Proteomic and Mutant Analysis of Hydrogenase Maturation Protein Gene hypE in Symbiotic Nitrogen Fixation of Mesorhizobium huakuii.

Hydrogenases catalyze the simple yet important redox reaction between protons and electrons and H2, thus mediating symbiotic interactions. The contribution of hydrogenase to this symbiosis and anti-oxidative damage was investigated using the M. huakuii hypE (encoding hydrogenase maturation protein) mutant. The hypE mutant grew a little faster than its parental 7653R and displayed decreased antioxidative capacity under H2O2-induced oxidative damage. Real-time quantitative PCR showed that hypE gene expression is significantly up-regulated in all the detected stages of nodule development. Although the hypE mutant can form nodules, the symbiotic ability was severely impaired, which led to an abnormal nodulation phenotype coupled to a 47% reduction in nitrogen fixation capacity. This phenotype was linked to the formation of smaller abnormal nodules containing disintegrating and prematurely senescent bacteroids. Proteomics analysis allowed a total of ninety differentially expressed proteins (fold change > 1.5 or <0.67, p < 0.05) to be identified. Of these proteins, 21 are related to stress response and virulence, 21 are involved in transporter activity, and 18 are involved in energy and nitrogen metabolism. Overall, the HypE protein is essential for symbiotic nitrogen fixation, playing independent roles in supplying energy and electrons, in bacterial detoxification, and in the control of bacteroid differentiation and senescence.

The [4Fe-4S]-Cluster of HydF is not Required for the Binding and Transfer of the Diiron Site of [FeFe]-Hydrogenases.

The active site of [FeFe]-hydrogenases contains a cubane [4Fe-4S]-cluster and a unique diiron cluster with biologically unusual CO and CN- ligands. The biogenesis of this diiron site, termed [2FeH ], requires the maturation proteins HydE, HydF and HydG. During the maturation process HydF serves as a scaffold protein for the final assembly steps and the subsequent transfer of the [2FeH ] precursor, termed [2FeP ], to the [FeFe]-hydrogenase. The binding site of [2FeP ] in HydF has not been elucidated, however, the [4Fe-4S]-cluster of HydF was considered as a possible binding partner of [2FeP ]. By targeting individual amino acids in HydF from Thermosipho melanesiensis using site directed mutagenesis, we examined the postulated binding mechanism as well as the importance and putative involvement of the [4Fe-4S]-cluster for binding and transferring [2FeP ]. Surprisingly, our results suggest that binding or transfer of [2FeP ] does not involve the proposed binding mechanism or the presence of a [4Fe-4S]-cluster at all.

A redox-active HybG-HypD scaffold complex is required for optimal ATPase activity during [NiFe]-hydrogenase maturation in Escherichia coli.

Four Hyp proteins build a scaffold complex upon which the Fe(CN)2 CO group of the [NiFe]-cofactor of hydrogenases (Hyd) is made. Two of these Hyp proteins, the redox-active, [4Fe-4S]-containing HypD protein and the HypC chaperone, form the basis of this scaffold complex. Two different scaffold complexes exist in Escherichia coli, HypCD, and the paralogous HybG-HypD complex, both of which exhibit ATPase activity. Apart from a Rossmann fold, there is no obvious ATP-binding site in HypD. The aim of this study, therefore, was to identify amino acid motifs in HypD that are required for the ATPase activity of the HybG-HypD scaffold complex. Amino acid-exchange variants in three conserved motifs within HypD were generated. Variants in which individual cysteine residues coordinating the iron-sulfur ([4Fe-4S]) cluster were exchanged abolished Hyd enzyme activity and reduced ATPase activity but also destabilized the complex. Two conserved cysteine residues, C69 and C72, form part of HypD's Rossmann fold and play a role in HypD's thiol-disulfide exchange activity. Substitution of these two residues individually with alanine also abolished hydrogenase activity and strongly reduced ATPase activity, particularly the C72A exchange. Residues in a further conserved GFETT motif were exchanged, but neither hydrogenase enzyme activity nor ATPase activity of the isolated HybG-HypD complexes was significantly affected. Together, our findings identify a strong correlation between the redox activity of HypD, ATPase activity, and the ability of the complex to mature Hyd enzymes. These results further highlight the important role of thiol residues in the HybG-HypD scaffold complex during [NiFe]-cofactor biosynthesis.

[FeFe]-Hydrogenase: Defined Lysate-Free Maturation Reveals a Key Role for Lipoyl-H-Protein in DTMA Ligand Biosynthesis.

