The two transmembrane helices of CcoP are sufficient for assembly of the cbb3-type heme-copper oxygen reductase from Vibrio cholerae

Young O. Ahn, Hyun Ju Lee, Daniel Kaluka, Syun Ru Yeh, Denis L. Rousseau, Pia Ädelroth, Robert B Gennis

Research output: Contribution to journalArticle

Abstract

The C-family (cbb3) of heme-copper oxygen reductases are proton-pumping enzymes terminating the aerobic respiratory chains of many bacteria, including a number of human pathogens. The most common form of these enzymes contains one copy each of 4 subunits encoded by the ccoNOQP operon. In the cbb3 from Rhodobacter capsulatus, the enzyme is assembled in a stepwise manner, with an essential role played by an assembly protein CcoH. Importantly, it has been proposed that a transient interaction between the transmembrane domains of CcoP and CcoH is essential for assembly. Here, we test this proposal by showing that a genetically engineered form of cbb3 from Vibrio cholerae (CcoNOQPX) that lacks the hydrophilic domain of CcoP, where the two heme c moieties are present, is fully assembled and stable. Single-turnover kinetics of the reaction between the fully reduced CcoNOQPX and O2 are essentially the same as the wild type enzyme in oxidizing the 4 remaining redox-active sites. The enzyme retains approximately 10% of the steady state oxidase activity using the artificial electron donor TMPD, but has no activity using the physiological electron donor cytochrome c4, since the docking site for this cytochrome is presumably located on the absent domain of CcoP. Residue E49 in the hydrophobic domain of CcoP is the entrance of the KC-channel for proton input, and the E49A mutation in the truncated enzyme further reduces the steady state activity to less than 3%. Hence, the same proton channel is used by both the wild type and truncated enzymes.

Original languageEnglish (US)
Pages (from-to)1231-1239
Number of pages9
JournalBiochimica et Biophysica Acta - Bioenergetics
Volume1847
Issue number10
DOIs
StatePublished - Jul 13 2015

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Vibrio cholerae
Heme
Copper
Oxidoreductases
Oxygen
Enzymes
Protons
Electrons
Rhodobacter capsulatus
Pathogens
Cytochromes
Operon
Electron Transport
Oxidation-Reduction
Catalytic Domain
Bacteria
Mutation
Kinetics

Keywords

  • Abbreviations TMPD N,N,N′,N′-tetramethyl-p-phenylenediamine
  • DDM n-dodecyl β-d-maltoside
  • HCO heme-copper oxygen reductase
  • PMS phenazine methosulphate

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

Cite this

The two transmembrane helices of CcoP are sufficient for assembly of the cbb3-type heme-copper oxygen reductase from Vibrio cholerae. / Ahn, Young O.; Lee, Hyun Ju; Kaluka, Daniel; Yeh, Syun Ru; Rousseau, Denis L.; Ädelroth, Pia; Gennis, Robert B.

In: Biochimica et Biophysica Acta - Bioenergetics, Vol. 1847, No. 10, 13.07.2015, p. 1231-1239.

Research output: Contribution to journalArticle

Ahn, Young O. ; Lee, Hyun Ju ; Kaluka, Daniel ; Yeh, Syun Ru ; Rousseau, Denis L. ; Ädelroth, Pia ; Gennis, Robert B. / The two transmembrane helices of CcoP are sufficient for assembly of the cbb3-type heme-copper oxygen reductase from Vibrio cholerae. In: Biochimica et Biophysica Acta - Bioenergetics. 2015 ; Vol. 1847, No. 10. pp. 1231-1239.
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abstract = "The C-family (cbb3) of heme-copper oxygen reductases are proton-pumping enzymes terminating the aerobic respiratory chains of many bacteria, including a number of human pathogens. The most common form of these enzymes contains one copy each of 4 subunits encoded by the ccoNOQP operon. In the cbb3 from Rhodobacter capsulatus, the enzyme is assembled in a stepwise manner, with an essential role played by an assembly protein CcoH. Importantly, it has been proposed that a transient interaction between the transmembrane domains of CcoP and CcoH is essential for assembly. Here, we test this proposal by showing that a genetically engineered form of cbb3 from Vibrio cholerae (CcoNOQPX) that lacks the hydrophilic domain of CcoP, where the two heme c moieties are present, is fully assembled and stable. Single-turnover kinetics of the reaction between the fully reduced CcoNOQPX and O2 are essentially the same as the wild type enzyme in oxidizing the 4 remaining redox-active sites. The enzyme retains approximately 10{\%} of the steady state oxidase activity using the artificial electron donor TMPD, but has no activity using the physiological electron donor cytochrome c4, since the docking site for this cytochrome is presumably located on the absent domain of CcoP. Residue E49 in the hydrophobic domain of CcoP is the entrance of the KC-channel for proton input, and the E49A mutation in the truncated enzyme further reduces the steady state activity to less than 3{\%}. Hence, the same proton channel is used by both the wild type and truncated enzymes.",
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T1 - The two transmembrane helices of CcoP are sufficient for assembly of the cbb3-type heme-copper oxygen reductase from Vibrio cholerae

