Many bioactive peptides require amidation of their carboxy terminus to exhibit full biological activity. Peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3), the enzyme that catalyzes the first of the two steps of this reaction, is composed of two domains, each of which binds one copper atom (CuH and CuM). The CuM site includes Met(314) and two His residues as ligands. Mutation of Met(314) to Ile inactivates PHM, but has only a minimal effect on the EXAFS spectrum of the oxidized enzyme, implying that it contributes only marginally to stabilization of the CuM site. To characterize the role of Met(314) as a CuM ligand, we determined the structure of the Met(314)Ile-PHM mutant. Since the mutant protein failed to crystallize in the conditions of the original wild-type protein, this structure determination required finding a new crystal form. The Met(314)Ile-PHM mutant structure confirms that the mutation does not abolish CuM binding to the enzyme, but causes other structural perturbations that affect the overall stability of the enzyme and the integrity of the CuH site. To eliminate possible effects of crystal contacts, we redetermined the structure of wt-PHM in the Met(314)Ile-PHM crystal form and showed that it does not differ from the structure of wild-type (wt)-PHM in the original crystals. Met(314)Ile-PHM was also shown to be less stable than wt-PHM by differential scanning calorimetry. Both structural and calorimetric studies point to a structural role for the CuM site, in addition to its established catalytic role.

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 non-equivalent Cu sites (CuM and CuH) separated by ~ 11 Å. In the resting state of the enzyme, CuM 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 CuH has a planar T-shaped geometry with three histidines residues and a vacant position that could be potentially occupied by a fourth ligand. Although the catalytic mechanism of PHM and the role of the metals are still being debated, CuM is identified as the metal involved in catalysis while CuH 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 20 hours soaking in mother liquor supplemented with 300 mM NaNO2, shows that nitrite anion coordinates CuM in an asymmetric bidentate fashion. Surprisingly, nitrite does not bind CuH despite the high concentration used in the experiments (nitrite/protein > 1000). Similarly, azide and carbon monoxide coordinate CuM but not CuH in the PHMcc crystal structures obtained by co-crystallization with 40 mM NaN3 and by soaking CO under 3 atm of pressure for 30 minutes. This lack of reactivity at the CuH is also observed in the reduced form of the enzyme: CO binds CuM but not CuH in the structure of PHMcc obtained by exposure of a crystal to 3 atm CO for 15 minutes, in the presence of 5 mM ascorbic acid (reductant). The necessity of CuH to maintain its redox potential in a narrow range compatible to its role as an electron-transfer site seems to explain the lack of coordination of small molecules to CuH; coordination of any external ligand will certainly modify its redox potential.