Intact Functional Fourteen-Subunit Respiratory Membrane Bound [NiFe]-Hydrogenase Complex of the Hyperthermophilic Archaeon Pyrococcus furiosus [Membrane Biology]

May 23rd, 2014 by McTernan, P. M., Chandrayan, S. K., Wu, C.-H., Vaccaro, B. J., Lancaster, W. A., Yang, Q., Fu, D., Hura, G. L., Tainer, J. A., Adams, M. W. W.

The archaeon Pyrococcus furiosus grows optimally at 100 degrees 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 Fe:Ni ratio of 15:1, resembling the predicted value of 13:1. The as-purified enzyme exhibited a rhombic EPR signal characteristic of the ready Ni-B 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 hr in air), but the purified enzyme was more thermolabile (half-lives at 90 degrees C of 1 hr and 25 hr, respectively). Structural analysis of purified MBH by small angle x-ray scattering (SAXS) indicated a Z-shaped structure with a mass of 310 kDa, resembling the predicted value (298 kDa). The SAXS 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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