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

Soboh, Basem; Lindenstrauß, Ute; Granich, Claudia; Javed, Mahwish; Herzberg, Martin; Thomas, Claudia; Stripp, Sven T.

doi:10.1042/bj20140485

Cited by 23 publications

(37 citation statements)

References 57 publications

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“…Although HypE is supplied to the reaction mix in the form of the GDE complex, without the addition of extra His HypE (lane 2) to the reaction assay, only ϳ60% cleavage was detected. The cleavage of ϳ20% of HybC in the absence of His HypF (lane 3) can be explained by the presence of Fe(CN) 2 CO on the GDE complex (14). Notably, the maximal cleavage shown in lane 1 was achieved by the addition of equimolar ratio of purified Involved in acquisition and insertion of the nickel into large subunit HybC (53).…”

Section: The In Vitro System To Study [Nife]-hydrogenase Maturationmentioning

confidence: 99%

“…Fig. 1 presents our current working model for hydrogenase maturation and suggests the following sequence of events: (i) synthesis of CO and CN, ligation to iron, and incorporation of the fully coordinated iron into the large subunit pro-protein by the coordinated activity of HypCDEF (12); (ii) nickel insertion by concerted activity of HypA, HypB, and SlyD (6, 13) (these accessory proteins are not required at high Ni 2ϩ concentrations) (5,14); (iii) nickel acting as recognition site for a specific endopeptidase (15); cleavage of the C-terminal extension of the pro-protein that harbors the [NiFe]-cofactor, inducing conformational changes; and (iv) complex formation with the small subunit to yield active [NiFe]-hydrogenase (16,17).…”

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confidence: 99%

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Proteolytic cleavage orchestrates cofactor insertion and protein assembly in [NiFe]-hydrogenase biosynthesis

Senger

Stripp

Soboh

2017

Journal of Biological Chemistry

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Metalloenzymes catalyze complex and essential processes, such as photosynthesis, respiration, and nitrogen fixation. For example, bacteria and archaea use [NiFe]-hydrogenases to catalyze the uptake and release of molecular hydrogen (H). [NiFe]-hydrogenases are redox enzymes composed of a large subunit that harbors a NiFe(CN)CO metallo-center and a small subunit with three iron-sulfur clusters. The large subunit is synthesized with a C-terminal extension, cleaved off by a specific endopeptidase during maturation. The exact role of the C-terminal extension has remained elusive; however, cleavage takes place exclusively after assembly of the [NiFe]-cofactor and before large and small subunits form the catalytically active heterodimer. To unravel the functional role of the C-terminal extension, we used an enzymatic maturation assay that allows synthesizing functional [NiFe]-hydrogenase-2 of from purified components. The maturation process included formation and insertion of the NiFe(CN)CO cofactor into the large subunit, endoproteolytic cleavage of the C-terminal extension, and dimerization with the small subunit. Biochemical and spectroscopic analysis indicated that the C-terminal extension of the large subunit is essential for recognition by the maturation machinery. Only upon completion of cofactor insertion was removal of the C-terminal extension observed. Our results indicate that endoproteolytic cleavage is a central checkpoint in the maturation process. Here, cleavage temporally orchestrates cofactor insertion and protein assembly and ensures that only cofactor-containing protein can continue along the assembly line toward functional [NiFe]-hydrogenase.

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Section: The In Vitro System To Study [Nife]-hydrogenase Maturationmentioning

confidence: 99%

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confidence: 99%

Proteolytic cleavage orchestrates cofactor insertion and protein assembly in [NiFe]-hydrogenase biosynthesis

Senger

Stripp

Soboh

2017

Journal of Biological Chemistry

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“…It was also shown that the HypC paralog HybG binds to the E. coli Hyd-2 large subunit precursor, which we term here pro-HybC (30,31). HybG is required for the maturation of hydrogen-oxidizing Hyd-2 and also functions in the maturation of the other hydrogen-oxidizing enzyme, Hyd-1, in the bacterium (30,32).…”

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Coordination of Synthesis and Assembly of a Modular Membrane-Associated [NiFe]-Hydrogenase Is Determined by Cleavage of the C-Terminal Peptide

