Maturation and assembly of [NiFe]-hydrogenases in Escherichia coli
Final Report Abstract
[NiFe]-hydrogenases are complex multi-subunit metalloenzymes that catalyze the reversible oxidation of H2. In E. coli all are membrane-associated enzymes. The maturation and assembly of these enzymes requires the concerted action of several accessory proteins, which perform various functions in biosynthesis of the cofactor-containing large and small subunits. The large, catalytic subunit of [NiFe]-hydrogenases has an active site with unusual diatomic CN- and CO ligands attached to the Fe atom. The metabolic precursor of the CN- ligand is carbamoylphosphate. The metabolic precursor of the CO ligand is unknown. Iron is also important in the formation of the iron-sulphur (FeS) cluster-containing small subunit, which is involved in electron transfer to and from the active site. The Isc (iron sulphur cluster) machinery is required for FeS-cluster biosynthesis and their subsequent insertion into the hydrogenase small subunit. The two major aims of our research in the current funding period were: 1. To try to determine the metabolic source of the CO diatomic ligand and determine where the Fe ion in the active site originates; and 2, to identify the routes taken by, and the proteins involved in, delivery of the FeS-clusters to the small subunits. Regarding the first aim, we demonstrated the following: 1. Infrared spectroscopy identified a Fe(I)-CO2 signal associated with HypC-like chaperone proteins. The identified signal was also found associated with the HypCD scaffold complex which carries synthesizes and delivers the Fe(CN)2CO moiety of the cofactor, but not with HypD on its own. The Fe(I)-CO2 signal is O2-sensitive and is suggested to be the direct precursor of the diatomic CO ligand. 2. No single mutation, or combination of mutations in genes encoding decarboxylases, carboxylases or carbonic anhydrase resulted in abolition of CO ligand biosynthesis. Thus, the source of the intracellular Fe(I)-CO2 remains unknown. However, the results suggest a redundant function of an enzyme or enzymes that interact with both iron and intracellularly produced CO2. Future studies will focus on identifying such an enzyme(s). 3. The large subunit, HybC, of Hyd-2 can only interact with the small subunit HybO after NiFe(CN)2CO cofactor synthesis has been completed. Protelolytic removal of a 14-amino acid C-terminal peptide from the large subunit occurs after cofactor insertion. 4. The large-small subunit heterodimer is only able to interact with the Tat translocon after successful cofactor insertion. This complete process ensures that only completely folded and active enzyme, with its correct cofactor complement, is translocated into the membrane. 5. Genetic removal of the C-terminal peptide from the large subunit generated a Hyd-2 enzyme lacking a NiFe(CN)2CO cofactor in its active site. Nevertheless, this ‚inactive’ enzyme was translocated across the membrane by the Tat machinery, indicating that the large subunit adopts a native conformation regardless of whether or not the active site has a cofactor inside. 6. The function of the cleaved C-terminal peptide is to maintain the 'empty' active site cavity in an open conformation to allow insertion of the Fe(CN)2CO group by the HypD-HybG scaffold complex. HypD-HybG only interacts with the HybC apo-large subunit, not with the C-terminally processed form of the subunit, indicating inaccessibility after conformational closure of the active site. Regarding the second aim, we demonstrated the following: 1. The cysteine desulfurylase, IscS, is essential for maturation and assembly of all hydrogenases. 2. A mutation in fdx, hscA, hscB or IscU, encoding ferredoxin, the Hsp-70 like chaperone, its co-chaperone and the FeS scaffold protein, respectively, revealed that the gene products are essential for H2-oxidizing enzyme activities but not H2-producing activity. H2-evolving activity therefore takes precedence over H2-oxidizing activity. 3. No strong hydrogenase phenotype could be identified when the genes encoding the proposed iron-trafficking proteins CyaY and YggX were deleted. These results indicate that a further route must be present in E. coli cells to deliver Fe ions both for FeS biosynthesis and for NiFe(CN)2CO cofactor biosynthesis. A total of 26 papers (15 when overview articles are substracted) have been published so far from results derived directly or indirectly by this project in the current funding period.
Publications
- (2013) HypD is the scaffold protein for Fe–(CN)2CO cofactor assembly in [NiFe]–hydrogenase maturation. Biochemistry 52: 3289–3296
Stripp, S.T., Soboh, B., Lindenstrauss, U., Braussemann, M., Herzberg, M., Nies, D.H., Sawers, R.G., and Heberle, J.
(See online at https://doi.org/10.1021/bi400302v) - (2013) The [NiFe]-hydrogenase accessory chaperones HypC and HybG of Escherichia coli are iron- and carbon dioxide-binding proteins. FEBS Lett. 587, 2512–2516
Soboh, B., Stripp, S.T., Bielak, C., Lindenstrauß, U., Braussemann, M., Javaid, M., Hallensleben, M., Granich, C., Herzberg, M., Heberle, J., and Sawers, R.G.
(See online at https://doi.org/10.1016/j.febslet.2013.06.055) - (2013) The levels of control exerted by the Isc iron-sulphur cluster system on the biosynthesis of the formate hydrogenlyase complex. Microbiology 159, 1179-1189
Pinske, C., Jaroschinsky, M., and Sawers, R.G.
(See online at https://doi.org/10.1099/mic.0.066142-0) - (2014) Ferredoxin has a pivotal role in the biosynthesis of the hydrogen-oxidizing hydrogenases in Escherichia coli. Int. J. Hydrogen Energy 39, 18533-18542
Jaroschinsky, M., and Sawers, R.G.
(See online at https://doi.org/10.1016/j.ijhydene.2014.03.124) - (2014) The influence of oxygen on [NiFe]-hydrogenase cofactor biosynthesis and how ligation of carbon monoxide precedes cyanation. PLoS One 9, e107488
Stripp, S.T., Lindenstrauss, U., Granich, C., Sawers, R.G., and Soboh, B.
(See online at https://doi.org/10.1371/journal.pone.0107488) - (2015) Coordination of synthesis and assembly of a modular membrane-associated [NiFe]-hydrogenase is determined by cleavage of the C- terminal peptide. J. Bact. 197, 2989-2998
Thomas, C., Muhr, E., and Sawers, R.G.
(See online at https://doi.org/10.1128/JB.00437-15) - (2015) Physiology and bioenergetics of [NiFe]-hydrogenase 2-catalyzed H2-consuming and H2- producing reactions in Escherichia coli. J. Bacteriol. 197, 296-306
Pinske, C., Jaroschinsky, M., Linek, S., Kelly, C.L., Sargent, F., and Sawers, R.G.
(See online at https://doi.org/10.1128/JB.02335-14) - (2017) Differential effects of isc operon mutations on the biosynthesis and activity of key anaerobic metalloenzymes in Escherichia coli. Microbiology 163, 878-890
Jaroschinsky, M., Pinske, C., and Sawers, R.G.
(See online at https://doi.org/10.1099/mic.0.000481)
