The dioxygen reactivity of as eries of TMPA-based copper(I) complexes (TMPA = tris(2-pyridylmethyl)amine), with and without secondary-coordination-sphere hydrogenbonding moieties,w as studied at À135 8 8Ci n2 -methyltetrahydrofuran (MeTHF). Kinetic stabilization of the H-bonded [( ðX 1 ÞðX 2 Þ TMPA)Cu II (O 2 C À )] + cupric superoxide species was achieved, and they were characterized by resonance Raman (rR) spectroscopy. The structures and physical properties of [( ðX 1 ÞðX 2 Þ TMPA)Cu II (N 3 À )] + azido analogues were compared, and the O 2 C À reactivity of ligand-Cu I complexes when an Hbonding moiety is replaced by amethyl group was contrasted. Ad rastic enhancement in the reactivity of the cupric superoxide towards phenolic substrates as well as oxidation of substrates possessing moderate C À Hb ond-dissociation energies is observed, correlating with the number and strength of the H-bonding groups.Oxidation and oxygenation reactions are vital for biological and synthetic processes. [1][2][3][4][5][6][7][8][9] In biology,s ome copper-containing metalloenzymes are capable of performing these reactions. [2,10] Forexample,galactose oxidase (GO) is responsible for the oxidation of primary alcohols to aldehydes (Figure 1A); monooxygenases such as peptidylglycine a-hydroxylating monooxygenase (PHM), dopamine b-monooxygenase (DbM), and lytic polysaccharide monooxygenases (LMPOs) catalytically hydroxylate organic substrates containing strong (85-100 kcal mol À1 )C ÀHb ond dissociation energies (BDEs) using dioxygen ( Figure 1B). [2,[10][11][12] These reactions are important for biosynthesis of human prohormones and neurotransmitters in the former,a nd breaking down polysaccharides in the latter. Acupric superoxide species (that is,Cu II À O 2 C À ,formed from the reaction of copper(I) and dioxygen) is postulated to be involved in the catalytic cycles of each of these enzymes. [11] In GO,t his complex abstracts ah ydrogen atom from anearby Tyrresidue,thereby forming the catalytically active intermediate responsible for substrate oxidation.While the exact nature of the reactive intermediate in LPMOs is still widely debated, [12][13][14] ac onsensus in the literature for PHM and DbMi sthat acupric superoxide is responsible for the initial hydrogen-atom transfer (HAT) from ac arbon substrate. [11] Thus,t he study of synthetic cupric superoxide model complexes to further understand their structures, physical-spectroscopic properties,a nd correlated reactivity toward the hydroxylation of substrates containing strong CÀ Hb onds is of considerable interest in catalysis.Ty pically,t hese primary copper-dioxygen superoxide model species [11,15,16] are difficult to study due to their tendency to form secondary Cu 2 -O 2 adducts (that is, m-1,2peroxo-dicopper(II), side-on peroxodicopper(II), or bis-moxodicopper(III) complexes) in solution. Researchers have been able to prevent the formation of 2:1C u:O 2 adducts through ligand design;the addition of as econdary coordination sphere of sterically bulky groups [17][18][19][20][21...

