Eduardo E. Chufán

Heme-Copper-Dioxygen Complexes: Toward Understanding Ligand-Environmental Effects on the Coordination Geometry, Electronic Structure, and Reactivity

The nature of the ligand is an important aspect of controlling the structure and reactivity in coordination chemistry. In connection with our study of heme-copper-oxygen reactivity relevant to cytochrome c oxidase dioxygen-reduction chemistry, we compare the molecular and electronic structures of two high-spin heme-peroxo-copper [Fe(III)O(2)(2-)Cu(II)](+) complexes containing N(4) tetradentate (1) or N(3) tridentate (2) copper ligands. Combining previously reported and new resonance Raman and EXAFS data coupled to density functional theory calculations, we report a geometric structure and more complete electronic description of the high-spin heme-peroxo-copper complexes 1 and 2, which establish mu-(O(2)(2-)) side-on to the Fe(III) and end-on to Cu(II) (mu-eta(2):eta(1)) binding for the complex 1 but side-on/side-on (mu-eta(2):eta(2)) mu-peroxo coordination for the complex 2. We also compare and summarize the differences and similarities of these two complexes in their reactivity toward CO, PPh(3), acid, and phenols. The comparison of a new X-ray structure of mu-oxo complex 2a with the previously reported 1a X-ray structure, two thermal decomposition products respectively of 2 and 1, reveals a considerable difference in the Fe-O-Cu angle between the two mu-oxo complexes ( angleFe-O-Cu = 178.2 degrees in 1a and angleFe-O-Cu = 149.5 degrees in 2a). The reaction of 2 with 1 equiv of an exogenous nitrogen-donor axial base leads to the formation of a distinctive low-temperature-stable, low-spin heme-dioxygen-copper complex (2b), but under the same conditions, the addition of an axial base to 1 leads to the dissociation of the heme-peroxo-copper assembly and the release of O(2). 2b reacts with phenols performing H-atom (e(-) + H(+)) abstraction resulting in O-O bond cleavage and the formation of high-valent ferryl [Fe(IV)=O] complex (2c). The nature of 2c was confirmed by a comparison of its spectroscopic features and reactivity with those of an independently prepared ferryl complex. The phenoxyl radical generated by the H-atom abstraction was either (1) directly detected by electron paramagnetic resonance spectroscopy using phenols that produce stable radicals or (2) indirectly detected by the coupling product of two phenoxyl radicals.

Amidation of Bioactive Peptides: The Structure of the Lyase Domain of the Amidating Enzyme

Many neuropeptides and peptide hormones require amidation of their carboxy terminal for full biological activity. The enzyme peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL; EC 4.3.2.5) catalyzes the second and last step of this reaction, N-dealkylation of the peptidyl-alpha-hydroxyglycine to generate the alpha-amidated peptide and glyoxylate. Here we report the X-ray crystal structure of the PAL catalytic core (PALcc) alone and in complex with the nonpeptidic substrate alpha-hydroxyhippuric acid. The structures show that PAL folds as a six-bladed beta-propeller. The active site is formed by a Zn(II) ion coordinated by three histidine residues; the substrate binds to this site with its alpha-hydroxyl group coordinated to the Zn(II) ion. The structures also reveal a tyrosine residue (Tyr(654)) at the active site as the catalytic base for hydroxyl deprotonation, an unusual role for tyrosine. A reaction mechanism is proposed based on this structural data and validated by biochemical analysis of site-directed PALcc mutants.

Differential reactivity between two copper sites in Peptidylglycine α-hydroxylating monooxygenase

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 nonequivalent Cu sites (Cu(M) and Cu(H)) separated by ∼11 Å. In the resting state of the enzyme, Cu(M) 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 Cu(H) has a planar T-shaped geometry with three histidines residues and a vacant position that could potentially be occupied by a fourth ligand. Although the catalytic mechanism of PHM and the role of the metals are still being debated, Cu(M) is identified as the metal involved in catalysis, while Cu(H) is associated with electron transfer. To further probe the role of the metals, we studied how small molecules such as nitrite (NO(2)(-)), azide (N(3)(-)), and carbon monoxide (CO) interact with the PHM copper ions. The crystal structure of an oxidized nitrite-soaked PHMcc, obtained by soaking for 20 h in mother liquor supplemented with 300 mM NaNO(2), shows that nitrite anion coordinates Cu(M) in an asymmetric bidentate fashion. Surprisingly, nitrite does not bind Cu(H), despite the high concentration used in the experiments (nitrite/protein > 1000). Similarly, azide and carbon monoxide coordinate Cu(M) but not Cu(H) in the PHMcc crystal structures obtained by cocrystallization with 40 mM NaN(3) and by soaking CO under 3 atm of pressure for 30 min. This lack of reactivity at the Cu(H) is also observed in the reduced form of the enzyme: CO binds Cu(M) but not Cu(H) in the structure of PHMcc obtained by exposure of a crystal to 3 atm CO for 15 min in the presence of 5 mM ascorbic acid (reductant). The necessity of Cu(H) to maintain its redox potential in a narrow range compatible with its role as an electron-transfer site seems to explain the lack of coordination of small molecules to Cu(H); coordination of any external ligand will certainly modify its redox potential.

Peptidylglycine α-Hydroxylating Monooxygenase (PHM)

Numerous peptides function as hormones, neurotransmitters, and growth factors. Enzymatic α-amidation is a biologically important posttranslational modification of the C-terminus of many of these peptides. This modification alters the biological properties and enhances the stability of the peptides toward digestion by carboxypeptidases. Peptidylglycine α-hydroxylating monooxygenase (PHM) is an ascorbate and copper-dependent catalytic domain of an α-amidating enzyme (peptidylglycine α-amidating monooxygenase, PAM) that catalyzes the stereospecific hydroxylation of an α-carbon of a terminal glycine residue, the first step in the amidation reaction. This reaction is followed by cleavage of the glycine N Cα bond, which is carried out by the second PAM catalytic domain, peptidyl-α-hydroxyglycine α-amidating lyase (PAL). Detailed structural studies of PHM revealed that its catalytic core binds two copper ions that support the oxygenation reaction by cycling through Cu(II)/Cu(I) oxidation states. These two Cu ions (CuH and CuM) are located 11 A apart and are separated by a solvent-accessible cleft. The monooxygenation reaction requires the two-electron activation of molecular oxygen, which is achieved by the binding of O2 to a single Cu(I) center (CuM). Formation of this complex is dependent upon the presence of a peptidylglycine substrate and a reducing agent (ascorbate). Since the resting state of the enzyme contains two Cu(II) ions, the catalytic reaction requires transfer of two electrons from the reducing agent to the metal centers, and from the reduced copper ions to dioxygen. The reduced oxygen species then carries out the stereospecific hydroxylation of glycine after abstraction of the pro-S hydrogen from Cα. Although, the structure and function of PHM have been broadly studied, the pathway of its electron transfer, the nature of the metal-oxygen species, and details of the mechanism are still being investigated. 3D Structure Keywords: peptidylglycine α-hydroxylating monooxygenase (PHM); peptidylglycine α-amidating monooxygenase (PAM); amidation of peptides; copper proteins