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 journalArticlepeer-review

26 Scopus citations


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
Issue number28
StatePublished - Jul 11 2014

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


Dive into the research topics of 'Intact functional fourteen-subunit respiratory membrane-bound [NiFe]-hydrogenase complex of the hyperthermophilic archaeon Pyrococcus furiosus'. Together they form a unique fingerprint.

Cite this