
Hoffmeier, Klaus1, Author
Harrenga, Axel2, Author           
Kannt, Aimo2, Author           
Michel, Hartmut2, Author           
Bamberg, Ernst3, Author           
Ludwig, Bernd1, Author
Affiliations:
1Molekulare Genetik, Institut für Biochemie, Biozentrum, Johann Wolfgang Goethe-Universität, 60439 Frankfurt, Germany, ou_persistent22              
2Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068290              
3Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society, ou_2068289              

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Free keywords: Bioinorganic chemistry; Peptides and proteins; Genetics; Monomers; Molecules
 Abstract: Heme−copper terminal oxidases use the free energy of oxygen reduction to establish a transmembrane proton gradient. While the molecular mechanism of coupling electron transfer to proton pumping is still under debate, recent structure determinations and mutagenesis studies have provided evidence for two pathways for protons within subunit I of this class of enzymes. Here, we probe the D-pathway by mutagenesis of the cytochrome c oxidase of the bacterium Paracoccus denitrificans; amino acid replacements were selected with the rationale of interfering with the hydrophilic lining of the pathway, in particular its assumed chain of water molecules. Proton pumping was assayed in the reconstituted vesicle system by a stopped-flow spectroscopic approach, allowing a reliable assessment of proton translocation efficiency even at low turnover rates. Several mutations at positions above the cytoplasmic 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, N131D, exhibited an ideal decoupled phenotype, with a turnover comparable to that of the wild-type enzyme. Since sets of mutations in other positions along the presumed course of the pathway showed normal proton translocation stoichiometries, we conclude that the D-pathway is too wide in most areas above positions 131/199 to be disturbed by single amino acid replacements.

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Language(s): eng - English
 Dates: 2000-03-221999-09-242000-05-182000-06-01
 Publication Status: Published in print
 Pages: 7
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1021/bi992235x
PMID: 10841754
 Degree: -

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Title: Biochemistry
Source Genre: Journal
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Publ. Info: Columbus, Ohio : American Chemical Society
Pages: - Volume / Issue: 39 (23) Sequence Number: - Start / End Page: 6756 - 6762 Identifier: ISSN: 0006-2960
CoNE: https://pure.mpg.de/cone/journals/resource/954925384103