2015

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During biosynthesis of [NiFe]-hydrogenase 2 (Hyd-2) of Escherichia coli, a 15-amino-acid C-terminal peptide is cleaved from the catalytic large subunit precursor, pro-HybC. This peptide is removed only after NiFe(CN) 2 CO cofactor insertion by the Hyp accessory protein machinery has been completed, suggesting that it has a regulatory function during enzyme maturation. We show here that in hyp mutants that fail to synthesize and insert the NiFe cofactor, and therefore retain the peptide, the Tat (twin-arginine translocon) signal peptide on the small subunit HybO is not removed and the subunit is degraded. In a mutant lacking the large subunit, the Tat signal peptide was also not removed from pre-HybO, indicating that the mature large subunit must actively engage the small subunit to elicit Tat transport. We validated the proposed regulatory role of the C-terminal peptide in controlling enzyme assembly by genetically removing it from the precursor of HybC, which allowed assembly and Tat-dependent membrane association of a HybC-HybO heterodimer lacking the NiFe(CN) 2 CO cofactor. Finally, genetic transfer of the C-terminal peptide from pro-HyaB, the large subunit of Hyd-1, onto HybC did not influence its dependence on the accessory protein HybG, a HypC paralog, or the specific protease HybD. This indicates that the C-terminal peptide per se is not required for interaction with the Hyp machinery but rather suggests a role of the peptide in maintaining a conformation of the protein suitable for cofactor insertion. Together, our results demonstrate that the C-terminal peptide on the catalytic subunit controls biosynthesis, assembly, and membrane association of Hyd-2. IMPORTANCE[NiFe]-hydrogenases are multisubunit enzymes with a catalytic subunit containing a NiFe(CN) 2 CO cofactor. Results of previous studies suggested that after synthesis and insertion of the cofactor by the Hyp accessory proteins, this large subunit changes conformation upon proteolytic removal of a short peptide from its C terminus. We show that removal of this peptide is necessary to allow the cleavage of the Tat signal peptide from the small subunit with concomitant membrane association of the heterodimer to occur. Genetic removal of the C-terminal peptide from the large subunit allowed productive interaction with the small subunit and Tat-dependent membrane insertion of a NiFe cofactor-free enzyme. Results based on swapping of C-terminal peptides between hydrogenases suggest that this peptide governs enzyme assembly via a conformational switch. Biosynthesis of complex metal cofactor-containing, multisubunit enzymes requires strict coordination of cofactor biosynthesis, subunit recruitment, and targeting of the protein to its final cellular location. This is particularly important for the numerous redox enzymes present in bacterial systems, many of which are membrane associated or indeed transported across the cytoplasmic membrane (1). Coordination of biosynthesis and assembly is important because cofactors are often highly complex and ma...

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“…Following this notion, the more crucial events with respect to the overall reaction rate arguably take place after formation of the HypCD‐complex, including the HypEF‐mediated, ATP‐dependent transcarbamoylation and dehydration reactions, the binding of cyanylated HypE to a preformed HypCD‐complex and the transfer of the Fe‐(CN) 2 CO‐group to HoxH. The molecular events leading to the assembly of the iron‐cofactor are currently under debate on the basis of the E. coli Hyp‐machinery (Soboh et al, ; Stripp et al, ; Stripp et al, ). Following entry of “modified” HypE into a functional ternary complex with the HypCD‐ or the homologous HybG‐HypD‐complex, redox chemistry affords transfer of the cyanide ligands, yielding the fully assembled Fe‐(CN) 2 CO‐group.…”

Section: Resultsmentioning

confidence: 99%

A flexible toolbox to study protein‐assisted metalloenzyme assembly in vitro

Schiffels

Selmer²

2015

Biotech & Bioengineering

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A number of metalloenzymes harbor unique cofactors, which are incorporated into the apo-enzymes via protein-assisted maturation. In the case of [NiFe]-hydrogenases, minimally seven maturation factors (HypABCDEF and a specific endopeptidase) are involved, making these enzymes an excellent example for studying metallocenter assembly in general. Here, we describe an innovative toolbox to study maturation involving multiple putative gene products. The two core elements of the system are a modular, combinatorial cloning system and a cell-free maturation system, which is based on recombinant Escherichia coli extracts and/or purified maturases. Taking maturation of the soluble, oxygen-tolerant [NiFe]-hydrogenase (SH) from Cupriavidus necator as an example, the capacities of the toolbox are illustrated. In total 18 genes from C. necator were analyzed, including four SH-structural genes, the SH-dedicated hyp-genes and a second set of hyp-genes putatively involved in maturation of the Actinobacterium-like hydrogenase (AH). The two hyp-sets were either expressed in their entirety from single vectors or split into functional modules, which enabled flexible approaches to investigate limitations, specificities and the capabilities of individual constituents to functionally substitute each other. Affinity-tagged Hyp-Proteins were used in pull-down experiments to demonstrate direct interactions between dedicated or non-related constituents. The dedicated Hyp-set from C. necator exhibited the highest maturation efficiency in vitro. Constituents of non-related maturation machineries were found to interact with and to accomplish partial activation of SH. In contrast to homologues of the Hyp-family, omission of the SH-specific endopeptidase HoxW completely abolished in vitro maturation. We detected stoichiometric imbalances inside the recombinant production system, which point to limitations by the cyanylation complex HypEF and the premature subunit HoxH. Purification of HoxW revealed strong indications for the presence of a putative [4Fe-4S]-cluster, which is unique among this class of maturases. Results are discussed in the context of [NiFe]-hydrogenase maturation, and in light of the capacity of the novel toolbox.

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

Cited by 23 publications

References 57 publications

Proteolytic cleavage orchestrates cofactor insertion and protein assembly in [NiFe]-hydrogenase biosynthesis

Proteolytic cleavage orchestrates cofactor insertion and protein assembly in [NiFe]-hydrogenase biosynthesis

Coordination of Synthesis and Assembly of a Modular Membrane-Associated [NiFe]-Hydrogenase Is Determined by Cleavage of the C-Terminal Peptide

A flexible toolbox to study protein‐assisted metalloenzyme assembly in vitro

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