Complex enzymes containing Fe-S clusters are ubiquitous in nature, where they are involved in a number of fundamental processes including carbon dioxide fixation, nitrogen fixation and hydrogen metabolism. Hydrogen metabolism is facilitated by the activity of three evolutionarily and structurally unrelated enzymes: the [NiFe]-hydrogenases, [FeFe]-hydrogenases and [Fe]-hydrogenases (Hmd). The catalytic core of the [FeFe]-hydrogenase (HydA), termed the H-cluster, exists as a [4Fe-4S] subcluster linked by a cysteine thiolate to a modified 2Fe subcluster with unique non-protein ligands. The 2Fe subcluster and non-protein ligands are synthesized by the hydrogenase maturation enzymes HydE, HydF and HydG; however, the mechanism, synthesis and means of insertion of H-cluster components remain unclear. Here we show the structure of HydA(DeltaEFG) (HydA expressed in a genetic background devoid of the active site H-cluster biosynthetic genes hydE, hydF and hydG) revealing the presence of a [4Fe-4S] cluster and an open pocket for the 2Fe subcluster. The structure indicates that H-cluster synthesis occurs in a stepwise manner, first with synthesis and insertion of the [4Fe-4S] subcluster by generalized host-cell machinery and then with synthesis and insertion of the 2Fe subcluster by specialized hydE-, hydF- and hydG-encoded maturation machinery. Insertion of the 2Fe subcluster presumably occurs through a cationically charged channel that collapses following incorporation, as a result of conformational changes in two conserved loop regions. The structure, together with phylogenetic analysis, indicates that HydA emerged within bacteria most likely from a Nar1-like ancestor lacking the 2Fe subcluster, and that this was followed by acquisition in several unicellular eukaryotes.

The L protein (BchL) of the dark-operative protochlorophyllide reductase (DPOR) from Rhodobacter sphaeroides has been purified from an Azotobacter vinelandii expression system; its interaction with nucleotides has been examined, and the X-ray structure of the protein has been determined with bound MgADP to 1.6 A resolution. DPOR catalyzes the reduction of protochlorophyllide to chlorophyllide, a reaction critical to the biosynthesis of bacteriochlorophylls. The DPOR holoenzyme is comprised of two component proteins, the dimeric BchL protein and the heterotetrameric BchN/BchB protein. The DPOR component proteins share significant overall similarities with the nitrogenase Fe protein (NifH) and the MoFe (NifDK) protein, the enzyme system responsible for reduction of dinitrogen to ammonia. Here, BchL was expressed in A. vinelandii and purified to homogeneity using an engineered polyhistidine tag. The purified, recombinant BchL was found to contain 3.6 mol of Fe/mol of BchL homodimer, consistent with the presence of a [4Fe-4S] cluster and analogous to the [4Fe-4S] cluster present in the Fe protein. The MgATP- and MgADP-induced conformational changes in BchL were examined by an Fe chelation assay and found to be distinctly different from the nucleotide-stimulated Fe release observed for the Fe protein. The recombinant BchL was crystallized with bound MgADP, and the structure was determined to 1.6 A resolution. BchL is found to share overall structural similarity with the nitrogenase Fe protein, including the subunit bridging [4Fe-4S] cluster and nucleotide binding sites. Despite the high level of structural similarity, however, BchL is found to be incapable of substituting for the Fe protein in a nitrogenase substrate reduction assay. The newly determined structure of BchL and its comparison to its close homologue, the nitrogenase Fe protein, provide the basis for understanding how these highly related proteins can discriminate between their respective functions in microbial systems where each must function simultaneously.

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BackgroundDog dander extract used for diagnosis and allergen-specific immunotherapy is often of variable and of poor quality.ObjectiveTo assemble four well-established dog allergen components into one recombinant folded protein for improved diagnosis and vaccination of allergy to dog.MethodsA linked molecule, comprising the four dog lipocalin allergens Can f 1, Can f 2, Can f 4 and Can f 6 was constructed. The tetrameric protein was structurally characterized by small angle X-ray scattering, and compared with each single recombinant lipocalin allergen or an equimolar mix of the four allergens by analytical size exclusion chromatography, circular dichroism, allergen-specific IgE in serum by ELISA and allergen-dependent capacity to activate basophils. The immunogenicity of the fusion protein was evaluated in immunized mice by assessing splenocyte proliferation and antibody production.ResultsThe linked tetrameric construct was produced as a soluble fusion protein, with the specific folds of the four individual allergens conserved. This multi-allergen molecule was significantly more efficient (p<0.001) than each single recombinant allergen in binding to dog-specific IgE, and the epitope spectrum was unaffected compared to an equimolar mix of the four allergens. Basophil degranulation revealed that the biologic activity of the linked molecule was retained. Immunization of mice with the linked construct induced comparable allergen-specific IgG responses with blocking capacity towards all included allergens and generated comparably low T-cell responses.ConclusionWe provide the first evidence for a linked recombinant molecule covering the major dog allergens for potential use in diagnostics and allergy vaccination of dog allergic patients.