Basem Soboh

In vitro synthesis of the iron–molybdenum cofactor of nitrogenase from iron, sulfur, molybdenum, and homocitrate using purified proteins

Biological nitrogen fixation, the conversion of atmospheric N2 to NH3, is an essential process in the global biogeochemical cycle of nitrogen that supports life on Earth. Most of the biological nitrogen fixation is catalyzed by the molybdenum nitrogenase, which contains at its active site one of the most complex metal cofactors known to date, the iron–molybdenum cofactor (FeMo-co). FeMo-co is composed of 7Fe, 9S, Mo, R-homocitrate, and one unidentified light atom. Here we demonstrate the complete in vitro synthesis of FeMo-co from Fe2+, S2−, MoO42−, and R-homocitrate using only purified Nif proteins. This synthesis provides direct biochemical support to the current model of FeMo-co biosynthesis. A minimal in vitro system, containing NifB, NifEN, and NifH proteins, together with Fe2+, S2−, MoO42−, R-homocitrate, S-adenosyl methionine, and Mg-ATP, is sufficient for the synthesis of FeMo-co and the activation of apo-dinitrogenase under anaerobic-reducing conditions. This in vitro system also provides a biochemical approach to further study the function of accessory proteins involved in nitrogenase maturation (as shown here for NifX and NafY). The significance of these findings in the understanding of the complete FeMo-co biosynthetic pathway and in the study of other complex Fe-S cluster biosyntheses is discussed.

The respiratory molybdo-selenoprotein formate dehydrogenases of Escherichia coli have hydrogen: benzyl viologen oxidoreductase activity.

BackgroundEscherichia coli synthesizes three membrane-bound molybdenum- and selenocysteine-containing formate dehydrogenases, as well as up to four membrane-bound [NiFe]-hydrogenases. Two of the formate dehydrogenases (Fdh-N and Fdh-O) and two of the hydrogenases (Hyd-1 and Hyd-2) have their respective catalytic subunits located in the periplasm and these enzymes have been shown previously to oxidize formate and hydrogen, respectively, and thus function in energy metabolism. Mutants unable to synthesize the [NiFe]-hydrogenases retain a H2: benzyl viologen oxidoreductase activity. The aim of this study was to identify the enzyme or enzymes responsible for this activity.ResultsHere we report the identification of a new H2: benzyl viologen oxidoreductase enzyme activity in E. coli that is independent of the [NiFe]-hydrogenases. This enzyme activity was originally identified after non-denaturing polyacrylamide gel electrophoresis and visualization of hydrogen-oxidizing activity by specific staining. Analysis of a crude extract derived from a variety of E. coli mutants unable to synthesize any [NiFe]-hydrogenase-associated enzyme activity revealed that the mutants retained this specific hydrogen-oxidizing activity. Enrichment of this enzyme activity from solubilised membrane fractions of the hydrogenase-negative mutant FTD147 by ion-exchange, hydrophobic interaction and size-exclusion chromatographies followed by mass spectrometric analysis identified the enzymes Fdh-N and Fdh-O. Analysis of defined mutants devoid of selenocysteine biosynthetic capacity or carrying deletions in the genes encoding the catalytic subunits of Fdh-N and Fdh-O demonstrated that both enzymes catalyze hydrogen activation. Fdh-N and Fdh-O can also transfer the electrons derived from oxidation of hydrogen to other redox dyes.ConclusionsThe related respiratory molybdo-selenoproteins Fdh-N and Fdh-O of Escherichia coli have hydrogen-oxidizing activity. These findings demonstrate that the energy-conserving selenium- and molybdenum-dependent formate dehydrogenases Fdh-N and Fdh-O exhibit a degree of promiscuity with respect to the electron donor they use and identify a new class of dihydrogen-oxidizing enzyme.

