[NiFe]‐hydrogenase maturation: Isolation of a HypC–HypD complex carrying diatomic CO and CN<sup>−</sup> ligands

Soboh, Basem; Stripp, Sven T.; Muhr, Enrico; Granich, Claudia; Braussemann, Mario; Herzberg, Martin; Heberle, Joachim; Sawers, R. Gary

doi:10.1016/j.febslet.2012.09.019

Cited by 41 publications

(99 citation statements)

References 36 publications

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“…Such a mechanism would ensure that in wild-type cells only a large subunit with a complete NiFe cofactor proceeds along the maturation pathway. After synthesis and insertion of the Fe(CN) 2 CO moiety into the apo-large subunit by the HybG-HypDEF machinery (26,27), nickel insertion occurs (16,19). This step signifies completion of cofactor biosynthesis.…”

Section: Discussionmentioning

confidence: 99%

“…A first indication that this C-terminal peptide might be important during enzyme maturation was originally demonstrated for enzymes from both bacterial and archaeal sources (20,21,24). It was subsequently shown that the HypC protein, which is proposed to have an important role in delivering the completed Fe(CN) 2 CO moiety of the active site cofactor to the apo-large subunit (3,(25)(26)(27), interacts with the precursor of the HycE protein, the large subunit of Escherichia coli Hyd-3 (17,18,28). Similar observations were made for Hyd enzymes from Ralstonia eutropha (19,29).…”

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confidence: 99%

“…The multiprotein complex that was identified also included the HypD and HypE accessory proteins, which are involved in NiFe cofactor biosynthesis. The HypE-HypF complex (33) synthesizes the CN Ϫ ligands of the cofactor (34,35), while HypD forms the core of the maturation protein complex binding HypC (25)(26)(27) and HybG (30,31). The importance of the C terminus on the HybC precursor (pro-HybC in Fig.…”

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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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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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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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“…NiFe(CN) 2 CO biosynthesis requires specific maturation machinery, in which six Hyp proteins (HypA-HypF) play key roles (6,7). Four Hyp proteins (HypC-HypF) are involved in the biosynthesis and incorporation of the Fe(CN) 2 CO group (8)(9)(10)(11)(12)(13)(14)(15). After Fe insertion, HypA and HypB insert the Ni ion into the hydrogenase large subunit (16).…”

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Structural basis of a Ni acquisition cycle for [NiFe] hydrogenase by Ni-metallochaperone HypA and its enhancer

Watanabe

Kawashima

Nishitani

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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The Ni atom at the catalytic center of [NiFe] hydrogenases is incorporated by a Ni-metallochaperone, HypA, and a GTPase/ATPase, HypB. We report the crystal structures of the transient complex formed between HypA and ATPase-type HypB (HypB AT ) with Ni ions. Transient association between HypA and HypB AT is controlled by the ATP hydrolysis cycle of HypB AT, which is accelerated by HypA. Only the ATP-bound form of HypB AT can interact with HypA and induces drastic conformational changes of HypA. Consequently, upon complex formation, a conserved His residue of HypA comes close to the N-terminal conserved motif of HypA and forms a Ni-binding site, to which a Ni ion is bound with a nearly square-planar geometry. The Ni binding site in the HypAB AT complex has a nanomolar affinity (K d = 7 nM), which is in contrast to the micromolar affinity (K d = 4 μM) observed with the isolated HypA. The ATP hydrolysis and Ni binding cause conformational changes of HypB AT , affecting its association with HypA. These findings indicate that HypA and HypB AT constitute an ATP-dependent Ni acquisition cycle for [NiFe]-hydrogenase maturation, wherein HypB AT functions as a metallochaperone enhancer and considerably increases the Ni-binding affinity of HypA.X-ray crystallography | metalloprotein | transient complex | metallochaperone A pproximately one-half of all cellular proteins require specific metal ions for proper function, which are delivered by specific metallochaperones (1, 2). However, the mechanisms of correct acquisition and delivery to target proteins of many metallochaperones remain poorly understood.[NiFe] hydrogenases harbor a complex metal cofactor, NiFe(CN) 2 CO, in their active sites (3). This cofactor catalyzes reversible H 2 production. The Ni atom in the NiFe(CN) 2 CO cofactor is bound to four thiolate groups, two of which also bridge the Fe(CN) 2 CO group (4, 5). NiFe(CN) 2 CO biosynthesis requires specific maturation machinery, in which six Hyp proteins (HypA-HypF) play key roles (6, 7). Four Hyp proteins (HypC-HypF) are involved in the biosynthesis and incorporation of the Fe(CN) 2 CO group (8-15). After Fe insertion, HypA and HypB insert the Ni ion into the hydrogenase large subunit (16).HypA is a Ni-metallochaperone that binds to a Ni ion with micromolar affinity (17-19), and its structure consists of a Ni-binding domain (NiBD) and a Zn-binding domain (ZnBD) (20, 21). The NiBD contains a highly conserved MHE motif that is essential for Ni binding at the N terminus (20,22). HypB consists of a common GTPase domain and a less conserved metal-binding region (23)(24)(25). Recently, ATPase-type HypB (HypB AT , previously abbreviated as mmHypB) proteins were identified from Thermococcales (26). GTPase and ATPase types of HypB belong to the SIMIBI class NTPase family and share a similar architecture, despite their low sequence similarity (27). HypA and HypB form a transient complex in the Ni insertion process (17,22,28,29). In the Escherichia coli system, Ni transfer occurs from HypB to HypA (30). However, the functi...

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“…Ezek alapján kijelenthető, hogy legalább a HypA, HypB, HypC, HypD, HypE, HypF és egy specifikus endopeptidáz szükséges az érett nagyalegység kialakításához (2. ábra) (Soboh 2012, Bürstel 2012.…”

Section: Hmd Vagy [Fe] Hidrogenázokunclassified

Fotobiohidrogén termelésében szerepet játszó enzimek bioszintézise és metabolikus kontextusa

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

Cited by 41 publications

References 36 publications

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

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

Structural basis of a Ni acquisition cycle for [NiFe] hydrogenase by Ni-metallochaperone HypA and its enhancer

Fotobiohidrogén termelésében szerepet játszó enzimek bioszintézise és metabolikus kontextusa

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

Cited by 41 publications

References 36 publications

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

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

Structural basis of a Ni acquisition cycle for [NiFe] hydrogenase by Ni-metallochaperone HypA and its enhancer

Fotobiohidrogén termelésében szerepet játszó enzimek bioszintézise és metabolikus kontextusa

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