A Labile Regulatory Copper Ion Lies Near the T1 Copper Site in the Multicopper Oxidase CueO

Abstract

CueO, a multicopper oxidase, is part of the copperregulatory cue operon in Escherichia coli, is expressed under conditions of copper stress and shows enhanced oxidase activity when additional copper is present. The 1.7-Å resolution structure of a crystal soaked in CuCl 2 reveals a Cu(II) ion bound to the protein 7.5 Å from the T1 copper site in a region rich in methionine residues. The trigonal bipyramidal coordination sphere is unusual, containing two methionine sulfur atoms, two aspartate carboxylate oxygen atoms, and a water molecule. Asp-439 both ligates the labile copper and hydrogen-bonds to His-443, which ligates the T1 copper. This arrangement may mediate electron transfer from substrates to the T1 copper. Mutation of residues bound to the labile copper results in loss of oxidase activity and of copper tolerance, confirming a regulatory role for this site. The methionine-rich portion of the protein, which is similar to that of other proteins involved in copper homeostasis, does not display additional copper binding. The type 3 copper atoms of the trinuclear cluster in the structure are bridged by a chloride ion that completes a square planar coordination sphere for the T2 copper atom but does not affect oxidase activity.

Molecular dynamics study of the structural basis of dysfunction and the modulation of reactive oxygen species generation by pathogenic mutants of human dihydrolipoamide dehydrogenase

“…These findings suggest that LADH might display some of its moonlighting functions as an independent enzyme (see also [25,26]). Protein-protein interactions and conditions used for crystallography often shift conformational equilibria of proteins and stabilize the thermodynamically favored conformation under the existing conditions [59][60][61][62][63]. This conformation may considerably deviate from the uncomplexed or the solution structure [64][65][66][67][68].…”

“…As there is no experimental data available yet on the diaphorase conformation of LADH, which could be used as structural restraints in the MD simulations, the accuracy of the presented structures cannot compete with a high-resolution crystal or NMR structure (although we applied the same force fields and simulation approaches which are routinely used in NMR and X-ray structure determination [47,63,65,67,68]). Nevertheless, we…”

Crystal Structures of Multicopper Oxidase CueO Bound to Copper(I) and Silver(I)

“…The T1 copper in CueO, unlike with laccases (22), is buried in the protein interior (23,24) (Fig. 1).…”

“…1). A 45-residue insert (residues 355-399) containing 14 methionines and five histidines, blocks solvent access to the T1 site and contributes ligands to an additional copper-binding site that must be occupied for full CueO activity (24). Organic compounds and Fe(II) are therefore only substrates for CueO in the presence of excess copper.…”

“…Organic compounds and Fe(II) are therefore only substrates for CueO in the presence of excess copper. We originally termed this additional site the regulatory copper site (rCu) for its role in stimulating CueO activity (24), but have renamed it the substrate copper site (sCu) in view of its Cu(I) substrate binding role. Much of the methionine-rich insert has been disordered and unseen in previous CueO structures (23)(24)(25).…”