NifX and NifEN exchange NifB cofactor and the VK‐cluster, a newly isolated intermediate of the iron‐molybdenum cofactor biosynthetic pathway

The iron‐molybdenum cofactor of nitrogenase (FeMo‐co) is synthesized in a multistep process catalysed by several Nif proteins and is finally inserted into a pre‐synthesized apo‐dinitrogenase to generate mature dinitrogenase protein. The NifEN complex serves as scaffold for some steps of this synthesis, while NifX belongs to a family of small proteins that bind either FeMo‐co precursors or FeMo‐co during cofactor synthesis. In this work, the binding of FeMo‐co precursors and their transfer between purified Azotobacter vinelandii NifX and NifEN proteins was studied to shed light on the role of NifX on FeMo‐co synthesis. Purified NifX binds NifB cofactor (NifB‐co), a precursor to FeMo‐co, with high affinity and is able to transfer it to the NifEN complex. In addition, NifEN and NifX exchange another [Fe‐S] cluster that serves as a FeMo‐co precursor, and we have designated it as the VK‐cluster. In contrast to NifB‐co, the VK‐cluster is electronic paramagnetic resonance (EPR)‐active in the reduced and the oxidized states. The NifX/VK‐cluster complex is unable to support in vitro FeMo‐co synthesis in the absence of NifEN because further processing of the VK‐cluster into FeMo‐co requires the simultaneous activities of NifEN and NifH. Our in vitro studies suggest that the role of NifX in vivo is to serve as transient reservoir of FeMo‐co precursors and thus help control their flux during FeMo‐co synthesis.

HypD is the scaffold protein for Fe-(CN)2CO cofactor assembly in [NiFe]-hydrogenase maturation.

[NiFe]-hydrogenases bind a NiFe-(CN)2CO cofactor in their catalytic large subunit. The iron-sulfur protein HypD and the small accessory protein HypC play a central role in the generation of the CO and CN(-) ligands. Infrared spectroscopy identified signatures on an anaerobically isolated HypCD complex that are reminiscent of those in the hydrogenase active site, suggesting that this complex is the assembly site of the Fe-(CN)2CO moiety of the cofactor prior to its transfer to the hydrogenase large subunit. Here, we report that HypD isolated in the absence of HypC shows infrared bands at 1956 cm(-1), 2072 cm(-1), and 2092 cm(-1) that can be assigned to CO, CN(1), and CN(2), respectively, and which are indistinguishable from those observed for the HypCD complex. HypC could not be isolated with CO or CN(-) ligand contribution. Treatment of HypD with EDTA led to the concomitant loss of Fe and the CO and CN(-) signatures, while oxidation by H2O2 resulted in a positive shift of the CO and CN(-) bands by 35 cm(-1) and 20 cm(-1), respectively, indicative of the ferrous iron as an immediate ligation site for the diatomic ligands. Analysis of HypD amino acid variants identified cysteines 41, 69, and 72 to be essential for maturation of the cofactor. We propose a refined model for the ligation of Fe-(CN)2CO to HypD and the role of HypC in [NiFe]-hydrogenase maturation.

Efficient electron transfer from hydrogen to benzyl viologen by the [NiFe]-hydrogenases of Escherichia coli is dependent on the coexpression of the iron-sulfur cluster-containing small subunit.

Escherichia coli can both oxidize hydrogen and reduce protons. These activities involve three distinct [NiFe]-hydrogenases, termed Hyd-1, Hyd-2, and Hyd-3, each minimally comprising heterodimers of a large subunit, containing the [NiFe] active site, and a small subunit, bearing iron–sulfur clusters. Dihydrogen-oxidizing activity can be determined using redox dyes like benzyl viologen (BV); however, it is unclear whether electron transfer to BV occurs directly at the active site, or via an iron–sulfur center in the small subunit. Plasmids encoding Strep-tagged derivatives of the large subunits of the three E. coli [NiFe]-hydrogenases restored activity of the respective hydrogenase to strain FTD147, which carries in-frame deletions in the hyaB, hybC, and hycE genes encoding the large subunits of Hyd-1, Hyd-2, and Hyd-3, respectively. Purified Strep-HyaB was associated with the Hyd-1 small subunit (HyaA), and purified Strep-HybC was associated with the Hyd-2 small subunit (HybO), and a second iron–sulfur protein, HybA. However, Strep-HybC isolated from a hybO mutant had no other associated subunits and lacked BV-dependent hydrogenase activity. Mutants deleted separately for hyaA, hybO, or hycG (Hyd-3 small subunit) lacked BV-linked hydrogenase activity, despite the Hyd-1 and Hyd-2 large subunits being processed. These findings demonstrate that hydrogenase-dependent reduction of BV requires the small subunit.