AU - Ahn, Young O.

AU - Lee, Hyun Ju

AU - Kaluka, Daniel

AU - Yeh, Syun Ru

AU - Rousseau, Denis L.

AU - Ädelroth, Pia

AU - Gennis, Robert B

PY - 2015/7/13

Y1 - 2015/7/13

N2 - The C-family (cbb3) of heme-copper oxygen reductases are proton-pumping enzymes terminating the aerobic respiratory chains of many bacteria, including a number of human pathogens. The most common form of these enzymes contains one copy each of 4 subunits encoded by the ccoNOQP operon. In the cbb3 from Rhodobacter capsulatus, the enzyme is assembled in a stepwise manner, with an essential role played by an assembly protein CcoH. Importantly, it has been proposed that a transient interaction between the transmembrane domains of CcoP and CcoH is essential for assembly. Here, we test this proposal by showing that a genetically engineered form of cbb3 from Vibrio cholerae (CcoNOQPX) that lacks the hydrophilic domain of CcoP, where the two heme c moieties are present, is fully assembled and stable. Single-turnover kinetics of the reaction between the fully reduced CcoNOQPX and O2 are essentially the same as the wild type enzyme in oxidizing the 4 remaining redox-active sites. The enzyme retains approximately 10% of the steady state oxidase activity using the artificial electron donor TMPD, but has no activity using the physiological electron donor cytochrome c4, since the docking site for this cytochrome is presumably located on the absent domain of CcoP. Residue E49 in the hydrophobic domain of CcoP is the entrance of the KC-channel for proton input, and the E49A mutation in the truncated enzyme further reduces the steady state activity to less than 3%. Hence, the same proton channel is used by both the wild type and truncated enzymes.

AB - The C-family (cbb3) of heme-copper oxygen reductases are proton-pumping enzymes terminating the aerobic respiratory chains of many bacteria, including a number of human pathogens. The most common form of these enzymes contains one copy each of 4 subunits encoded by the ccoNOQP operon. In the cbb3 from Rhodobacter capsulatus, the enzyme is assembled in a stepwise manner, with an essential role played by an assembly protein CcoH. Importantly, it has been proposed that a transient interaction between the transmembrane domains of CcoP and CcoH is essential for assembly. Here, we test this proposal by showing that a genetically engineered form of cbb3 from Vibrio cholerae (CcoNOQPX) that lacks the hydrophilic domain of CcoP, where the two heme c moieties are present, is fully assembled and stable. Single-turnover kinetics of the reaction between the fully reduced CcoNOQPX and O2 are essentially the same as the wild type enzyme in oxidizing the 4 remaining redox-active sites. The enzyme retains approximately 10% of the steady state oxidase activity using the artificial electron donor TMPD, but has no activity using the physiological electron donor cytochrome c4, since the docking site for this cytochrome is presumably located on the absent domain of CcoP. Residue E49 in the hydrophobic domain of CcoP is the entrance of the KC-channel for proton input, and the E49A mutation in the truncated enzyme further reduces the steady state activity to less than 3%. Hence, the same proton channel is used by both the wild type and truncated enzymes.

KW - Abbreviations TMPD N,N,N′,N′-tetramethyl-p-phenylenediamine

KW - DDM n-dodecyl β-d-maltoside

KW - HCO heme-copper oxygen reductase

KW - PMS phenazine methosulphate

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