Basem Soboh
Dr. Basem Soboh has moved to:
Freie Universität Berlin
Fachbereich Physik
Research interests: In vitro biosynthesis of complex Fe-S cluster cofactors of metalloenzymes
Understanding how complex metal-based cofactors including iron-sulphur (Fe-S) clusters are biochemically synthesized and assembled, can make an important contribution for the production of biocatalysts with optimal selectivity and activity as well as for the development of anticancer, antibacterial and antiviral agents. The major focus of our current work is to optimize an in vitro system to facilitate the study of the biosynthesis and maturation of [NiFe] hydrogenase using only purified components.
Research projects in more detail:
The active site of [NiFe]-hydrogenase has one carbon monoxide and two cyanide ligands coordinated to the iron atom, a feature that is to date unique in biology. In order to learn from hydrogenases to develop a robust and stable catalyst for hydrogen production, we must first understand at the molecular level the biosynthesis and assembly of these enzymes.
The core of these enzymes comprises a heterodimer of a large subunit with a unique NiFe(CN)2CO metallocofactor and an electron-transferring small subunit containing three iron-sulfur clusters. The nickel ion of the bimetallic active site is coordinated via four highly conserved cysteinyl thiolates, of which two are also coordinated to the Fe ion. The Fe ion carries one carbon monoxide and two cyanide ligands. These diatomic unique ligands are proposed to maintain a low-spin Fe(II) state that is important for H2 activation.
Minimally six accessory proteins (HypA through HypF) are necessary for NiFe(CN)2CO cofactor biosynthesis in E. coli. To understand how this unique active site cofactor of [NiFe]-hydrogenase is assembled and inserted into apo-hydrogenase, we have successfully isolated and characterized most of proteins and intermediates along the path of NiFe(CN)2CO cofactor biosynthesis using a multi-disciplinary approach combining biochemical and diverse spectroscopic techniques.
Recently, we could demonstrate for the first time the in vitro synthesis and maturation of active [NiFe]-hydrogenase 2 of E. coli using a defined system with purified components. The maturation process included (I) formation of the Fe(CN)2CO moiety, (II) its insertion into the pro-protein of a large subunit of hydrogenase 2 of E. coli (Pro-HybC) (III) followed by the insertion of nickel, (IV) removal of the 15 amino acid long C-terminal extension by endoproteolytic cleavage of pro-protein harboring the [NiFe]-center and complex formation of matured HybC with the small subunit (HybO) to yield active [NiFe]-hydrogenase. This finding allows us to exert much greater control over the in vitro maturation system, it allows studying maturation steps individually. This study includes determination of the temporal sequence of events and analysis of the compositions of functional complexes at each maturation step along the path of NiFe(CN)2CO biosynthesis. A more thorough spectroscopic and structural investigation of an all-pure-component-based in vitro system is required to conclusively trace the intermediates of the assembly process of [NiFe]-hydrogenase.
Publications
Lindenstrauss U., Lilie H. and Soboh B. (2015) Pro-protein cleavage is an important checkpoint during maturation of [NiFe]-hydrogenases (submitted to JBC)
Stripp ST., Lindenstrauss U., Sawers RG. and Soboh B. (2015) Identification of an Isothiocyanate on the HypEF Complex Suggests a Route for Efficient Cyanyl-Group Channeling during [NiFe]-Hydrogenase Cofactor Generation. PLoS One e0133118. doi: 10.1371/journal.pone.0133118.
Stripp ST., Lindenstrauss U., Granich C., Sawers RG. and Soboh B. (2014) The influence of oxygen on [NiFe]-hydrogenase cofactor biosynthesis and how ligation of carbon monoxide precedes cyanation. Plos ONE 9 e107488. doi: 10.1371/journal.pone.0107488.
Soboh B., Lindenstrauss U., Granich C., Javaid M., Herzberg M., Claudia T. and Stripp ST. (2014) [NiFe]-hydrogenase maturation in vitro: analysis of the roles of the HybG and HypD accessory proteins. Biochemical journal. 1;464(2):169-77.
Soboh B., Stripp ST., Bielak C., Lindenstrauß U., Braussemann M., Javaid M., Hallensleben M., Granich C., Herzberg M., Heberle J. and Sawers RG. (2013) The [NiFe]-hydrogenase accessory chaperones HypC and HybG of Escherichia coli are iron- and carbon dioxide-binding proteins. FEBS Lett. 19;587(16):2512-6.
Soboh B. and Sawers RG. (2013) [NiFe]-hydrogenase cofactor assembly. In: Encyclopedia of Inorganic and Bioorganic Chemistry - Metals in Cells, Chapter eibc2154 ISBN: 9781119951438. doi: 10.1002/9781119951438.eibc2154.
