Spectroscopic methods, density functional calculations, and ligand field analyses are combined to define the geometric models and electronic structure descriptions of the Cu(M) and Cu(H) sites in the oxidized form of the noncoupled binuclear copper protein peptidylglycine alpha-hydroxylating monooxygenase (PHM). The Cu(M) site has a square pyramidal geometry with a long axial Cu-methionine bond and two histidines, H(2)O, and OH(-) as equatorial ligands. The Cu(H) site has a slightly D(2)(d) distorted square planar geometry with three histidines and H(2)O ligands. The structurally inequivalent Cu(M) and Cu(H) sites do not exhibit measurable differences in optical and electron paramagnetic resonance (EPR) spectra, which result from their similar ligand field transition energies and ground-state Cu covalencies. The additional axial methionine ligand interaction and associated square pyramidal distortion of the Cu(M) site have the opposite effect of the strong equatorial OH(-) donor ligand on the Cu d orbital splitting pattern relative to the Cu(H) site leading to similar ligand field transition energies for both sites. The small molecule NO(2)(-) binds in different coordination modes to the Cu(M) and Cu(H) site because of differences in their exchangeable coordination positions resulting in these Cu(II) sites being spectroscopically distinguishable. Azide binding to PHM is used as a spectroscopic and electronic structure analogue to OOH(-) binding to provide a starting point for developing a geometric and electronic structural model for the putative Cu(II)(M)-OOH intermediate in the H-atom abstraction reaction of PHM. Possible electronic structure contributions of the Cu(II)(M)-OOH intermediate to reactivity are considered by correlation to the well-studied L3Cu(II)-OOH model complex (L3 = [HB[3-tBu-5-iPrpz](3)]). The Met-S ligand of the Cu(M) site is found to contribute to the stabilization of the Cu(II)(M)-oxyl species, which would be a product of Cu(II)(M)-OOH H-atom abstraction reaction. This Met-S contribution could have a significant effect on the energetics of a H-atom abstraction reaction by the Cu(II)(M)-OOH intermediate.

Bioactive peptides frequently terminate with an essential alpha-amide that is generated from a COOH-terminal Gly in a two-step enzymatic process occurring within the lumen of the secretory pathway. The first enzyme, peptidylglycine alpha-hydroxylating monooxygenase, is a member of the copper- and ascorbate-dependent monooxygenase family. The second enzyme, peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL, EC 4.3.2.5), has no known homologues. Examination of the catalytic core of PAL (PALcc) using trypsin, BNPS skatole, and COOH-terminally truncated proteins failed to identify stable subdomains. Treatment of PALcc with divalent metal ion chelators inactivated the enzyme and increased its protease and thermal sensitivity, suggesting a structural role for bound metal. Purified PALcc contained 0.7 +/- 0.4 mol of zinc/mol of enzyme. Since the four Cys residues in PALcc form two disulfide bonds, potential Zn ligands include conserved Asp, Glu, and His residues. The secretion and activity of PALcc bearing mutations in each conserved Asp, Glu, and His residue were evaluated. Mutation of three conserved Asp residues and two conserved His residues yielded a protein that could not be secreted, suggesting that these residues play a structural role. Analysis of mutants that were efficiently secreted identified three His residues along with single Asp residue that may play a role in catalysis. These essential residues occur in a pattern unique to PAL.

The biosynthesis of the majority of biologically active peptides ends with an obligatory alpha-amidation step that is catalyzed only by peptidylglycine alpha-hydroxylating monooxygenase (PHM). The utility of two mechanisms proposed for this copper- and ascorbate-dependent monooxygenase was examined using site-directed mutagenesis and intrinsic tryptophan fluorescence. Retention of full activity by PHMccGln(170)Ala and -Asn eliminates a critical role for Gln(170) in a substrate-mediated electron transfer pathway. The 20-fold reduction in V(max) observed for PHMccGln(170)Glu and -Leu is consistent with a key role for conformational changes in this region. Mutation of Tyr(79), situated near Cu(A), to Trp reduced V(max) 200-fold. Measurement of changes in intrinsic fluorescence allowed determination of a K(d) for copper (0.06 microM) and for a peptidylglycine substrate, Phe-Gly-Phe-Gly (0.8 microM). Although the peptidylglycine substrate bound more tightly at pH 7.0 than at pH 5.5, V(max) decreased 25-fold at neutral pH. Total quenching of the signal from Trp(79) in apoPHMccTyr(79)Trp along with its greatly reduced V(max) defines a critical role for Cu(A) in the rate-limiting step of the reaction. Taking into account our data and the results of kinetic, spectroscopic, and crystallographic studies, we propose a mechanism in which substrate-mediated activation of molecular oxygen binding at Cu(A) completes a pathway for electron transfer from Cu(B).

Bifunctional peptidylglycine alpha-amidating enzyme (alpha-AE) catalyzes the two-step conversion of C-terminal glycine-extended peptides to C-terminal alpha-amidated peptides and glyoxylate. The first step is the ascorbate-, O2-, and copper-dependent hydroxylation of the alpha-carbon of the glycyl residue, producing an alpha-hydroxyglycine-extended peptide. The second step is the ascorbate-, O2-, and copper-independent dealkylation of the carbinolamide intermediate. We show that alpha-AE requires 1.1 +/- 0. 2 mol of zinc/mol of enzyme for maximal (S)-N-dansyl-Tyr-Val-alpha-hydroxyglycine dealkylation activity. Treatment of the enzyme with EDTA abolishes both the peptide hydroxylation and the carbinolamide dealkylation activities. Addition of Zn(II), Co(II), Cd(II), and Mn(II) partially restores carbinolamide dealkylation activity to the EDTA-treated enzyme. Addition of Co(II) produces the greatest restoration of dealkylation activity, 32% relative to a control not treated with EDTA, while Mn(II) addition results in the smallest restoration of dealkylation activity, only 3% relative to an untreated control. The structure and coordination of the zinc center has been investigated by X-ray absorption spectroscopy. EXAFS data are best interpreted by an average coordination of 2-3 histidine ligands and 1-2 non-histidine O/N ligands. Since catalytic zinc centers in other zinc metalloenzymes generally exhibit only O/N ligands to the zinc atom, a zinc-bound water or hydroxide may serve as a general base for the abstraction of the hydroxyl proton from the carbinolamide intermediate. Alternatively, the zinc may function in a structural role.