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J Biol Chem. 2014 Jul 11;289(28):19364-72. doi: 10.1074/jbc.M114.567255. Epub 2014 May 23.

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

Author information

1
From the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-7229.
2
the Department of Physiology, John Hopkins University School of Medicine, Baltimore, Maryland 21205, and.
3
the Physical Bioscience Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720.
4
From the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-7229, adams@bmb.uga.edu.

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. MBH is encoded by a 14-gene operon with both hydrogenase and Na(+)/H(+) antiporter modules. Herein a His-tagged MBH was 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.

KEYWORDS:

Affinity Purification; Bioenergetics; Electron Transfer; Hydrogen; Hydrogenase; Membrane Enzyme; Respiration; Solubilization; Thermophile

PMID:
24860091
PMCID:
PMC4094048
DOI:
10.1074/jbc.M114.567255
[Indexed for MEDLINE]
Free PMC Article
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