title: An improved purification procedure for the soluble [NiFe]-hydrogenase of Ralstonia eutropha: new insights into its (in)stability and spectroscopic properties.
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 (HoxFUYHI(2)) with aerobic specific H(2)-NAD(+) activities of 150-185 mumol/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.
authors.affiliation: Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands.
authors.forename: Eddy
authors.initials: E
authors.lastname: van der Linden
authors.forename: Tanja
authors.initials: T
authors.lastname: Burgdorf
authors.forename: Antonio L
authors.initials: AL
authors.lastname: de Lacey
authors.forename: Thorsten
authors.initials: T
authors.lastname: Buhrke
authors.forename: Marcel
authors.initials: M
authors.lastname: Scholte
authors.forename: Victor M
authors.initials: VM
authors.lastname: Fernandez
authors.forename: Bärbel
authors.initials: B
authors.lastname: Friedrich
authors.forename: Simon P J
authors.initials: SP
authors.lastname: Albracht
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D001665:Binding Sites; D020118:Cupriavidus necator / Q000201:enzymology*; D004795:Enzyme Stability; D006864:Hydrogenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015394:Molecular Structure; D010084:Oxidation-Reduction; D013057:Spectrum Analysis
chemical_list: D001426:Bacterial Proteins; C043296:nickel-iron hydrogenase; D006864:Hydrogenase