A Universal Scaffold for Synthesis of the Fe(CN)2(CO) Moiety of [NiFe] Hydrogenase

• Published in: The Journal of biological chemistry 2012-11, Vol.287 (46), p.38845-38853
• Main Authors: Bürstel, Ingmar, Siebert, Elisabeth, Winter, Gordon, Hummel, Philipp, Zebger, Ingo, Friedrich, Bärbel, Lenz, Oliver
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Bioenergetics; Carbon Monoxide; Carbon Monoxide - chemistry; Catalytic Domain; Cupriavidus necator - metabolism; Cyanides - chemistry; Cysteine - chemistry; DNA Mutational Analysis; Energy Metabolism; Enzyme Catalysis; Enzyme Maturation; Escherichia coli - metabolism; Genetic Engineering - methods; Hydrogenase; Hydrogenase - chemistry; Infrared Spectroscopy; Ions; Iron - chemistry; Ligands; Metalloenzymes; Metals - chemistry; Molecular Biology; Mutagenesis Site-specific; Proteins - chemistry; Spectroscopy
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.376947

Hydrogen-cycling [NiFe] hydrogenases harbor a dinuclear catalytic center composed of nickel and iron ions, which are coordinated by four cysteine residues. Three unusual diatomic ligands in the form of two cyanides (CN−) and one carbon monoxide (CO) are bound to the iron and apparently account for the complexity of the cofactor assembly process, which involves the function of at least six auxiliary proteins, designated HypA, -B, -C, -D, -E, and -F. It has been demonstrated previously that the HypC, -D, -E, and -F proteins participate in cyanide synthesis and transfer. Here, we show by infrared spectroscopic analysis that the purified HypCD complexes from Ralstonia eutropha and Escherichia coli carry in addition to both cyanides the CO ligand. We present experimental evidence that in vivo the attachment of the CN− ligands is a prerequisite for subsequent CO binding. With the aid of genetic engineering and subsequent mutant analysis, the functional role of conserved cysteine residues in HypD from R. eutropha was investigated. Our results demonstrate that the HypCD complex serves as a scaffold for the assembly of the Fe(CN)2(CO) entity of [NiFe] hydrogenase. Background: The active site iron of [NiFe] hydrogenases is equipped with a carbonyl ligand (CO) and two cyanides (CN−). Results: A complex of the hydrogenase accessory proteins HypC and HypD contains both CN− and CO ligands. Conclusion: The entire Fe(CN)2(CO) moiety is assembled on a scaffold and subsequently transferred to apo-hydrogenase. Significance: Scaffold-assisted cofactor assembly is a common trait of hydrogenases and other metalloenzymes.