Probing the Origin of the Metabolic Precursor of the CO Ligand in the Catalytic Center of [NiFe] Hydrogenase

• Published in: The Journal of biological chemistry 2011-12, Vol.286 (52), p.44937-44944
• Main Authors: Bürstel, Ingmar, Hummel, Philipp, Siebert, Elisabeth, Wisitruangsakul, Nattawadee, Zebger, Ingo, Friedrich, Bärbel, Lenz, Oliver
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Bioenergetics; Biophysics; Carbon Monoxide; Carbon Monoxide - chemistry; Catalytic Domain; Cupriavidus necator - enzymology; Energy Metabolism; Enzyme Maturation; Enzyme Processing; FTIR Spectroscopy; Hydrogenase - chemistry; Ligands; Metalloenzymes; Palladium - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.309351

The O2-tolerant [NiFe] hydrogenases of Ralstonia eutropha are capable of H2 conversion in the presence of ambient O2. 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 13CO serves as precursor of the carbonyl group of the regulatory [NiFe] hydrogenase in R. eutropha. Corresponding 13CO 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 PdCl2, cells cultivated on H2, CO2, and O2 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 PdCl2 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. The active site iron of [NiFe] hydrogenases is equipped with a carbonyl ligand of undetermined origin. The carbonyl ligand derives exclusively from the cellular metabolism, and the CO scavenger PdCl2 mediates severe retardation of hydrogenase-driven growth. The data indicate multiple, growth mode-dependent biosynthetic pathways for the carbonyl ligand. Understanding the intricate cofactor assembly of [NiFe] hydrogenase is crucial for hydrogen-based biotechnology.