Metal Binding Activity of the Escherichia coli Hydrogenase Maturation Factor HypB

Abstract

The formation of the [NiFe] metallocenter of Escherichia coli hydrogenase 3 requires the participation of proteins encoded by the hydrogenase pleiotropy operon hypABCDEF. The insertion of Ni(II) into the precursor enzyme follows the incorporation of the iron center and is the function of HypA, a Zn(II)-binding protein, and HypB, a GTPase. The Ni(II) donor and the mechanism of transfer of Ni(II) into the hydrogenase precursor protein are not known. In this study, we demonstrate that HypB is a nickel-binding protein capable of binding 1 equiv of Ni(II) with a K(d) in the sub-picomolar range. In addition, HypB has a weaker metal-binding site that is not specific for Ni(II) over Zn(II). Examination of the isolated C-terminal GTPase domain revealed that the high-affinity metal binding capability was severely abrogated but the low-affinity site was intact. By mutating conserved cysteine and histidine residues in E. coli HypB, we have localized the high-affinity Ni(II)-binding site to an N-terminal CXXCGC motif and the low-affinity metal-binding site to the GTPase domain. A model for the function of HypB during the Ni(II) loading of hydrogenase is proposed.

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

“…The most informative system for identifying the roles of these accessory proteins has been Escherichia coli, where the accessory proteins play roles in maturation of all three NiFe hydrogenases (the term hyp is derived from hydrogenase pleiotropic) and the currently-assigned roles are based primarily on the work of A.Böck and colleagues (Blokesch and Bock 2002;Blokesch et al 2004a;Hube et al 2002;Reissmann et al2003). 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).…”

“…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.…”

“…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. This high affinity site would serve as the nickel donor for hydrogenase.…”

“…HypA would direct Ni(II)-bound HypB to the iron-loaded site of the hydrogenase large subunit, and facilitate the metal transfer. The model takes into account the weaker Ni-binding ability of HypA (compared to HypB), as important in facilitating the Ni-transfer step (Leach et al 2005). The authors also suggested a feasible role for the low-affinity metal binding site of HypB; it may detect the nickel status of the enzyme in order to sense when to activate the GTPase.…”

Effects of Metal on the Biochemical Properties of Helicobacter pylori HypB, a Maturation Factor of [NiFe]-Hydrogenase and Urease

“…A few HypB proteins have sequences rich in histidine residues that contribute to the binding of multiple nickel ions and are thought to function in nickel storage (18,53,57). Furthermore, Escherichia coli HypB (EcHypB) binds stoichiometric nickel with subpicomolar affinity to a three-cysteine motif at the N terminus of the protein (8,36). Although this latter nickel site is essential for hydrogenase production in E. coli (13), it is not found in all HypB homologs.…”