Intact functional fourteen-subunit respiratory membrane-bound [NiFe]-hydrogenase complex of the hyperthermophilic archaeon Pyrococcus furiosus

Patrick M. McTernan, Sanjeev K. Chandrayan, Chang Hao Wu, Brian J. Vaccaro, W. Andrew Lancaster, Qingyuan Yang, Dax Fu, Greg L. Hura, John A. Tainer, Michael W W Adams

Research output: Contribution to journalArticle

Abstract

The archaeon Pyrococcus furiosus grows optimally at 100°C by converting carbohydrates to acetate, CO2, and H2, obtaining energy from a respiratory membrane-bound hydrogenase (MBH). This conserves energy by coupling H2 production to oxidation of reduced ferredoxin with generation of a sodium ion gradient.MBHis encoded by a 14-gene operon with both hydrogenase and Na+/H+ antiporter modules. Herein a His-tagged MBHwas expressed in P. furiosus and the detergent-solubilized complex purified under anaerobic conditions by affinity chromatography. Purified MBH contains all 14 subunits by electrophoretic analysis (13 subunits were also identified by mass spectrometry) and had a measured iron:nickel ratio of 15:1, resembling the predicted value of 13:1. The as-purified enzyme exhibited a rhombic EPR signal characteristic of the ready nickel- boron state. The purified and membrane-bound forms of MBH both preferentially evolved H2 with the physiological donor (reduced ferredoxin) as well as with standard dyes. The O2 sensitivities of the two forms were similar (half-lives of ∼15 h in air), but the purified enzyme was more thermolabile (half-lives at 90°C of 1 and 25 h, respectively). Structural analysis of purified MBH by small angle x-ray scattering indicated a Z-shaped structure with a mass of 310 kDa, resembling the predicted value (298 kDa). The angle x-ray scattering analyses reinforce and extend the conserved sequence relationships of group 4 enzymes and complex I (NADH quinone oxidoreductase). This is the first report on the properties of a solubilized form of an intact respiratory MBH complex that is proposed to evolve H2 and pump Na+ ions.

Original languageEnglish (US)
Pages (from-to)19364-19372
Number of pages9
JournalJournal of Biological Chemistry
Volume289
Issue number28
DOIs
StatePublished - Jul 11 2014

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Pyrococcus furiosus
Hydrogenase
Archaea
Membranes
Ferredoxins
Nickel
Enzymes
Ion Pumps
X-Rays
Scattering
Ions
Affinity chromatography
X rays
Sodium-Hydrogen Antiporter
Boron
Conserved Sequence
Operon
Affinity Chromatography
Structural analysis
Detergents

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

McTernan, P. M., Chandrayan, S. K., Wu, C. H., Vaccaro, B. J., Lancaster, W. A., Yang, Q., ... Adams, M. W. W. (2014). Intact functional fourteen-subunit respiratory membrane-bound [NiFe]-hydrogenase complex of the hyperthermophilic archaeon Pyrococcus furiosus. Journal of Biological Chemistry, 289(28), 19364-19372. https://doi.org/10.1074/jbc.M114.567255

Intact functional fourteen-subunit respiratory membrane-bound [NiFe]-hydrogenase complex of the hyperthermophilic archaeon Pyrococcus furiosus. / McTernan, Patrick M.; Chandrayan, Sanjeev K.; Wu, Chang Hao; Vaccaro, Brian J.; Lancaster, W. Andrew; Yang, Qingyuan; Fu, Dax; Hura, Greg L.; Tainer, John A.; Adams, Michael W W.

In: Journal of Biological Chemistry, Vol. 289, No. 28, 11.07.2014, p. 19364-19372.

Research output: Contribution to journalArticle

McTernan, PM, Chandrayan, SK, Wu, CH, Vaccaro, BJ, Lancaster, WA, Yang, Q, Fu, D, Hura, GL, Tainer, JA & Adams, MWW 2014, 'Intact functional fourteen-subunit respiratory membrane-bound [NiFe]-hydrogenase complex of the hyperthermophilic archaeon Pyrococcus furiosus', Journal of Biological Chemistry, vol. 289, no. 28, pp. 19364-19372. https://doi.org/10.1074/jbc.M114.567255
McTernan, Patrick M. ; Chandrayan, Sanjeev K. ; Wu, Chang Hao ; Vaccaro, Brian J. ; Lancaster, W. Andrew ; Yang, Qingyuan ; Fu, Dax ; Hura, Greg L. ; Tainer, John A. ; Adams, Michael W W. / Intact functional fourteen-subunit respiratory membrane-bound [NiFe]-hydrogenase complex of the hyperthermophilic archaeon Pyrococcus furiosus. In: Journal of Biological Chemistry. 2014 ; Vol. 289, No. 28. pp. 19364-19372.
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abstract = "The archaeon Pyrococcus furiosus grows optimally at 100°C by converting carbohydrates to acetate, CO2, and H2, obtaining energy from a respiratory membrane-bound hydrogenase (MBH). This conserves energy by coupling H2 production to oxidation of reduced ferredoxin with generation of a sodium ion gradient.MBHis encoded by a 14-gene operon with both hydrogenase and Na+/H+ antiporter modules. Herein a His-tagged MBHwas expressed in P. furiosus and the detergent-solubilized complex purified under anaerobic conditions by affinity chromatography. Purified MBH contains all 14 subunits by electrophoretic analysis (13 subunits were also identified by mass spectrometry) and had a measured iron:nickel ratio of 15:1, resembling the predicted value of 13:1. The as-purified enzyme exhibited a rhombic EPR signal characteristic of the ready nickel- boron state. The purified and membrane-bound forms of MBH both preferentially evolved H2 with the physiological donor (reduced ferredoxin) as well as with standard dyes. The O2 sensitivities of the two forms were similar (half-lives of ∼15 h in air), but the purified enzyme was more thermolabile (half-lives at 90°C of 1 and 25 h, respectively). Structural analysis of purified MBH by small angle x-ray scattering indicated a Z-shaped structure with a mass of 310 kDa, resembling the predicted value (298 kDa). The angle x-ray scattering analyses reinforce and extend the conserved sequence relationships of group 4 enzymes and complex I (NADH quinone oxidoreductase). This is the first report on the properties of a solubilized form of an intact respiratory MBH complex that is proposed to evolve H2 and pump Na+ ions.",
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AU - Wu, Chang Hao

AU - Vaccaro, Brian J.

AU - Lancaster, W. Andrew

AU - Yang, Qingyuan

AU - Fu, Dax

AU - Hura, Greg L.

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AU - Adams, Michael W W

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