Affinity gradients drive copper to cellular destinations

Abstract

Copper is an essential trace element for eukaryotes and most prokaryotes. However, intracellular free copper must be strictly limited because of its toxic side effects. Complex systems for copper trafficking evolved to satisfy cellular requirements while minimizing toxicity. The factors driving the copper transfer between protein partners along cellular copper routes are, however, not fully rationalized. Until now, inconsistent, scattered and incomparable data on the copper-binding affinities of copper proteins have been reported. Here we determine, through a unified electrospray ionization mass spectrometry (ESI-MS)-based strategy, in an environment that mimics the cellular redox milieu, the apparent Cu(I)-binding affinities for a representative set of intracellular copper proteins involved in enzymatic redox catalysis, in copper trafficking to and within various cellular compartments, and in copper storage. The resulting thermodynamic data show that copper is drawn to the enzymes that require it by passing from one copper protein site to another, exploiting gradients of increasing copper-binding affinity. This result complements the finding that fast copper-transfer pathways require metal-mediated protein-protein interactions and therefore protein-protein specific recognition. Together with Cu,Zn-SOD1, metallothioneins have the highest affinity for copper(I), and may play special roles in the regulation of cellular copper distribution; however, for kinetic reasons they cannot demetallate copper enzymes. Our study provides the thermodynamic basis for the kinetic processes that lead to the distribution of cellular copper.

Unification of the Copper(I) Binding Affinities of the Metallo-chaperones Atx1, Atox1, and Related Proteins

“…At a higher Bcs concentration (Ͼ200 M) where Atox1-type proteins compete strongly for Cu I , GSH cannot compete (even at millimolar concentration; supplemental Tables S3 and S4). This result contrasts with that from gas phase experiments that concluded that the affinity of GSH for Cu I is similar to that of Dtt and that it can compete for Cu I with the Atox1 protein (16). The current uncertainties associated with ESI-MS detection are addressed under the "Discussion.…”

“…The absolute dissociation constant log K D ϭ Ϫ15.3 for the 1:1 complex was estimated via potentiometric titration of Cu I -Dtt samples prepared in situ via Dtt reduction of Cu 2ϩ (39). As Dtt has been used previously as both a competing ligand and an affinity standard for estimation of the Cu I affinities of proteins (16,40), its Cu I binding stoichiometry and affinity were re-investigated here to provide a unified scale.…”

“…Consequently, these two ligands in combination are able to buffer free Cu ϩ concentrations over the wide range from 10 Ϫ12 to 10 Ϫ19 M. They constitute a pair of complementary and versatile probes for Cu I . Dtt has been used as both a competing ligand and an affinity standard in work employing ESI-MS as an indirect detector of solution phase equilibria (16,40). Its Cu I binding stoichiometry and affinity were re-investigated here to provide a unified scale.…”

“…Yet reported K D values are scattered widely as highlighted by those of Atox1-type proteins, which differ by more than 10 orders of magnitude (K D ϳ10 Ϫ5 , 10 Ϫ10 , 10 Ϫ14 , and 10 Ϫ18 M) even though the structures and metal-binding sites of these proteins essentially superimpose (11)(12)(13)(14)(15)(16). The various values were determined via different experimental approaches with different ligand probes and affinity standards.…”

Loop recognition and copper-mediated disulfide reduction underpin metal site assembly of Cu A in human cytochrome oxidase

“…At the same time, these features make free copper ions toxic by outcompeting other metal ions in binding the target metalloproteins and as a source of reactive oxygen species (1). Living organisms thus resort to different strategies aimed to transport copper ions and regulate their levels in the cell, such as the use of metallochaperones, which bind copper ions and deliver them specifically to the target proteins (2)(3)(4)(5)(6).…”