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 (NO₂ ^-), azide (N₃^-), 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 NaNO₂, 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 NaN₃ 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.