Pam heterozygous mice reveal essential role for Cu in amygdalar behavioral and synaptic function.

Copper (Cu) is an essential element with many biological roles, but its roles in the mammalian nervous system are poorly understood. Mice deficient in the cuproenzyme peptidylglycine α-amidating monooxygenase (Pam(+/-) mice) were initially generated to study neuropeptide amidation. Pam(+/-) mice exhibit profound deficits in a few behavioral tasks, including enhancements in innate fear along with deficits in acquired fear. Interestingly, several Pam(+/-) phenotypes were recapitulated in Cu-restricted wild-type mice and rescued in Cu-supplemented Pam(+/-) mice. These behaviors correspond to enhanced excitability and deficient synaptic plasticity in the amygdala of Pam(+/-) mice, which are also rescued by Cu supplementation. Cu and ATP7A are present at synapses, in key positions to respond to and influence synaptic activity. Further study demonstrated that extracellular Cu is necessary for wild-type synaptic plasticity and sufficient to induce long-term potentiation. These experiments support roles for PAM in Cu homeostasis and for synaptic Cu in amygdalar function.

Differential reactivity between two copper sites in peptidylglycine α-hydroxylating monooxygenase.

Peptidylglycine α-hydroxylating monooxygenase (PHM) catalyzes the stereospecific hydroxylation of the Cα of C-terminal glycine-extended peptides and proteins, the first step in the activation of many peptide hormones, growth factors, and neurotransmitters. The crystal structure of the enzyme revealed two nonequivalent Cu sites (Cu(M) and Cu(H)) separated by ∼11 Å. In the resting state of the enzyme, Cu(M) is coordinated in a distorted tetrahedral geometry by one methionine, two histidines, and a water molecule. The coordination site of the water molecule is the position where external ligands bind. The Cu(H) has a planar T-shaped geometry with three histidines residues and a vacant position that could potentially be occupied by a fourth ligand. Although the catalytic mechanism of PHM and the role of the metals are still being debated, Cu(M) is identified as the metal involved in catalysis, while Cu(H) is associated with electron transfer. To further probe the role of the metals, we studied how small molecules such as nitrite (NO(2)(-)), azide (N(3)(-)), and carbon monoxide (CO) interact with the PHM copper ions. The crystal structure of an oxidized nitrite-soaked PHMcc, obtained by soaking for 20 h in mother liquor supplemented with 300 mM NaNO(2), shows that nitrite anion coordinates Cu(M) in an asymmetric bidentate fashion. Surprisingly, nitrite does not bind Cu(H), despite the high concentration used in the experiments (nitrite/protein > 1000). Similarly, azide and carbon monoxide coordinate Cu(M) but not Cu(H) in the PHMcc crystal structures obtained by cocrystallization with 40 mM NaN(3) and by soaking CO under 3 atm of pressure for 30 min. This lack of reactivity at the Cu(H) is also observed in the reduced form of the enzyme: CO binds Cu(M) but not Cu(H) in the structure of PHMcc obtained by exposure of a crystal to 3 atm CO for 15 min in the presence of 5 mM ascorbic acid (reductant). The necessity of Cu(H) to maintain its redox potential in a narrow range compatible with its role as an electron-transfer site seems to explain the lack of coordination of small molecules to Cu(H); coordination of any external ligand will certainly modify its redox potential.

Interactions of peptide amidation and copper: novel biomarkers and mechanisms of neural dysfunction.

Mammalian genomes encode only a small number of cuproenzymes. The many genes involved in coordinating copper uptake, distribution, storage and efflux make gene/nutrient interactions especially important for these cuproenzymes. Copper deficiency and copper excess both disrupt neural function. Using mice heterozygous for peptidylglycine alpha-amidating monooxygenase (PAM), a cuproenzyme essential for the synthesis of many neuropeptides, we identified alterations in anxiety-like behavior, thermoregulation and seizure sensitivity. Dietary copper supplementation reversed a subset of these deficits. Wildtype mice maintained on a marginally copper-deficient diet exhibited some of the same deficits observed in PAM(+/-) mice and displayed alterations in PAM metabolism. Altered copper homeostasis in PAM(+/-) mice suggested a role for PAM in the cell type specific regulation of copper metabolism. Physiological functions sensitive to genetic limitations of PAM that are reversed by supplemental copper and mimicked by copper deficiency may serve as indicators of marginal copper deficiency.