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

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.