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.

Reversible Active Site Sulfoxygenation Can Explain the Oxygen Tolerance of a NAD+-Reducing [NiFe] Hydrogenase and Its Unusual Infrared Spectroscopic Properties

“…As reported previously, 48,50,53,54 the corresponding IR spectrum, henceforth called "split-spectrum", exhibits one intense CO stretching band at about 1957 cm −1 and four corresponding CN stretching bands at approximately 2070, 2081, 2090, and 2098 cm −1 ( Figure 3A). In addition, a weak CO stretching band can be observed at ca.…”

Overexpression, Isolation, and Spectroscopic Characterization of the Bidirectional [NiFe] Hydrogenase from Synechocystis sp. PCC 6803

“…in the also bidirectional, but oxygen tolerant, soluble hydrogenase from R. eutropha H16 (15,16,39). In this hydrogenase, however, four instead of two CN stretching vibrational bands were detected besides one CO absorbance band.…”

“…Under such conditions, no evidence of paramagnetic nickel species could be detected by EPR spectroscopy (16). However, in some enzyme preparations significant amounts of Ni a -C could also be induced electrochemically or by an excess of NADH or dithionite (39).…”

“…For this enzyme a non-standard coordination of the active [NiFe] site was proposed with one additional cyanide ligand bound to each iron and nickel (15,16,39). Such a ligation would possibly protect the catalytic center from binding of oxygen and the related inactivation.…”

The Hydrogenase Subcomplex of the NAD+‐Reducing [NiFe] Hydrogenase from Ralstonia eutropha – Insights into Catalysis and Redox Interconversions

“…[19] In the SH holoenzyme, the NiFe catalytic center of the hydrogenase module is connected to the NAD + -binding site of HoxF by a series of iron-sulfur clusters and two flavin mononucleotides, Figure 1. [20] One of the flavin units, designated as FMN-b, is a constituent of the HoxFU module, whereas the second flavin, FMN-a, has been suggested to be bound to the hydrogenase small subunit HoxY. [21] This assignment was based on the observation that the N-terminal region of HoxY is very similar to the FMN-binding region of flavodoxin.…”