The Complex Between Hydrogenase-maturation Proteins HypC and HypD is an Intermediate in the Supply of Cyanide to the Active Site Iron of [NiFe]-Hydrogenases
| Autor: | Berthold F. Matzanke, Simon P. J. Albracht, August Böck, Nikola Drapal, Alexander Jacobi, Melanie Blokesch |
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| Přispěvatelé: | Molecular Microbial Physiology (SILS, FNWI) |
| Rok vydání: | 2004 |
| Předmět: |
Hydrogenase
Macromolecular Substances Stereochemistry Iron Cyanide medicine.disease_cause law.invention Spectroscopy Mossbauer chemistry.chemical_compound Biosynthesis Structural Biology law Catalytic Domain Escherichia coli medicine Electron paramagnetic resonance Molecular Biology Cyanides biology Ligand Escherichia coli Proteins Electron Spin Resonance Spectroscopy Proteins Active site N-terminus chemistry Biochemistry biology.protein |
| Zdroj: | Journal of Molecular Biology, 344, 155-167. Academic Press Inc. |
| ISSN: | 0022-2836 |
| DOI: | 10.1016/j.jmb.2004.09.040 |
| Popis: | Carbamoylphosphate has been shown to be the educt for the synthesis ofthe CN ligands of the NiFe metal centre of hydrogenases from Escherichiacoli. In the absence of carbamoylphosphate, cells accumulate a complex oftwo hydrogenase maturation proteins, namely HypC and HypD for thesynthesis of hydrogenase 3. A procedure for the purification of wild-typeHypD protein or of a biologically active derivative carrying the Strep-tagIIwat the N terminus has been developed. HypD is a monomeric proteinpossessing about 4 mol of iron per mol of protein. Electron paramagneticresonance (EPR) and Mo¿ssbauer spectroscopy demonstrated that the iron ispresent as a diamagnetic [4Fe¿4S]2C cluster. The complex between HypCand HypD can be cross-linked by a number of thiol and primary aminespecificlinkers. When HypD and HypC were overproduced side-by-sidewith HypE, the HypC¿HypD complex contained substoichiometricamounts of HypE whose proportion in the complex could be augmentedwhen HypF was also overproduced. HypE trapped in this complex couldbe carbamoylated by protein HypF and after dehydration transferred thecyano group to the HypC¿HypD part of the complex. Free HypC andHypD were not cyanated by HypE-CN. An active HypC¿HypD complexfrom anaerobic cells was inactivated by incubation with K3[Fe(CN)6] butnot with K4[Fe(CN)6]. The results suggest the existence of a dynamiccomplex between the hydrogenase maturation proteins HypD, HypC,HypE and HypF, which is the site of ligand biosynthesis and attachment tothe iron atom of the NiFe site in hydrogenase 3. |
| Databáze: | OpenAIRE |
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