Maturation of [FeFe]-hydrogenase (HydA) involves synthesis of a CO, CN- , and dithiomethylamine (DTMA)-coordinated 2Fe subcluster that is inserted into HydA to make the active hydrogenase. This process requires three maturation enzymes: the radical S-adenosyl-l-methionine (SAM) enzymes HydE and HydG, and the GTPase HydF. In vitro maturation with purified maturation enzymes has been possible only when clarified cell lysate was added, with the lysate presumably providing essential components for DTMA synthesis and delivery. Here we report maturation of [FeFe]-hydrogenase using a fully defined system that includes components of the glycine cleavage system (GCS), but no cell lysate. Our results reveal for the first time an essential role for the aminomethyl-lipoyl-H-protein of the GCS in hydrogenase maturation and the synthesis of the DTMA ligand of the H-cluster. In addition, we show that ammonia is the source of the bridgehead nitrogen of DTMA.

Inactivation of the uptake hydrogenase in the purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus CBS enables a biological water-gas shift platform for H2 production.

Biological H2 production has potential to address energy security and environmental concerns if produced from renewable or waste sources. The purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus CBS produces H2 while oxidizing CO, a component of synthesis gas (Syngas). CO-linked H2 production is facilitated by an energy-converting hydrogenase (Ech), while a subsequent H2 oxidation reaction is catalyzed by a membrane-bound hydrogenase (MBH). Both hydrogenases contain [NiFe] active sites requiring 6 maturation factors (HypA-F) for assembly, but it is unclear which of the two annotated sets of hyp genes are required for each in R. gelatinosus CBS. Herein, we report correlated expression of hyp1 genes with Ech genes and hyp2 expression with MBH genes. Moreover, we find that while Ech H2 evolving activity is only delayed when hyp1 is deleted, hyp2 deletion completely disrupts MBH H2 uptake, providing a platform for a biologically driven water-gas shift reaction to produce H2 from CO.

Complex formation between the Escherichia coli [NiFe]-hydrogenase nickel maturation factors.

The biosynthesis of the dinuclear metal cluster at the active sites of the [NiFe]-hydrogenase enzymes is a multi-step process executed by a suite of accessory proteins. Nickel insertion during maturation of Escherichia coli [NiFe]-hydrogenase 3 is achieved by the metallochaperones HypA, SlyD and the GTPase HypB, but how these proteins cooperate to ensure nickel delivery is not known. In this study, the complexes formed between the individual purified proteins were examined by using several methods. Size exclusion chromatography (SEC) indicated that SlyD and HypB interact primarily in a 1:1 complex. The affinity of HypB-SlyD was measured by using surface plasmon resonance, which revealed a KD of 24 ± 10 nM in the absence of nucleotide and an interaction several fold tighter in the presence of GDP. A ternary complex between all three proteins was not detected, and instead SlyD blocked the interaction of HypA with HypB in competitive binding experiments. Furthermore, cross-linking experiments suggest a weak interaction between HypA and SlyD, which is not detectable by SEC. Electrochemical analysis confirmed each of the pairwise interactions and that the relative affinities of these complexes are on the order of HypB-SlyD > HypB-HypA > HypA-SlyD. These results indicate a hierarchy of interactions, as opposed to a single multiprotein complex, and provide insight into the nickel delivery process during hydrogenase enzyme maturation.

Crystal structures of a [NiFe] hydrogenase large subunit HyhL in an immature state in complex with a Ni chaperone HypA.

Ni-Fe clusters are inserted into the large subunit of [NiFe] hydrogenases by maturation proteins such as the Ni chaperone HypA via an unknown mechanism. We determined crystal structures of an immature large subunit HyhL complexed with HypA from Thermococcus kodakarensis Structure analysis revealed that the N-terminal region of HyhL extends outwards and interacts with the Ni-binding domain of HypA. Intriguingly, the C-terminal extension of immature HyhL, which is cleaved in the mature form, adopts a ß-strand adjacent to its N-terminal ß-strands. The position of the C-terminal extension corresponds to that of the N-terminal extension of a mature large subunit, preventing the access of endopeptidases to the cleavage site of HyhL. These findings suggest that Ni insertion into the active site induces spatial rearrangement of both the N- and C-terminal tails of HyhL, which function as a key checkpoint for the completion of the Ni-Fe cluster assembly.

Genetic analyses of the functions of [NiFe]-hydrogenase maturation endopeptidases in the hyperthermophilic archaeon Thermococcus kodakarensis.

