[NiFe] hydrogenases from the hyperthermophilic bacterium Aquifex aeolicus: properties, function, and phylogenetics

Abstract

Genes potentially coding for three distinct [NiFe] hydrogenases are present in the genome of Aquifex aeolicus. We have demonstrated that all three hydrogenases are expressed under standard growth conditions of the organism. Two hydrogenases were further purified to homogeneity. A periplasmically oriented hydrogenase was obtained in two forms, i.e., as a soluble enzyme containing only the two essential subunits and as a detergent-solubilized complex additionally containing a membrane-integral b-type cytochrome. The second hydrogenase purified was identified as a soluble cytoplasmic enzyme. The isolated enzymes were characterized with respect to biochemical/biophysical parameters, activity, thermostability, and substrate specificity. The phylogenetic positioning of all three hydrogenases was analyzed. A model for the metabolic roles of the three enzymes is proposed on the basis of the obtained results.

Characterization of a unique [FeS] cluster in the electron transfer chain of the oxygen tolerant [NiFe] hydrogenase from Aquifex aeolicus

“…2, dashed line, Table S1) (6,14). Double integration of the iron-sulfur cluster signals accounted for 1.9 AE 0.3 spins∕mol of protein, indicating the presence of two S ¼ 1∕2 centers.…”

“…Hydrogenase I was purified at room temperature under semianaerobic conditions in a 50 mM Tris-HCl buffer pH 7.0, in the presence of 5-10% glycerol and 0.01% n-dodecyl-β-D-maltoside (DDM) as previously described (6). Redox potentials were determined by cw EPR potentiometric titrations.…”

“…This microorganism harbors three distinct [NiFe] hydrogenases, among which Hase I is located in the aerobic respiration pathway and attached to the membrane via a diheme cytb (6). Hase I consists of two subunits; the large subunit contains the hetero-bimetallic nickel-iron site and the small subunit the electron transfer cofactors, namely iron-sulfur clusters (6). Based on spectroelectrochemical studies, this enzyme exhibits enhanced thermostability and tolerance for inhibitors (e.g., O 2 and CO) (4,7).…”

“…The molecular mechanism and structural determinants for this increased oxygen tolerance remain to be understood. Recent EPR and FTIR studies have shown that the [NiFe] center of Hase I has a geometric and electronic structure similar to standard hydrogenases (6,7). The as-isolated enzyme is in the Ni-B state, a dormant form known to carry a bridging hydroxyl ligand between the two metals.…”

Molecular Biology of Microbial Hydrogenases

“…These enzymes, which are induced under N 2 fi xing conditions, are localised on the cytoplasmic side of either the cytoplasmic or thylakoid membrane (Appel and Schulz, 1998;Tamagnini et al, 2002). The third H 2 ase of A. aeolicus, a soluble enzyme which belongs to group 2 has been proposed to provide reductant to the reductive TCA cycle for CO 2 fi xation (Brugna-Guiral et al, 2003).…”