Search

Peptidylglycine monooxygenase (PHM) catalyzes the final step in the biosynthesis of amidated peptides that serve as important signaling molecules in numerous endocrine pathways. The catalytic mechanism has attracted much attention because of a number of unique attributes, including the presence of a pair of uncoupled copper centers separated by 11 Å (termed CuH and CuM), an unusual Cu(I)SMet interaction at the oxygen binding M-site, and the postulated Cu(II)-superoxo intermediate. Understanding the mechanism requires determining the catalytic roles of the individual copper centers and how they change during catalysis, a task made more difficult by the overlapping spectral signals from each copper center in the wild-type (WT) protein. To aid in this effort, we constructed and characterized two PHM variants that bound metal at only one site. The H242A variant bound copper at the H-center, while the H107AH108A double mutant bound copper at the M-center; both mutants were devoid of catalytic activity. Oxidized Cu(II) forms showed electron paramagnetic resonance and extended X-ray absorption fine structure (EXAFS) spectra consistent with their previously determined Cu(II)His3O and Cu(II)His2O2 ligand sets for the H- and M-centers, respectively. Cu(I) forms, on the other hand, showed unique chemistry. The M-center bound two histidines and a methionine at all pHs, while the H-center was two-coordinate at neutral pH but coordinated a new methionine S ligand at low pH. Fourier transform infrared studies confirmed and extended previous assignments of CO binding and showed unambiguously that the 2092 cm(-1) absorbing species observed in the WT and many variant forms is an M-site Cu(I)-CO adduct. Silver binding was also investigated. When H107AH108A and M109I (a WT analogue with both sites intact) were incubated with excess AgNO3, each variant bound a single Ag(I) ion, from which it was inferred that Ag(I) binds selectively at the M-center with little or no affinity for the H-center. EXAFS at the Ag K-edge established a strong degree of similarity between the ligand sets of Cu and Ag bound at the M-center. These studies validate previous spectral assignments and provide new insights into the detailed chemistry of each metal site.

The present study uses CO as a surrogate for oxygen to probe how substrate binding triggers oxygen activation in peptidylglycine monooygenase (PHM). Infrared stretching frequencies (ν(C≡O)) of the carbonyl (CO) adducts of copper proteins are sensitive markers of Cu(I) coordination, and are useful in probing oxygen reactivity since the electronic properties of O2 and CO are similar. The carbonyl chemistry has been explored using PHM WT and a number of active site variants in the absence and presence of peptidyl substrates. We have determined that upon carbonylation (i) a major CO band at 2092 cm−1 and a second minor CO band at 2063 cm−1 are observed in the absence of peptide substrate Ac-YVG; (ii) the presence of peptide substrate amplifies the minor CO band and causes it to partially interconvert with the CO band at 2092 cm−1; (iii) the substrate induced CO band is associated with a second conformer at CuM; and (iv) the CuH-site mutants which are inactive, fail to generate any substrate-induced CO bands. The total intensity of both bands is constant suggesting that the Cu(I)M site partitions between the two carbonylated enzyme states. Together, these data provide evidence for two conformers at CuM, one of which is induced by binding of the peptide substrate, with the implication that this represents the conformation that also allows binding and activation of O2.

Cuproproteins such as PHM and DBM mature in late endosomal vesicles of the mammalian secretory pathway where changes in vesicle pH are employed for sorting and post-translational processing. Colocation with the P1B-type ATPase ATP7A suggests that the latter is the source of copper and supports a mechanism where selectivity in metal transfer is achieved by spatial colocation of partner proteins in their specific organelles or vesicles. In previous work we have suggested that a lumenal loop sequence located between trans-membrane helices TM1 and TM2 of the ATPase, and containing five histidines and four methionines, acts as an organelle-specific chaperone for metallation of the cuproproteins. The hypothesis posits that the pH of the vesicle regulates copper ligation and loop conformation via a mechanism which involves His to Met ligand switching induced by histidine protonation. Here we report the effect of pH on the HM loop copper coordination using X-ray absorption spectroscopy (XAS), and show via selenium substitution of the Met residues that the HM loop undergoes similar conformational switching to that found earlier for its partner PHM. We hypothesize that in the absence of specific chaperones, HM motifs provide a template for building a flexible, pH-sensitive transfer site whose structure and function can be regulated to accommodate the different active site structural elements and pH environments of its partner proteins.

