The bifunctional roles of nickel ions in Helicobacter pylori [NiFe]-hydrogenase maturation pathway

Abstract

[NiFe]-hydrogenases are widely produced by many types of bacteria and involved in hydrogen metabolism. The large subunit of the hydrogenase contains a dinuclear [NiFe] metal center, which is required for enzyme activity. The insertion of nickel ion into the metalocenter requires the participation of two chaperones, HypA and HypB, a metal-binding GTPase. Although extensive studies have been performed, the detailed mechanism of nickel insertion carried out by these two proteins remains unknown. Herein, we purified and characterize the HypB protein from Helicobacter pylori. The protein showed the capacity to bind both Zn2+ and Ni2+ with a stoichiometry of one metal ion per monomer. Apo-form and Zn2+-bound HypB have intrinsic weak GTPase activity. However, Ni2+ binding to HypB significantly enhanced its GTPase activity and caused the dimerization of the protein. These data clearly demonstrated the bifunctional roles of Ni2+ in the hydrogenase maturation pathway, which served as delivery cargos as well as a regulator for HypB GTPase activity. Particually, direct Ni2+ transfer from HypA to HypB protein was observed. The HypA-HypB complex interfaces on HypA protein were mapped based on chemical shift perturbation data and a HypA-HypB complex model was built up using homolog modeling and docking methods. Based on all the data, a GTPase regulated Ni2+ delivery mechanism was proposed to elucidate the detailed functions performed by the two Ni2+ chaperones.