title: Probing the origin of the metabolic precursor of the CO ligand in the catalytic center of [NiFe] hydrogenase.
abstract: The O(2)-tolerant [NiFe] hydrogenases of Ralstonia eutropha are capable of H(2) conversion in the presence of ambient O(2). Oxygen represents not only a challenge for catalysis but also for the complex assembling process of the [NiFe] active site. Apart from nickel and iron, the catalytic center contains unusual diatomic ligands, namely two cyanides (CN(-)) and one carbon monoxide (CO), which are coordinated to the iron. One of the open questions of the maturation process concerns the origin and biosynthesis of the CO group. Isotope labeling in combination with infrared spectroscopy revealed that externally supplied gaseous (13)CO serves as precursor of the carbonyl group of the regulatory [NiFe] hydrogenase in R. eutropha. Corresponding (13)CO titration experiments showed that a concentration 130-fold higher than ambient CO (0.1 ppmv) caused a 50% labeling of the carbonyl ligand in the [NiFe] hydrogenase, leading to the conclusion that the carbonyl ligand originates from an intracellular metabolite. A novel setup allowed us to the study effects of CO depletion on maturation in vivo. Upon induction of CO depletion by addition of the CO scavenger PdCl(2), cells cultivated on H(2), CO(2), and O(2) showed severe growth retardation at low cell concentrations, which was on the basis of partially arrested hydrogenase maturation, leading to reduced hydrogenase activity. This suggests gaseous CO as a metabolic precursor under these conditions. The addition of PdCl(2) to cells cultivated heterotrophically on organic substrates had no effect on hydrogenase maturation. These results indicate at least two different pathways for biosynthesis of the CO ligand of [NiFe] hydrogenase.
authors: Humboldt-Universität zu Berlin, Institut für Biologie/Mikrobiologie, Chausseestrasse 117, 10115 Berlin, Germany.
authors: Ingmar
authors: I
authors: Bürstel
authors: Philipp
authors: P
authors: Hummel
authors: Elisabeth
authors: E
authors: Siebert
authors: Nattawadee
authors: N
authors: Wisitruangsakul
authors: Ingo
authors: I
authors: Zebger
authors: Bärbel
authors: B
authors: Friedrich
authors: Oliver
authors: O
authors: Lenz
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D002248:Carbon Monoxide / Q000737:chemistry*; D020134:Catalytic Domain; D020118:Cupriavidus necator / Q000201:enzymology*; D006864:Hydrogenase / Q000737:chemistry*; D008024:Ligands; D010165:Palladium / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D008024:Ligands; D010165:Palladium; D002248:Carbon Monoxide; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; C008756:palladium chloride