Peptidylglycine monooxygenase is a copper-containing enzyme that catalyzes the amidation of neuropeptides hormones, the first step of which is the conversion of a glycine-extended pro-peptide to its α-hydroxyglcine intermediate. The enzyme contains two mononuclear Cu centers termed CuM (ligated to imidazole nitrogens of H242, H244 and the thioether S of M314) and CuH (ligated to imidazole nitrogens of H107, H108, and H172) with a Cu-Cu separation of 11 Å. During catalysis, the M site binds oxygen and substrate, and the H site donates the second electron required for hydroxylation. The WT enzyme shows maximum catalytic activity at pH 5.8 and undergoes loss of activity at lower pHs due to a protonation event with a pKA of 4.6. Low pH also causes a unique structural transition in which a new S ligand coordinates to copper with an identical pKA, manifest by a large increase in Cu-S intensity in the X- ray absorption spectroscopy. In previous work (Bauman, A. T., Broers, B. A., Kline, C. D., and Blackburn, N. J. (2011) Biochemistry 50, 10819-10828), we tentatively assigned the new Cu-S interaction to binding of M109 to the H-site (part of an HHM conserved motif common to all but one member of the family). Here we follow up on these findings via studies on the catalytic activity, pH-activity profiles, and spectroscopic (electron paramagnetic resonance, XAS, and Fourier transform infrared) properties of a number of H-site variants, including H107A, H108A, H172A, and M109I. Our results establish that M109 is indeed the coordinating ligand and confirm the prediction that the low pH structural transition with associated loss of activity is abrogated when the M109 thioether is absent. The histidine mutants show more complex behavior, but the almost complete lack of activity in all three variants coupled with only minor differences in their spectroscopic properties suggests that unique structural elements at H are critical for functionality. The data suggest a more general utility for the HHM motif as a copper- and pH-dependent conformational switch.

Peptidylglycine monooxygenase (PHM)1 catalyses the first step in the amidation of neuropeptides hormones, converting the glycine-extended propeptide to its α-hydroxyglcine intermediate (1). The catalytic core (residues 42 – 356) termed PHMcc is homologous to that of other copper monooxygenases involved in neurotransmitter biosynthesis such as dopamine β-monooxygenase (DBM) (2) and tyramine β-monooxygenase (TBM) (3). Spectroscopic studies have established structural similarities between their active sites (4-8), but PHMcc is the only member of the group for which crystal structures are available (9-12). Unlike the coupled binuclear centers common to hemocyanins, tyrosinases, catechol oxidases (13-16) and oxygen activating dicopper model complexes (17), the copper centers in PHM are mononuclear, and are separated by 11 A of solvent-filled channel. CuM is coordinated by two histidines and a methionine while CuH is coordinated to three histidine residues (Figure 1). Figure 1 Top: view of the active site of PHM showing the CuH (right) coordinated to three histidines and the CuM 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. ... The detailed mechanism of substrate hydroxylation has been the subject of much debate. It is generally accepted that the enzyme cycles through a reductive phase in which the two copper centers are reduced to Cu(I), and an oxidative phase in which O2 is activated and subsequently hydroxylates the substrate. A structure of the reduced enzyme co-crystalized with a slow substrate has revealed the presence of a dioxygen molecule bound at the CuM center, the bond length of which is consistent with a Cu(II)-superoxo species. A superoxo intermediate is supported by other biochemical (18, 19) and theoretical (15, 20) studies that implicate Cu(II)-O•−2 as the reactive oxygen species. It has been further suggested that the large spatial separation of the Cu centers in PHM prevents formation of the peroxide, and thus allows the potent electrophilic reactivity of the mononuclear Cu(II)-superoxo species to be fully expressed in the form of H-atom abstraction from the substrate (15, 18, 20) to form a mononuclear hydroperoxo species at CuM. Subsequent steps in the reaction pathway are less clear and alternate mechanisms have been proposed that involve long-range electron transfer from CuH either before (18) or after (20) the transfer of an OH group to the substrate radical to form product (17). Early site-directed mutagenic studies on PHMcc M314X substitutions reported undetectable catalytic activity in the Ile, His, Asp and Cys variants (21, 22), while more recent mutagenic analysis of the homologous Met ligand in TBM (M471) showed similarly low or undetectable activity for His, Asp, and Cys variants(4). These studies established an essential role for M314 in PHM catalysis. Extensive characterization of the Cu-S bond by EXAFS (5-7, 23, 24), led to the finding that the interaction was unusually weak at pHs at or above the pH optimum (5.8), but became much more intense at lower pH. This led to the suggestion that the dynamics of the Cu-S(Met) interaction were controlling catalytic activity via a “Met-on” (inactive) to “Met-off ”(active) structural transition. (24). In the present paper we explore further the relationship between catalytic activity and the Cu(I)-methionine interactions as a function of pH. Using a complex buffer system containing formate, MES, HEPES and CHES, we have determined the complete pH-activity profile between pH 3 and 8, and correlated this with changes in the strength or shell occupancy of the Cu-S interaction in the EXAFS. These results suggest that the increase in Cu-S interaction is due to coordination of an additional S ligand at low pH mediated by conformational change at one of the Cu centers, which brings a new S-donor residue into a favorable orientation for coordination to copper, thereby generating the inactive form. Cys coordination is unlikely since all Cys residues in PHM are engaged in disulfide crosslinks. Sequence comparison with the PHM homologues TBM and DBM suggest that M109 (adjacent to the H-site ligands H107 and H108) is the most likely candidate. A model is presented in which H108 protonates with a pKA of 4.6 to generate the inactive low-pH form with CuH coordinated by M109, H107 and H172.