Stripp ST., Soboh B., Lindenstrauss U., Braussemann M., Herzberg M., Nies DH. , Sawers RG. and Heberle J. (2013) HypD is the Scaffold Protein for Fe-(CN)2CO Cofactor Assembly in [NiFe]-Hydrogenase Maturation. Biochemistry, 52 (19), 3289–32962
Trchounian K., Soboh B., Sawers RG. and Trchounian A. (2013) Contribution of hydrogenase 2 to stationary phase H2 production by Escherichia coli during fermentation of glycerol. Cell. Biochem. Biophys. 66-(1)103-108.
Soboh B., Stripp ST., Muhr E., Granich C., Braussemann M., Herzberg M., Heberle J. and Sawers RG. (2012) [NiFe]-hydrogenase maturation: isolation of a HypC-HypD complex carrying diatomic CO and CN- ligands.FEBS Lett., 586(21) 3882-3887
Soboh B., Kuhns M., Braussemann M., Waclawek M., Muhr E., Pierik AJ. and Sawers RG. (2012) Evidence for an oxygen-sensitive iron-sulfur cluster in an immature large subunit species of Escherichia coli [NiFe]-hydrogenase 2. Biochem Biophys Res Commun. 424(1),158-163
Petkun S., Shi R., Li Y., Asinas A., Munger C., Zhang L., Waclawek M., Soboh B., Sawers RG. and Cygler M. (2011) Structure of Hydrogenase Maturation Protein HypF with Reaction Intermediates Shows Two Active Sites. Structure 19 (12), 1773–1783
Pinske C., Krüger S., Soboh B., Ihling C., Kuhns M., Braussemann M., Jaroschinsky M., Sauer C., Sargent F., Sinz A. and Sawers RG. (2011) Efficient electron transfer from hydrogen to benzyl viologen by the [NiFe]-hydrogenases of Escherichia coli is dependent on the coexpression of the iron-sulphur cluster-containing small subunit. Arch. Microbiol.193(12),893-903
Soboh B., Pinske C., Kuhns M., Waclawek M., Ihling C., Trchounian K., Trchounian A., Sinz. A., and Sawers RG. (2011) The respiratory molybdo-selenoprotein formate dehydrogenases of Escherichia coli have hydrogen: benzyl viologen oxidoreductase activity. BMCMicrobiol.11:173
Soboh B., Krüger S., Kuhns M., Pinske C., Lehmann A. and Sawers RG. (2010) Development of a cell-free system reveals an oxygen-labile step in the maturation of [NiFe]-hydrogenase 2 of E. coli.FEBS Lett. 584 (18), 4109-4114
Soboh B , Boyd ES., Zhao D, Peters JW. and Rubio LM. ( 2010) Substrate specificity and evolutionary implications of a NifDK enzyme carrying NifB-co at its active site. FEBSLett. 584(8),1487-92
Rubio LM., Hernández JA., Soboh B., Zhao D., Igarashi RY. , Curatti L. and Ludden PW. (2008). The Role of Nif Proteins in Nitrogenase Maturation. Plant Science and Biotechnology in Agriculture, Book: Biological Nitrogen Fixation, Volume 42, pp 325-328
Curatti L., Hernandez JA., Igarashi RY., Soboh B., Zhao D. and Rubio LM. (2007). In vitro synthesis of the iron-molybdenum cofactor of nitrogenase from iron, sulfur, molybdenum and homocitrate using purified proteins. Proc. Natl. Acad. Sci. 104 (45), 17626-31
George SJ., Igarashi RY., Piamonteze C., Soboh B., Cramer SP. and Rubio LM. (2007) Identification of a Mo-Fe-S cluster on NifEN by Mo K-edge EXAFS. J. Am. Chem. Soc.,129(11),3060-3061
Hernández JA., Igarashi RY., Soboh B., Curatti L., Dean DR., Ludden PW. and Rubio, LM. (2006) NifX and NifEN exchange biosynthetic precursors of the iron-molybdenum cofactor of nitrogenase. Mol. Microbiol.,63 (1),177-92
Soboh B., Igarashi RY., Hernandez JA. and Rubio LM. (2006) Purification of a NifEN protein complex that contains bound Mo and a FeMo-co precursor from an Azotobacter vinelandii ΔnifHDK strain. J. Biol. Chem., 281, 36701-36709
Soboh B., Linder D. and Hedderich R. (2004) A multisubunit membrane-bound [NiFe] hydrogenase and a NADH-dependent Fe-only hydrogenase in the fermenting bacterium Thermoanaerobacter tengcongensis Microbiology 150, 2451-2463
Soboh B., Linder D. and Hedderich R. (2002) Purification and catalytic properties of a CO-oxidizing:H2 evolving enzyme complex from Carboxydothermus hydrogenoformans Eur.J. Biochem. 269, 5712-21.