Functional Analysis by Site-Directed Mutagenesis of the NAD ⁺ -Reducing Hydrogenase from Ralstonia eutropha

Abstract

The tetrameric cytoplasmic [NiFe] hydrogenase (SH) of Ralstonia eutropha couples the oxidation of hydrogen to the reduction of NAD؉ under aerobic conditions. In the catalytic subunit HoxH, all six conserved motifs surrounding the [NiFe] site are present. Five of these motifs were altered by site-directed mutagenesis in order to dissect the molecular mechanism of hydrogen activation. Based on phenotypic characterizations, 27 mutants were grouped into four different classes. Mutants of the major class, class I, failed to grow on hydrogen and were devoid of H 2 -oxidizing activity. The enzyme hydrogenase catalyzes the reversible cleavage of H 2 into 2 H ϩ ions and 2 electrons. Hydrogenases are found in almost all phylogenetic lines of prokaryotes and in a few unicellular eukaryotes. The physiological function of a given hydrogenase is dependent on its cellular location and its capacity to interact with various redox partners. Based on their metal contents, two major classes are distinguished: the [NiFe] and Fe-only hydrogenases. Fe-only hydrogenases are extremely sensitive to O 2 and so far have been found only in obligate anaerobes, where they occur either as a monomer in the cytoplasm or as a heterodimer in the periplasm (1, 45). [NiFe] hydrogenases are composed of at least two heterologous polypeptides, a large subunit of approximately 60 kDa and a small subunit that is relatively diverse in size and cofactor composition (40).Crystallographic data are available for both classes of hydrogenases (12,15,16,25,27,42 Electron paramagnetic resonance (EPR) spectroscopy and Xray absorption spectroscopy (XAS) show that H 2 activation occurs close to the nickel atom, suggesting a change in its oxidation state during the catalytic cycle (14,26,39).Mutant proteins are useful tools for mechanistic studies. A complex protein-assisted maturation pathway is involved in the assembly of the [NiFe] active site (7,17,19). Almost no information is available on these posttranslational processes in strictly anaerobic organisms, whose hydrogenase structure is well defined. On the other hand, elaborate efforts to crystallize [NiFe] hydrogenases from aerobic organisms, for which hydrogenase biosynthesis has been extensively studied, have not yet been successful.More than 100 hydrogenase gene sequences are now available in the database. Inspection of these sequences shows that despite functional and structural diversity, core signatures are highly conserved in the catalytic subunit, in particular in the environment of the [NiFe] active site (3,43). Therefore, analysis of mutant proteins, in correlation with the available atomic structures, should provide further insight into the reaction mechanism of [NiFe] hydrogenases.In this study we selected the cytoplasmic [NiFe] hydrogenase (soluble hydrogenase [SH]) of Ralstonia eutropha as a model. This hydrogenase consists of a heterodimeric hydrogenase module together with a flavin mononucleotide (FMN)-containing iron-sulfur protein, in which the capacity to couple H 2 oxidation with the redu...

[NiFe]-Hydrogenases of <i>Ralstonia eutropha</i> H16: Modular Enzymes for Oxygen-Tolerant Biological Hydrogen Oxidation

“…In HoxH(R60Q), another conserved arginine residue located close to the proximal cluster in the D. gigas hydrogenase (R63) was exchanged. This led to decreased H 2 oxidation activity and, even more notably, to O 2 sensitivity suggesting that Arg60 is involved not only in subunit interaction but also contributes to the stability of the SH in the presence of O 2 [Burgdorf et al, 2002]. From these results it can be concluded that both the active site structure and its protein environment have a signifi cant impact on the behavior of the catalyst towards O 2 .…”

“…Using site-directed mutagenesis, the roles of amino acids located in six conserved motifs surrounding the NiFe-active site of SH were investigated [Burgdorf et al, 2002;Massanz and Friedrich, 1999]. Only a few of these mutants will be discussed here.…”

A Glutamate Is the Essential Proton Transfer Gate during the Catalytic Cycle of the [NiFe] Hydrogenase

“…It is worth noting that this work provides a better understanding on previous observations performed in Ralstonia eutropha-soluble [NiFe] hydrogenase, in which a similar mutant to E25Q had lost its H 2 uptake and D 2 /H ϩ activities (45). The cellular system provided by D. fructosovorans made possible the purification and the complete kinetic and spectroscopic characterizations of the hydrogenase mutants.…”

The Soluble NAD+-Reducing [NiFe]-Hydrogenase fromRalstonia eutrophaH16 Consists of Six Subunits and Can Be Specifically Activated by NADPH

“…The HoxH subunit of the SH contains six conserved motifs, which are typical of [NiFe] hydrogenases. The amino acids identified as conserved signatures, among which are two pairs of cysteines, surround the Ni-Fe active site (15,40). The HoxY subunit of the SH is a truncated version of the small subunit of standard [NiFe]-hydrogenases and presumably accommodates only the proximal [4Fe-4S] cluster.…”