A copper-methionine interaction controls the pH-dependent activation of peptidylglycine monooxygenase

Abstract

The pH dependence of native peptidylglycine monooxygenase (PHM) and its M314H variant has been studied in detail. For wild-type (WT) PHM, the intensity of the Cu-S interaction visible in the Cu(I) extended X-ray absorption fine structure (EXAFS) data is inversely proportional to catalytic activity over the pH range of 3-8. A previous model based on more limited data was interpreted in terms of two protein conformations involving an inactive Met-on form and an active flexible Met-off form [Bauman, A. T., et al. (2006) Biochemistry 45, 11140-11150] that derived its catalytic activity from the ability to couple into vibrational modes critical for proton tunneling. The new studies comparing the WT and M314H variant have led to the evolution of this model, in which the Met-on form has been found to be derived from coordination of an additional Met residue, rather than a more rigid conformer of M314 as previously proposed. The catalytic activity of the mutant decreased by 96% because of effects on both k(cat) and K(M), but it displayed the same activity-pH profile with a maximum around pH 6. At pH 8, the reduced Cu(I) form gave spectra that could be simulated by replacement of the Cu(M) Cu-S(Met) interaction with a Cu-N/O interaction, but the data did not unambiguously assign the ligand to the imidazole side chain of H314. At pH 3.5, the EXAFS still showed the presence of a strong Cu-S interaction, establishing that the Met-on form observed at low pH in WT cannot be due to a strengthening of the Cu(M)-methionine interaction but must arise from a different Cu-S interaction. Therefore, lowering the pH causes a conformational change at one of the Cu centers that brings a new S donor residue into a favorable orientation for coordination to copper and generates an inactive form. Cys coordination is unlikely because all Cys residues in PHM are engaged in disulfide cross-links. Sequence comparison with the PHM homologues tyramine β-monooxygenase and dopamine β-monooxygenase suggests that M109 (adjacent to H site ligands H107 and H108) is the most likely candidate. A model is presented in which H108 is protonated with a pK(a) of 4.6 to generate the inactive low-pH form with Cu(H) coordinated by M109, H107, and H172.

Figure 1

Top: view of the active site of PHM showing the Cu_H (right) coordinated to three histidines and the Cu_M center (left) centers coordinated to two histidines and a methionine. A substrate molecule (di-iodo-YG) is bound in the site close to the M center. (Taken from pdb file 1OPM). Bottom: reactions catalyzed by the PHM and PAL domains of peptidylglycine α-amidating lyase (PAM).

Figure 2

Fourier transforms of x-ray absorption data for WT PHMcc between pH 3 and pH 8. Spectra correspond to the pHs (from the bottom) 3.5, 4.0, 4.5, 5.0, 5.5, and 7.0. The inset shows the change in the shell occupancy of the Cu-S interaction (simulated using parameters listed in Table S1 of the supporting information) plotted as a function of pH, and simulated using a pK_A=4.69.

Figure 3

Normalized activity versus pH profiles for (a) the WT and (b) M314H variant of PHM. Red squares are experimental data, black lines are simulated data. Activity data were measured using an O₂ electrode under saturating conditions of dansyl-YVG, ascorbate and atmospheric oxygen. Activity data were normalized for ease of comparison. The data were analyzed as described previously in reference (24), in terms of an inactive species S1 present at low pH which is transformed into the active state S2 (or active states S2 and S3 in the M314H variant) by deprotonantion steps with pK_A values as shown.

Figure 4

Top: pH dependence of the 1s → 4p transition on the absorption edge of WT PHMcc. Bottom: the fractional change in 8983 eV intensity (red squares) fitted to a pH titration curve with pK_A = 4.8.

Figure 5

Experimental and simulated Fourier transforms and EXAFS (insets) for the reduced forms of the M314H variants (a) at pH 7 and (b) at pH 3.5. Parameters used in the fits are listed in Table 1.

Figure 6

Comparison of the 1s → 4p absorption edge transition for WT and M314H PHMcc at pH 7 and 3.5.

Figure 7

Structural depiction of two different conformations of the H center in PHMcc showing differences in the orientation of H107, H108 and M109 residues. The H172 ligand has been omitted for clarity. (a) Conformation found in WT PHMcc (pdb 1OPM) with each His ligand coordinated by imidazole-Nδ; (b) conformation observed in the M314I variant (pdb 1YI9) with H107 dissociated.