Mechanism Of Copper Transfer Research Articles

Biochemical pathway for the biosynthesis of the CuA center in bacterial cytochrome c oxidase.

The di-copper center CuA is an essential metal cofactor in cytochrome oxidase (Cox) of mitochondria and many prokaryotes, mediating one-electron transfer from cytochrome c to the site for oxygen reduction. CuA is located in subunit II (CoxB) of Cox and protrudes into the periplasm of Gram-negative bacteria or the mitochondrial intermembrane space. How the two copper ions are brought together to build CoxB·CuA is the subject of this review. It had been known that the reductase TlpA and the metallochaperones ScoI and PcuC are required for CuA formation in bacteria, but the mechanism of copper transfer has emerged only recently for the Bradyrhizobiumdiazoefficiens system. It consists of the following steps: (a) TlpA keeps the active site cysteine pair of CoxB in its dithiol state as a prerequisite for metal insertion; (b) ScoI·Cu2+ rapidly forms a transient complex with apo-CoxB; (c) PcuC, loaded with Cu1+ and Cu2+ , dissociates this complex to CoxB·Cu2+ , and a second PcuC·Cu1+ ·Cu2+ transfers Cu1+ to CoxB·Cu2+ , yielding mature CoxB·CuA . Variants of this pathway might exist in other bacteria or mitochondria.

Trapping intermediates in metal transfer reactions of the CusCBAF export pump of Escherichia coli

Escherichia coli CusCBAF represents an important class of bacterial efflux pump exhibiting selectivity towards Cu(I) and Ag(I). The complex is comprised of three proteins: the CusA transmembrane pump, the CusB soluble adaptor protein, and the CusC outer-membrane pore, and additionally requires the periplasmic metallochaperone CusF. Here we used spectroscopic and kinetic tools to probe the mechanism of copper transfer between CusF and CusB using selenomethionine labeling of the metal-binding Met residues coupled to RFQ-XAS at the Se and Cu edges. The results indicate fast formation of a protein−protein complex followed by slower intra-complex metal transfer. An intermediate coordinated by ligands from each protein forms in 100 ms. Stopped-flow fluorescence of the capping CusF-W44 tryptophan that is quenched by metal transfer also supports this mechanism. The rate constants validate a process in which shared-ligand complex formation assists protein association, providing a driving force that raises the rate into the diffusion-limited regime.

Ctr1 Intracellular Loop Is Involved in the Copper Transfer Mechanism to the Atox1 Metallochaperone

Understanding the human copper cycle is essential to understand the role of metals in promoting neurological diseases and disorders. One of the cycles controlling the cellular concentration and distribution of copper involves the copper transporter, Ctr1; the metallochaperone, Atox1; and the ATP7B transporter. It has been shown that the C-terminus of Ctr1, specifically the last three amino acids, HCH, is involved in both copper coordination and the transfer mechanism to Atox1. In contrast, the role of the intracellular loop of Ctr1, which is an additional intracellular segment of Ctr1, in facilitating the copper transfer mechanism has not been investigated yet. Here, we combine various biophysical methods to explore the interaction between this Ctr1 segment and metallochaperone Atox1 and clearly demonstrate that the Ctr1 intracellular loop (1) can coordinate Cu(I) via interactions with the side chains of one histidine and two methionine residues and (2) closely interacts with the Atox1 metallochaperone. Our findings are another important step in elucidating the mechanistic details of the eukaryotic copper cycle.

EPR Spectroscopy Shows that the Blood Carrier Protein, Human Serum Albumin, Closely Interacts with the N-Terminal Domain of the Copper Transporter, Ctr1

Copper is an essential metal whose localization within the cells must be carefully controlled to avoid copper dependent redox cycling. Although most of the key proteins involved in cellular copper transfer have been identified, fundamental questions regarding the copper transfer mechanism have yet to be resolved. One of the blood carrier proteins believed to be involved in copper transfer to the cell is human serum albumin (HSA). However, direct evidence for close interaction between HSA and the extracellular domain of the copper transporter Ctr1 has not yet been found. By utilizing EPR spectroscopy, we show here that HSA closely interacts with the first 14 amino acids of the Ctr1, even without the presence of copper ions.

