Structural investigations of nickel metalloproteins: The Nik system (NikA and NikR), acireductone dioxygenase, and hydrogenase

Nickel, essential to many biological processes, is a component of the active sites of enzymes that catalyze both redox and non-redox reactions. Here the results of investigations aimed at understanding the structure and function of four nickel metalloproteins is presented. X-ray absorption spectroscopy (XAS) was used to explore the nickel sites in Escherichia coli NikA and NikR. NikA is a Ni-binding component of the NikA-E nickel specific permease, responsible for the uptake of Ni in E. coli. NikA also plays a role in the negative chemotaxis associated with toxic Ni concentrations. NikA plays a non-redox role, and XAS of its nickel site exhibited an octahedral environment composed of six N/O-ligands. NikR regulates Ni uptake and translocation by regulation of the nik operon. XAS of the high affinity nickel sites of NikR holoprotein, C-terminal domain, and a half-site DNA complex revealed a planar four coordinate, a five coordinate, and an octahedral site respectively. There is a predominance of N/O-ligation for the NikR samples as expected for Ni proteins with a non-redox role. Only the holoprotein possesses one sulfur ligand. The Ni coordination environments support a proposed role for NikR function where the N-terminus binds DNA specific subsites, and the C-terminal senses, and binds nickel and stabilizes the NikR-DNA complex.; XAS was used to probe the structure of the novel nickel containing acireductone dioxygenase (NiARD). Klebsiella pneumoniae produces two ARD's, involved in its methionine salvage pathway. They differ only in their metal content, but catalyze different reactions. NiARD provides a shunt to the methionine salvage pathway. XAS of resting NiARD and the enzyme substrate complex shows an octahedral resting NiARD with N/O-ligands, where 3 or 4 are histidines. Substrate binds to NiARD in a bidentate fashion displacing two ligands, at least one being a histidine.; Nickel also plays a key redox role in NiFe hydrogenases. Here circular dichroism and electronic spectroscopy were used to monitor conformational changes in Thiocapsa roseopersicina NiFe hydrogenase as a function of redox poise. Although changes may occur at the metal sites, the resulting changes in the protein secondary structure are small.