Peptidylglycine a-hydroxylating monooxygenase (PHM) catalyzes the stereospecific hydroxylation of the C alpha 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 similar to 11 angstrom. 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 (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 NaNO2, shows that nitrite anion coordinates Cum 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.