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  Exploring the entrance of proton pathways in cytochrome c oxidase from Paracoccus denitrificans: Surface charge, buffer capacity and redox-dependent polarity changes at the internal surface

Kirchberg, K., Michel, H., & Alexiev, U. (2013). Exploring the entrance of proton pathways in cytochrome c oxidase from Paracoccus denitrificans: Surface charge, buffer capacity and redox-dependent polarity changes at the internal surface. Biochimica et Biophysica Acta, Bioenergetics, 1827(3), 276-284. doi:10.1016/j.bbabio.2012.10.014.

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 Creators:
Kirchberg, Kristina1, 2, Author              
Michel, Hartmut1, Author              
Alexiev, Ulrike2, Author
Affiliations:
1Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068290              
2Physics Department, Freie Universität Berlin, Berlin, Germany, ou_persistent22              

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Free keywords: Cytochrome c oxidase; pH-indicator dye; Covalent surface labelling; Surface potential; Buffer capacity; Fluorescein
 Abstract: Cytochrome c oxidase (CcO), the terminal oxidase of cellular respiration, reduces molecular oxygen to water. The mechanism of proton pumping as well as the coupling of proton and electron transfer is still not understood in this redox-linked proton pump. Eleven residues at the aqueous-exposed surfaces of CcO from Paracoccus denitrificans have been exchanged to cysteines in a two-subunit base variant to yield single reactive cysteine variants. These variants are designed to provide unique labeling sites for probes to be used in spectroscopic experiments investigating the mechanism of proton pumping in CcO. To this end we have shown that all cysteine variants are enzymatically active. Cysteine positions at the negative (N-) side of the membrane are located close to the entrance of the D- and K-proton transfer pathways that connect the N-side with the catalytic oxygen reduction site. Labeling of the pH-indicator dye fluorescein to these sites allowed us to determine the surface potential at the cytoplasmic CcO surface, which corresponds to a surface charge density of −0.5 elementary charge/1000 Å2. In addition, acid–base titrations revealed values of CcO buffer capacity. Polarity measurements of the label environment at the N-side provided (i) site-specific values indicative of a hydrophilic and a more hydrophobic environment dependent on the label position, and (ii) information on a global change to a more apolar environment upon reduction of the enzyme. Thus, the redox state of the copper and heme centers inside the hydrophobic interior of CcO affect the properties at the cytoplasmic surface.

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Language(s): eng - English
 Dates: 2012-10-182012-08-062012-10-252012-10-312013-03
 Publication Status: Published in print
 Pages: 9
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1016/j.bbabio.2012.10.014
 Degree: -

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Title: Biochimica et Biophysica Acta, Bioenergetics
  Abbreviation : Biochim. Biophys. Acta, Bioenerg.
Source Genre: Journal
 Creator(s):
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Publ. Info: Amsterdam : Elsevier
Pages: - Volume / Issue: 1827 (3) Sequence Number: - Start / End Page: 276 - 284 Identifier: ISSN: 0005-2728
CoNE: https://pure.mpg.de/cone/journals/resource/954926938702_6