HybF, a Zinc-Containing Protein Involved in NiFe Hydrogenase Maturation

Blokesch, Melanie; Michaela, Rohrmoser; Rode, Sabine; Böck, August

doi:10.1128/jb.186.9.2603-2611.2004

Cited by 63 publications

(106 citation statements)

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“…HypA (also known as HybF) is a Zn-containing protein (Atanassova and Zamble 2005)) but also binds stoichiometric amounts of nickel, and HypB is a GTPase (Blokesch et al 2004a;Leach et al 2005). The present model is that HypA serves as a nickel chaperone, and HypB as a regulator that thermodynamically controls the donation of the metal to the hydrogenase apoprotein or release of the nickel-free chaperone (Blokesch et al 2004c;Leach et al 2005;Reissmann et al 2003). The present model for HypA/HypB function (Leach et al 2005) involves a key metal binding site in HypB beyond the usual (Ni-binding) polyhistidine stretch.…”

Section: Accessory Proteins For Hydrogenase Maturationmentioning

confidence: 99%

Nickel-binding and accessory proteins facilitating Ni-enzyme maturation in Helicobacter pylori

2007

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Helicobacter pylori colonizes the human gastric mucosa and this can lead to chronic gastritis, peptic and duodenal ulcers, and even gastric cancers. The bacterium colonizes over one-half of the worlds population. Nickel plays a major role in the bacteriums colonization and persistence attributes as two nickel enzyme sinks obligately contain the metal. Urease accounts for up to 10% of the total cellular protein made and is required for initial colonization processes, and the hydrogen oxidizing hydrogenase provides the bacterium a high-energy substrate yielding low potential electrons for energy generation. A battery of accessory proteins are needed for maturation or activation of each of the apoen-zymes. These include Ni-chaperones and GTPas-es, some of which are unique to each Ni-enzyme and others that are individually required for maturation of both the Ni-enzymes. H. pylori's need for some conventional hydrogenase maturation proteins playing roles in urease maturation may have to do with the poor nickel-sequestering ability of the UreE urease maturation protein compared to other systems. H. pylori also possesses a NixA nickel specific permease, a nickel dependent regulator (NikR), a recently identified nickel efflux system (CznABC), and a histidinerich heat shock protein, HspA. Based on mutant analysis approaches all these proteins have roles in nickel homeostasis, in urease expression, and in host colonization. The His-rich putative nickel storage proteins Hpn and Hpn-like play roles in nickel detoxification and may influence the levels of Ni-activated urease that can be achieved.

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Section: Accessory Proteins For Hydrogenase Maturationmentioning

confidence: 99%

Nickel-binding and accessory proteins facilitating Ni-enzyme maturation in Helicobacter pylori

2007

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“…In vitro analysis of H. pylori HypA revealed that it forms a homodimer that cooperatively binds two nickel ions and that the protein can associate with HypB (38). A Strep tag fusion of HybF from E. coli also binds nickel and/or zinc, but neither dimerization nor association with HypB was detected (7).…”

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“…SlyD can bind multiple nickel ions (23); however, the ⌬slyD strain of E. coli only has a partially deficient hydrogenase phenotype (54), suggesting that this protein is not the key nickel delivery factor. Instead, it has been proposed that HypA/HybF might serve as a source of nickel in E. coli and H. pylori at low metal concentrations (7,38).In order to understand more about the cellular functions of these proteins, we purified E. coli HypA and examined the biochemical properties of this protein. While this work was in…”

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Escherichia coli HypA Is a Zinc Metalloprotein with a Weak Affinity for Nickel

Atanassova

Zamble

2005

J Bacteriol

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The hyp operon encodes accessory proteins that are required for the maturation of the [NiFe] hydrogenase enzymes and, in some organisms, for the production of urease enzymes as well. HypA or a homologous protein is required for nickel insertion into the hydrogenase precursor proteins. In this study, recombinant HypA from Escherichia coli was purified and characterized in vitro. Metal analysis was used to demonstrate that HypA simultaneously binds stoichiometric Zn 2؉ and stoichiometric Ni 2؉ . Competition experiments with a metallochromic indicator reveal that HypA binds zinc with nanomolar affinity. Spectroscopic analysis of cobaltcontaining HypA provides evidence for a tetrathiolate coordination sphere, suggesting that the zinc site has a structural role. In addition, HypA can exist as several oligomeric complexes and the zinc content modulates the quaternary structure of the protein. Fluorescence titration experiments demonstrate that HypA binds nickel with micromolar affinity and that the presence of zinc does not dramatically affect the nickel-binding activity. Finally, complex formation between HypA and HypB, another accessory protein required for nickel insertion, was observed. These experiments suggest that HypA is an architectural component of the hydrogenase metallocenter assembly pathway and that it may also have a direct role in the delivery of nickel to the hydrogenase large subunit.Hydrogen metabolism is important in a variety of organisms that either consume hydrogen gas as an alternative source of energy or produce it in the dispersion of excess reducing equivalents (10,22,42,48). A key enzyme in these pathways is hydrogenase, which catalyzes the reversible formation of H 2 from protons and electrons (15,42). The mechanisms of action and the structures of hydrogenases have been extensively studied due to their unusual chemistry, as well as the potential for use in biotechnological applications (for examples of recent reviews, see references 10, 15, 22, and 49). The hydrogenases can be divided into three phylogenetically distinct classes on the basis of the active-site composition (48). Members of one of these classes, the [NiFe] hydrogenases, are widely distributed in Bacteria and Archaea (48) and contain one iron and one nickel in a deeply buried bimetallic active site (49).Biosynthesis of the [NiFe] hydrogenase molecular structures in vivo requires multiple accessory proteins that sequentially assemble and insert the active-site components (6,12,28,39,48). These proteins have been assigned the hydrogenase pleiotropy (Hyp) designation in many organisms. Although the details of hydrogenase metallocenter assembly are not yet clear, the sequence of events has been mapped out based on genetic and biochemical studies of several systems, including the biosynthesis of Escherichia coli hydrogenase 3. In the first phase, HypCDEF interact with the hydrogenase large subunit to prepare and insert the iron center with its unusual three CO and CN diatomic ligands. HypC then remains associated with the precurs...

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“…HypA is a Ni-metallochaperone that binds to a Ni ion with micromolar affinity (17)(18)(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).…”

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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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HybF, a Zinc-Containing Protein Involved in NiFe Hydrogenase Maturation

Cited by 63 publications

References 60 publications

Nickel-binding and accessory proteins facilitating Ni-enzyme maturation in Helicobacter pylori

Nickel-binding and accessory proteins facilitating Ni-enzyme maturation in Helicobacter pylori

Escherichia coli HypA Is a Zinc Metalloprotein with a Weak Affinity for Nickel

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

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