High-affinity metal binding by the Escherichia coli [NiFe]-hydrogenase accessory protein HypB is selectively modulated by SlyD

Abstract

[NiFe]-hydrogenase, which catalyzes the reversible conversion between hydrogen gas and protons, is a vital component of the metabolism of many pathogens. Maturation of [NiFe]-hydrogenase requires selective nickel insertion that is completed, in part, by the metallochaperones SlyD and HypB. Escherichia coli HypB binds nickel with sub-picomolar affinity, and the formation of the HypB-SlyD complex activates nickel release from the high-affinity site (HAS) of HypB. In this study, the metal selectivity of this process was investigated. Biochemical experiments revealed that the HAS of full length HypB can bind stoichiometric zinc. Moreover, in contrast to the acceleration of metal release observed with nickel-loaded HypB, SlyD blocks the release of zinc from the HypB HAS. X-ray absorption spectroscopy (XAS) demonstrated that SlyD does not impact the primary coordination sphere of nickel or zinc bound to the HAS of HypB. Instead, computational modeling and X-ray absorption spectroscopy of HypB loaded with nickel or zinc indicated that zinc binds to HypB with a different coordination sphere than nickel. The data suggested that Glu9, which is not a nickel ligand, directly coordinates zinc. These results were confirmed through the characterization of E9A-HypB, which afforded weakened zinc affinity compared to wild-type HypB but similar nickel affinity. This mutant HypB fully supports the production of [NiFe]-hydrogenase in E. coli. Altogether, these results are consistent with the model that the HAS of HypB functions as a nickel site during [NiFe]-hydrogenase enzyme maturation and that the metal selectivity is controlled by activation of metal release by SlyD.

Metalation calculators forE. colistrain JM109 (DE3): Aerobic, anaerobic and hydrogen peroxide exposed cells cultured in LB media

“…The HypB metallochaperone recruits Ni 2+ to the HypA/B complex and subsequently to hydrogenase in E. coli grown under anaerobic conditions 30 . Based on a Ni 2+ affinity of 6 x 10 -14 M and Zn 2+ affinity of 2.2 x 10 -11 M reported by Zamble and co-workers 31 , and excluding metals other than Ni 2+ or Zn 2+ (or using arbitrary weak HypB affinities for other metals in the spreadsheet), the anaerobic calculator (https://mib-nibb.webspace.durham.ac.uk/metalationcalculators/, Supplementary Spreadsheet 2) reassuringly predicts HypB to be predominantly metalated by Ni 2+ in this condition (57.4% Ni 2+ , 2.6% Zn 2+ ).…”

Principles and practice of determining metal–protein affinities

“…Notably, both the spectroscopic and metal-binding properties of Par are highly pH-dependent with a combination of different charge species present at physiological pH [ 27 ], thus particular care should be taken with pH control. Par also responds to several other divalent metal ions (including Fe(II), Co(II) and Ni(II) which each display 1 : 2 stoichiometry for metal : ligand binding) [ 27 , 78 , 79 ] and has been used, for example, to quantify Ni(II) release from proteins [ 80 , 81 ], but reported affinities determined in 50% dioxane/water solution [ 76 ] may differ in aqueous buffers. Slow ligand-exchange observed for Fe(II) complexes makes Par unsuitable as an equilibrium probe for Fe(II) (see a selected example in Figure 6 ) [ 78 ].…”

The carbon monoxide dehydrogenase accessory protein CooJ is a histidine-rich multidomain dimer containing an unexpected Ni(II)-binding site

“…With two independent types of Ni(II)-binding sites, RrCooJ thus resembles the known behavior of HypB and UreE nickelchaperones. Indeed, in addition to the nonconserved histidinerich cluster, two metal-binding sites have been identified in HypB: a conserved low-affinity Ni(II)/Zn(II) binding site, pro-posed to be responsible for the direct Ni(II) transfer to its physiological partner HypA (27), and a nonconserved high affinity Ni(II)-binding site at the N terminus (28). Similarly, the dimeric UreE presents a conserved metal-binding site at the dimer interface, involving a conserved His residue from each monomer located in the C-terminal domains, as well as a second His residue in the disordered C-terminal tail, in addition to other metal-binding sites located in the histidine-rich segment, found in some UreE homologs (29).…”