JBIC Journal of Biological Inorganic Chemistry

, Volume 11, Issue 2, pp 247–260

An improved purification procedure for the soluble [NiFe]-hydrogenase of Ralstonia eutropha: new insights into its (in)stability and spectroscopic properties

Authors

  • Eddy van der Linden
    • Swammerdam Institute for Life SciencesUniversity of Amsterdam
  • Tanja Burgdorf
    • Institut für Biologie/MikrobiologieHumboldt-Universität zu Berlin
  • Antonio L. de Lacey
    • Instituto de Catálisis, CSICCampus Universidad Autónoma
  • Thorsten Buhrke
    • Institut für Biologie/MikrobiologieHumboldt-Universität zu Berlin
  • Marcel Scholte
    • Swammerdam Institute for Life SciencesUniversity of Amsterdam
  • Victor M. Fernandez
    • Instituto de Catálisis, CSICCampus Universidad Autónoma
  • Bärbel Friedrich
    • Institut für Biologie/MikrobiologieHumboldt-Universität zu Berlin
    • Swammerdam Institute for Life SciencesUniversity of Amsterdam
Original Paper

DOI: 10.1007/s00775-005-0075-4

Cite this article as:
van der Linden, E., Burgdorf, T., de Lacey, A.L. et al. J Biol Inorg Chem (2006) 11: 247. doi:10.1007/s00775-005-0075-4

Abstract

Infrared (IR) spectra in combination with chemical analyses have recently shown that the active Ni–Fe site of the soluble NAD+-reducing [NiFe]-hydrogenase from Ralstonia eutropha contains four cyanide groups and one carbon monoxide as ligands. Experiments presented here confirm this result, but show that a variable percentage of enzyme molecules loses one or two of the cyanide ligands from the active site during routine purification. For this reason the redox conditions during the purification have been optimized yielding hexameric enzyme preparations (HoxFUYHI2) with aerobic specific H2–NAD+ activities of 150–185 μmol/min/mg of protein (up to 200% of the highest activity previously reported in the literature). The preparations were highly homogeneous in terms of the active site composition and showed superior IR spectra. IR spectro-electrochemical studies were consistent with the hypothesis that only reoxidation of the reduced enzyme with dioxygen leads to the inactive state, where it is believed that a peroxide group is bound to nickel. Electron paramagnetic resonance experiments showed that the radical signal from the NADH-reduced enzyme derives from the semiquinone form of the flavin (FMN-a) in the hydrogenase module (HoxYH dimer), but not of the flavin (FMN-b) in the NADH-dehydrogenase module (HoxFU dimer). It is further demonstrated that the hexameric enzyme remains active in the presence of NADPH and air, whereas NADH and air lead to rapid destruction of enzyme activity. It is proposed that the presence of NADPH in cells keeps the enzyme in the active state.

Keywords

Cyanide ligand[NiFe]-hydrogenaseOxygen sensitivityRalstonia eutrophaNADPH reductionHoxI subunitProtein purification

Abbreviations

BV

Benzyl viologen

EPR

Electron paramagnetic resonance

EXAFS

Extended X-ray absorption fine structure

IR

Infrared

KPi buffer

50 mM potassium phosphate buffer (pH 7.0)

SH

Soluble hydrogenase

XANES

X-ray absorption near edge structure

XAS

X-ray absorption spectroscopy

Copyright information

© SBIC 2006