Melanins are highly conjugated biopolymer pigments that provide photoprotection in a wide array of organisms, from bacteria to humans. The rate-limiting step in melanin biosynthesis, which is the ortho -hydroxylation of the amino acid L-tyrosine to L-DOPA, is catalyzed by the ubiquitous enzyme tyrosinase (Ty). Ty contains a coupled binuclear copper active site that binds O 2 to form a μ:η 2 :η 2 -peroxide dicopper(II) intermediate (oxy-Ty), capable of performing the regioselective monooxygenation of para -substituted monophenols to catechols. The mechanism of this critical monooxygenation reaction remains poorly understood despite extensive efforts. In this study, we have employed a combination of spectroscopic, kinetic, and computational methods to trap and characterize the elusive catalytic ternary intermediate (Ty/O 2 /monophenol) under single-turnover conditions and obtain molecular-level mechanistic insights into its monooxygenation reactivity. Our experimental results, coupled with quantum-mechanics/molecular-mechanics calculations, reveal that the monophenol substrate docks in the active-site pocket of oxy-Ty fully protonated, without coordination to a copper or cleavage of the μ:η 2 :η 2 -peroxide O-O bond. Formation of this ternary intermediate involves the displacement of active-site water molecules by the substrate and replacement of their H bonds to the μ:η 2 :η 2 -peroxide by a single H bond from the substrate hydroxyl group. This H-bonding interaction in the ternary intermediate enables the unprecedented monooxygenation mechanism, where the μ-η 2 :η 2 -peroxide O-O bond is cleaved to accept the phenolic proton, followed by substrate phenolate coordination to a copper site concomitant with its aromatic ortho -hydroxylation by the nonprotonated μ-oxo. This study provides insights into O 2 activation and reactivity by coupled binuclear copper active sites with fundamental implications in biocatalysis.

The factors that control the diverse reactivity of the μ-η2:η2-peroxide dicopper(II) oxy-intermediates in the coupled binuclear copper proteins remain elusive. Here, spectroscopic and computational methods reveal H-bonding interactions between active-site...

Tyrosinase is the most predominant member of the coupled binuclear copper (CBC) protein family. The recent trapping and spectroscopic definition of the elusive catalytic ternary intermediate (enzyme/O2/monophenol) of tyrosinase dictates a monooxygenation mechanism that revises previous proposals and involves cleavage of the μ‐η2:η2‐peroxide dicopper(II) O–O bond to accept the phenolic proton, followed by monophenolate coordination to copper concomitant with aromatic hydroxylation by the non‐protonated μ‐oxo. Here, we compare and contrast previously proposed and current mechanistic models for monophenol monooxygenation of tyrosinase. Next, we discuss how these recent insights provide new opportunities towards uncovering structure–function relationships in CBC enzymes, as well as understanding fundamental principles for O2 activation and reactivity by bioinorganic active sites.

We previously reported that a Pt(IV) complex, [Pt(IV)(dach)Cl4] [trans-d,l-1,2-diaminocyclohexanetetrachloroplatinum(IV)] binds to the N7 of 5'-dGMP (deoxyguanosine-5'-monophosphate) at a relatively fast rate and oxidizes it to 8-oxo-5'-dGMP. Here, we further studied the kinetics of the oxidation of 5'-dGMP by the Pt(IV) complex. The electron transfer rate constants between 5'-dGMP and Pt(IV) in [H8-5'-dGMP-Pt(IV)] and [D8-5'-dGMP-Pt(IV)] were similar, giving a small value of the kinetic isotope effect (KIE: 1.2 ± 0.2). This small KIE indicates that the deprotonation of H8 in [H8-5'-dGMP-Pt(IV)] is not involved in the rate-determining step in the electron transfer between guanine (G) and Pt(IV). We also studied the reaction of 5'-dGDP (deoxyguanosine-5'-diphosphate) and 5'-dGTP (deoxyguanosine-5'-triphosphate) with the Pt(IV) complex. Our results showed that [Pt(IV)(dach)Cl4] oxidized 5'-dGDP and 5'-dGTP to 8-oxo-5'-dGDP and 8-oxo-5'-dGTP, respectively, by the same mechanism and kinetics as for 5'-dGMP. The Pt(IV) complex binds to N7 followed by a two-electron inner sphere electron transfer from G to Pt(IV). The reaction was catalyzed by Pt(II) and occurred faster at higher pH. The electron transfer was initiated by either an intramolecular nucleophilic attack by any of the phosphate groups or an intermolecular nucleophilic attack by free OH(-) in the solution. The rates of reactions for the three nucleotides followed the order: 5'-dGMP > 5'-dGDP > 5'-dGTP, indicating that the bulkier the phosphate groups are, the slower the reaction is, due to the larger steric hindrance and rotational barrier of the phosphate groups.