Copper-Transfer Mechanism from the Human Chaperone Atox1 to a Metal-Binding Domain of Wilson Disease Protein

The molecular details of how copper (Cu) is transferred from the human Cu chaperone Atox1 to metal-binding domains (MBDs) of P(1B)-type ATPases are still unclear. Here, we use a computational approach, employing quantum mechanics/molecular mechanics (QM/MM) methods, to shed light on the reaction mechanism [probable intermediates, Cu(I) coordination geometries, activation barriers, and energetics] of Cu(I) transfer from Atox1 to the fourth MBD of Wilson disease protein (WD4). Both Atox1 and WD4 have solvent-exposed metal-binding motifs with two Cys residues that coordinate Cu(I). After assessing the existence of all possible 2-, 3- and 4-coordinate Cu-intermediate species, one dominant reaction path emerged. First, without activation barrier, WD4's Cys1 binds Cu(I) in Atox1 to form a 3-coordinated intermediate. Next, with an activation barrier of about 9.5 kcal/mol, a second 3-coordinated intermediate forms that involves both of the Cys residues in WD4 and Cys1 of Atox1. This species can then form the product by decoordination of Atox1's Cys1 (barrier of about 8 kcal/mol). Overall, the Cu-transfer reaction from Atox1 to WD4 appears to be kinetically accessible but less energetically favorable (DeltaE = 7.7 kcal/mol). Our results provide unique insights into the molecular mechanism of protein-mediated Cu(I) transfer in the secretory pathway and are in agreement with existing experimental data.

A strategy for the NMR characterization of type II copper(II) proteins: the case of the copper trafficking protein CopC from Pseudomonas Syringae.

CopC from Pseudomonas syringae was found to be a protein capable of binding both Cu(I) and Cu(II) at two different sites. The solution structure of the apo protein is available, and structural information has been obtained on the Cu(I) bound form. We attempt here to set the limits for the determination of the solution structure of a Cu(II) protein, such as the Cu(II) bound form of CopC, in which the Cu(II) ion takes a type II coordination. The electron relaxation time is estimated from NMRD measurements to be 3 ns which leads to a correlation time for the nuclear spin-electron spin dipolar interaction of 2 ns. This information allowed us to tailor the NMR experiments and to fully exploit purely heteronuclear spectroscopy to assign as many signals as possible. In this way, 37 (13)C and 11 (15)N signals that completely escape detection with conventional approaches were assigned. Paramagnetic based structural constraints were obtained by measuring paramagnetic longitudinal relaxation enhancements (rho(para)) which allowed us to precisely locate the copper ion within the protein frame. Pseudocontact shifts (pcs's) were also used as constraints for 83 (1)H and 18 (13)C nuclei. With them, together with other standard structural constraints, a structure is obtained (and submitted to PDB) where information is only missing in a sphere with a 6 A radius from the copper ion. If we borrow information from EXAFS data, which show evidence of two copper coordinated histidines, then His 1 and His 91 are unambiguously identified as copper ligands. EXAFS data indicate two more light donor atoms (O/N) which could be from Asp 27 and Glu 89, whereas the NMRD data indicate the presence of a semicoordinated water molecule at 2.8 A (Cu-O distance) roughly orthogonal to the plane identified by the other four ligands. This represents the most extensively characterized structure of a type II Cu(II) protein obtained employing the most advanced NMR methods and with the aid of EXAFS data. The knowledge of the location of the Cu(II) in the protein is important for the copper transfer mechanism.

Copper delivery by metallochaperone proteins.

Copper is an essential element in all living organisms, serving as a cofactor for many important proteins and enzymes. Metallochaperone proteins deliver copper ions to specific physiological partners by direct protein-protein interactions. The Atx1-like chaperones transfer copper to intracellular copper transporters, and the CCS chaperones shuttle copper to copper,zinc superoxide dismutase. Crystallographic studies of these two copper chaperone families have provided insights into metal binding and target recognition by metallochaperones and have led to detailed molecular models for the copper transfer mechanism.

Kinetics and Mechanism of Copper Transfer by Hydroxy-Oximes in Three-Phase Liquid Systems

Abstract A model of copper-facilitated transport in a three-liquid-phase pertraction system is proposed. The model takes into account the diffusional transport of copper species (ions and complexes) in all three liquids as well as the kinetics of chemical reactions. The latter, according to the model suggested, occurs in narrow reaction zones: aqueous layers adjacent to the donor membrane and acceptor-membrane interfaces. Experiments were carried out in a two-compartment agitated diffusion cell with a bulk liquid membrane: a 5% (vol) solution of the commercial copper extractant LIX 860 in C11-C13 normal paraffins. The donor solution used contained 1 g/L copper and the acceptor liquid was a 4.5 N aqueous solution of sulfuric acid. On the basis of experimentally obtained pertraction curves and the model suggested, it was found that the overall rate of the process is controlled by the diffusion of copper ions in the donor phase as well as by copper-complex decomposition. By applying an optimization procedure...