The maturation of [NiFe]-hydrogenases requires a number of accessory proteins, which include hydrogenase-specific endopeptidases. The endopeptidases carry out the final cleavage reaction of the C-terminal regions of [NiFe]-hydrogenase large subunit precursors. The hyperthermophilic archaeon Thermococcus kodakarensis harbors two [NiFe]-hydrogenases, a cytoplasmic Hyh and a membrane-bound Mbh, along with two putative hydrogenase-specific endopeptidase genes. In this study, we carried out a genetic examination on the two endopeptidase genes, TK2004 and TK2066. Disruption of TK2004 resulted in a strain that could not grow under conditions requiring hydrogen evolution. The Mbh large subunit precursor (pre-MbhL) in this strain was not processed at all whereas Hyh cleavage was not affected. On the other hand, disruption of TK2066 did not affect the growth of T. kodakarensis under the conditions examined. Cleavage of the Hyh large subunit precursor (pre-HyhL) was impaired, but could be observed to some extent. In a strain lacking both TK2004 and TK2066, cleavage of pre-HyhL could not be observed. Our results indicate that pre-MbhL cleavage is carried out solely by the endopeptidase encoded by TK2004. Pre-HyhL cleavage is mainly carried out by TK2066, but TK2004 can also play a minor role in this cleavage.

Deletion of a gene cluster for [Ni-Fe] hydrogenase maturation in the anaerobic hyperthermophilic bacterium Caldicellulosiruptor bescii identifies its role in hydrogen metabolism.

The anaerobic, hyperthermophlic, cellulolytic bacterium Caldicellulosiruptor bescii grows optimally at ∼80 °C and effectively degrades plant biomass without conventional pretreatment. It utilizes a variety of carbohydrate carbon sources, including both C5 and C6 sugars, released from plant biomass and produces lactate, acetate, CO2, and H2 as primary fermentation products. The C. bescii genome encodes two hydrogenases, a bifurcating [Fe-Fe] hydrogenase and a [Ni-Fe] hydrogenase. The [Ni-Fe] hydrogenase is the most widely distributed in nature and is predicted to catalyze hydrogen production and to pump protons across the cellular membrane creating proton motive force. Hydrogenases are the key enzymes in hydrogen metabolism and their crystal structure reveals complexity in the organization of their prosthetic groups suggesting extensive maturation of the primary protein. Here, we report the deletion of a cluster of genes, hypABFCDE, required for maturation of the [Ni-Fe] hydrogenase. These proteins are specific for the hydrogenases they modify and are required for hydrogenase activity. The deletion strain grew more slowly than the wild type or the parent strain and produced slightly less hydrogen overall, but more hydrogen per mole of cellobiose. Acetate yield per mole of cellobiose was increased ∼67 % and ethanol yield per mole of cellobiose was decreased ∼39 %. These data suggest that the primary role of the [Ni-Fe] hydrogenase is to generate a proton gradient in the membrane driving ATP synthesis and is not the primary enzyme for hydrogen catalysis. In its absence, ATP is generated from increased acetate production resulting in more hydrogen produced per mole of cellobiose.

Elimination of hydrogenase active site assembly blocks H2 production and increases ethanol yield in Clostridium thermocellum.

BACKGROUND: The native ability of Clostridium thermocellum to rapidly consume cellulose and produce ethanol makes it a leading candidate for a consolidated bioprocessing (CBP) biofuel production strategy. C. thermocellum also synthesizes lactate, formate, acetate, H2, and amino acids that compete with ethanol production for carbon and electrons. Elimination of H2 production could redirect carbon flux towards ethanol production by making more electrons available for acetyl coenzyme A reduction to ethanol. RESULTS: H2 production in C. thermocellum is encoded by four hydrogenases. Rather than delete each individually, we targeted hydrogenase maturase gene hydG, involved in converting the three [FeFe] hydrogenase apoenzymes into holoenzymes. Further deletion of the [NiFe] hydrogenase (ech) resulted in a mutant that functionally lacks all four hydrogenases. H2 production in ∆hydG∆ech was undetectable, and the ethanol yield nearly doubled to 64% of the maximum theoretical yield. Genomic analysis of ∆hydG revealed a mutation in adhE, resulting in a strain with both NADH- and NADPH-dependent alcohol dehydrogenase activities. While this same adhE mutation was found in ethanol-tolerant C. thermocellum strain E50C, ∆hydG and ∆hydG∆ech are not more ethanol tolerant than the wild type, illustrating the complicated interactions between redox balancing and ethanol tolerance in C. thermocellum. CONCLUSIONS: The dramatic increase in ethanol production suggests that targeting protein post-translational modification is a promising new approach for simultaneous inactivation of multiple enzymes.