[NiFe]-hydrogenase maturation: Isolation of a HypC–HypD complex carrying diatomic CO and CN− ligands

The HypC and HypD maturases are required for the biosynthesis of the Fe(CN)2CO cofactor in the large subunit of [NiFe]‐hydrogenases. Using infrared spectroscopy we demonstrate that an anaerobically purified, Strep‐tagged HypCD complex from Escherichia coli exhibits absorption bands characteristic of diatomic CO and CN− ligands as well as CO2. Metal and sulphide analyses revealed that along with the [4Fe–4S]2+ cluster in HypD, the complex has two additional oxygen‐labile Fe ions. We prove that HypD cysteine 41 is required for the coordination of all three ligands. These findings suggest that the HypCD complex carries minimally the Fe(CN)2CO cofactor.

Substrate specificity and evolutionary implications of a NifDK enzyme carrying NifB‐co at its active site

The in vitro reconstitution of molybdenum nitrogenase was manipulated to generate a chimeric enzyme in which the active site iron–molybdenum cofactor (FeMo‐co) is replaced by NifB‐co. The NifDK/NifB‐co enzyme was unable to reduce N2 to NH3, while exhibiting residual C2H4 and considerable H2 production activities. Production of H2 by NifDK/NifB‐co was stimulated by N2 and was dependent on NifH and ATP hydrolysis. Thus, NifDK/NifB‐co is a useful tool to gain insights into the catalytic mechanism of nitrogenase. Furthermore, phylogenetic analysis of D and K homologs indicates that several early emerging lineages, which contain NifB, NifH and NifDK encoding genes but which lack other genes required for processing NifB‐co into FeMo‐co, might encode an enzyme with similar catalytic properties to NifDK/NifB‐co.

The [NiFe]‐hydrogenase accessory chaperones HypC and HybG of Escherichia coli are iron‐ and carbon dioxide‐binding proteins

HybG and HybG bind by comigration in sds page (View interaction)

[NiFe]-hydrogenase maturation in vitro: analysis of the roles of the HybG and HypD accessory proteins

[NiFe]-hydrogenases (Hyd) bind a nickel-iron-based cofactor. The Fe ion of the cofactor is bound by two cyanide ligands and a single carbon monoxide ligand. Minimally six accessory proteins (HypA-HypF) are necessary for NiFe(CN)2CO cofactor biosynthesis in Escherichia coli. It has been shown that the anaerobically purified HypC-HypD-HypE scaffold complex carries the Fe(CN)2CO moiety of this cofactor. In the present study, we have purified the HybG-HypDE complex and used it to successfully reconstitute in vitro active Hyd from E. coli. HybG is a homologue of HypC that is specifically required for the maturation of Hyd-2 and also functions in the maturation of Hyd-1 of E. coli. Maturation of active Hyd-1 and Hyd-2 could be demonstrated in extracts derived from HybG- and HypD-deficient E. coli strains by adding anaerobically purified HybG-HypDE complex. In vitro maturation was dependent on ATP, carbamoylphosphate, nickel and reducing conditions. Hydrogenase maturation was prevented when the purified HybG-HypDE complex used in the maturation assay lacked a bound Fe(CN)2CO moiety. These findings demonstrate that it is possible to isolate incompletely processed intermediates on the maturation pathway and to use these to activate apo-forms of [NiFe]-hydrogenase large subunits.

Development of a cell-free system reveals an oxygen-labile step in the maturation of [NiFe]-hydrogenase 2 of Escherichia coli

By combining extracts from strains lacking genes encoding either the maturation enzymes or the large subunits of hydrogenases 1, 2 and 3 we could reconstitute in vitro under strictly anaerobic conditions 10–15% of the hydrogenase activity present in wild type Escherichia coli extracts. Purified, unprocessed Strep‐tagged variants of the hydrogenase 2 large subunit, HybC, isolated from either a ΔhybD (encoding the hydrogenase 2‐specific protease) mutant or a strain deficient in HypF could also be matured to active, processed enzyme using this system. These studies reveal that minimally one step early on the hydrogenase maturation pathway is oxygen‐labile.