Entrance of the proton pathway in cbb 3-type heme-copper oxidases

Hyun Ju Lee, Robert B. Gennis, Pia Ädelroth

Research output: Contribution to journalArticle

Abstract

Heme-copper oxidases (HCuOs) are the last components of the respiratory chain in mitochondria and many bacteria. They catalyze O 2 reduction and couple it to the maintenance of a proton-motive force across the membrane in which they are embedded. In the mitochondrial-like, A family of HCuOs, there are two well established proton transfer pathways leading from the cytosol to the active site, the D and the K pathways. In the C family (cbb 3) HCuOs, recent work indicated the use of only one pathway, analogous to the K pathway. In this work, we have studied the functional importance of the suggested entry point of this pathway, the Glu-25 (Rhodobacter sphaeroides cbb3 numbering) in the accessory subunit CcoP (E25 P). We show that catalytic turnover is severely slowed in variants lacking the protonatable Glu-25. Furthermore, proton uptake from solution during oxidation of the fully reduced cbb3 by O 2 is specifically and severely impaired when Glu-25 was exchanged for Ala or Gln, with rate constants 100-500 times slower than in wild type. Thus, our results support the role of E25 P as the entry point to the proton pathway in cbb 3 and that this pathway is the main proton pathway. This is in contrast to the A-type HCuOs, where the D (and not the K) pathway is used during O 2 reduction. The cbb 3 is in addition to O 2 reduction capable of NO reduction, an activity that was largely retained in the E25 P variants, consistent with a scenario where NO reduction in cbb 3 uses protons from the periplasmic side of the membrane.

Original languageEnglish (US)
Pages (from-to)17661-17666
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume108
Issue number43
DOIs
StatePublished - Oct 25 2011

Fingerprint

Heme
Protons
Rhodobacter sphaeroides
Proton-Motive Force
Membranes
Electron Transport
Cytosol
Catalytic Domain
Mitochondria
Maintenance
copper oxidase
Bacteria

Keywords

  • Glutamate
  • Nitric oxide
  • Oxygen reduction

ASJC Scopus subject areas

  • General

Cite this

Entrance of the proton pathway in cbb 3-type heme-copper oxidases. / Lee, Hyun Ju; Gennis, Robert B.; Ädelroth, Pia.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 108, No. 43, 25.10.2011, p. 17661-17666.

Research output: Contribution to journalArticle

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AB - Heme-copper oxidases (HCuOs) are the last components of the respiratory chain in mitochondria and many bacteria. They catalyze O 2 reduction and couple it to the maintenance of a proton-motive force across the membrane in which they are embedded. In the mitochondrial-like, A family of HCuOs, there are two well established proton transfer pathways leading from the cytosol to the active site, the D and the K pathways. In the C family (cbb 3) HCuOs, recent work indicated the use of only one pathway, analogous to the K pathway. In this work, we have studied the functional importance of the suggested entry point of this pathway, the Glu-25 (Rhodobacter sphaeroides cbb3 numbering) in the accessory subunit CcoP (E25 P). We show that catalytic turnover is severely slowed in variants lacking the protonatable Glu-25. Furthermore, proton uptake from solution during oxidation of the fully reduced cbb3 by O 2 is specifically and severely impaired when Glu-25 was exchanged for Ala or Gln, with rate constants 100-500 times slower than in wild type. Thus, our results support the role of E25 P as the entry point to the proton pathway in cbb 3 and that this pathway is the main proton pathway. This is in contrast to the A-type HCuOs, where the D (and not the K) pathway is used during O 2 reduction. The cbb 3 is in addition to O 2 reduction capable of NO reduction, an activity that was largely retained in the E25 P variants, consistent with a scenario where NO reduction in cbb 3 uses protons from the periplasmic side of the membrane.

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