Bovine serum amine oxidase (BSAO) catalyzes the oxidative deamination of primary amines, concomitant with the reduction of molecular oxygen to hydrogen peroxide via a ping-pong mechanism. A protocol has been developed for an analysis of chemical and kinetic mechanisms in the conversion of dioxygen to hydrogen peroxide. Steady-state kinetics show that two groups need to be deprotonated to facilitate the oxidative half-reaction. The pH dependence of Vmax/Km(O2) reveals pKa's of 6.2 +/- 0.3 and 7.0 +/- 0.2, respectively. A pKa of 7.2 +/- 0.1 has been obtained from a titration of anaerobically reduced BSAO using UV-vis spectrophotometry. The near identity of the pKa obtained from the reduced enzyme titration with the second pKa from steady-state kinetics suggests that this second pKa arises from the reduced cofactor. The assignment of pKa is supported by the observed pH dependence for formation of the cofactor semiquinone signal, detected by EPR spectroscopy under anaerobic conditions. To address the nature of rate-limiting steps in the oxidative half-reaction, the solvent isotope effect, viscosity effect, and O-18 isotope effect on Vmax/Km(O2) have been determined. The solvent isotope effect is indistinguishable from unity, ruling out a proton transfer as a rate-determining step. Use of glucose as a solvent viscosogen shows no viscosity effect, indicating that binding of oxygen is not in the rate-determining step. The O-18 kinetic isotope effect is independent of pH with an average value of 18(V/K) = 1.0097 +/- 0. 0010. This has been compared to calculated equilibrium O-18 isotope effects for various dioxygen intermediate species [Tian and Klinman (1993) J. Am. Chem. Soc. 115, 8891], leading to the conclusion that either the first electron transfer to dioxygen or the desorption of product peroxide from a Cu(II)-OOH complex could be the rate-limiting step. The distribution of steady-state enzyme species was, therefore, analyzed through a combination of stopped-flow experiments and analysis of DV and D(V/K) for benzylamine oxidation. We conclude that the major species accumulating in the steady state are the oxidized cofactor-substrate Schiff base complex and the reduced, aminoquinol form of cofactor. These data rule out a slow release of product hydroperoxide from the aminoquinone form of enzyme, leading to the conclusion that the first electron transfer from substrate-reduced cofactor to dioxygen is the rate-determining step in the oxidative half-reaction. This step is also estimated to be 40% rate-limiting in kcat. An important mechanistic conclusion from this study is that dioxygen binding is a separate step from the rate-limiting electron-transfer step to form superoxide. On the basis of a recently determined X-ray structure for the active form of a yeast amine oxidase from Hansenula polymorpha [Li et al. (1998) Structure 6, 293], a hydrophobic space has been identified near the O-2 position of reduced cofactor as the putative dioxygen binding site. Movement of superoxide from this site onto the Cu(II) at the act...

A recent report by Mills and Klinman [Mills, S. A., and Klinman, J. P. (2000) J. Am. Chem. Soc. 122, 9897-9904] described the preparation and initial characterization of a cobalt-substituted form of the copper amine oxidase from Hansenula polymorpha (HPAO). This enzyme was found to be fully catalytically active at saturating substrate concentrations, but with a K(m) for O(2) approximately 70-fold higher than that of the copper-containing, wild-type enzyme. Herein, we report a detailed analysis of the mechanism of catalysis for the wild-type and the cobalt-substituted forms of HPAO. Both forms of enzyme are concluded to utilize the same mechanism for oxygen reduction, involving initial, rate-limiting electron transfer from the reduced cofactor of the enzyme to prebound dioxygen. Superoxide formed in this manner is stabilized by the active site metal, facilitating the transfer of a second electron and two protons to form the product hydrogen peroxide. The elevated K(m) for O(2) at the dioxygen binding site in Co-substituted HPAO, relative to that of wild-type HPAO, is proposed to be due to a change in the net charge at the adjacent metal site from +1 (cupric hydroxide) in wild-type enzyme to +2 (cobaltous H(2)O) in cobalt-substituted HPAO.

Glucose oxidase catalyzes the oxidation of glucose by molecular dioxygen, forming gluconolactone and hydrogen peroxide. A series of probes have been applied to investigate the activation of dioxygen in the oxidative half-reaction, including pH dependence, viscosity effects, 18O isotope effects, and solvent isotope effects on the kinetic parameter Vmax/Km(O2). The pH profile of Vmax/Km(O2) exhibits a pKa of 7.9 +/- 0.1, with the protonated enzyme form more reactive by 2 orders of magnitude. The effect of viscosogen on Vmax/Km(O2) reveals the surprising fact that the faster reaction at low pH (1.6 x 10(6) M-1 s-1) is actually less diffusion-controlled than the slow reaction at high pH (1.4 x 10(4) M-1 s-1); dioxygen reduction is almost fully diffusion-controlled at pH 9.8, while the extent of diffusion control decreases to 88% at pH 9.0 and 32% at pH 5.0, suggesting a transition of the first irreversible step from dioxygen binding at high pH to a later step at low pH. The puzzle is resolved by 18O isotope effects. 18(Vmax/Km) has been determined to be 1.028 +/- 0.002 at pH 5.0 and 1.027 +/- 0.001 at pH 9.0, indicating that a significant O-O bond order decrease accompanies the steps from dioxygen binding up to the first irreversible step at either pH. The results at high pH lead to an unequivocal mechanism; the rate-limiting step in Vmax/Km(O2) for the deprotonated enzyme is the first electron transfer from the reduced flavin to dioxygen, and this step accompanies binding of molecular dioxygen to the active site. In combination with the published structural data, a model is presented in which a protonated active site histidine at low pH accelerates the second-order rate constant for one electron transfer to dioxygen through electrostatic stabilization of the superoxide anion intermediate. Consistent with the proposed mechanisms for both high and low pH, solvent isotope effects indicate that proton transfer steps occur after the rate-limiting step(s). Kinetic simulations show that the model that is presented, although apparently in conflict with previous models for glucose oxidase, is in good agreement with previously published kinetic data for glucose oxidase. A role for electrostatic stabilization of the superoxide anion intermediate, as a general catalytic strategy in dioxygen-utilizing enzymes, is discussed.

Two polypyridyl copper(II) complexes, [Cu(acac)(p-CPIP)(CH 3 OH)](NO 3 ) (1) and [Cu(acac)1,10-phenanthroline), have been synthesized and characterized by singlecrystal analysis, IR and electronic spectroscopy. X-ray analyses reveal that both 1 and 2 possess a triclinic crystal system with Pmn1 and mononuclear five-coordinated square pyramidal geometry, which were further linked to a one-dimensional structure through p-p stacking and intermolecular hydrogen bonds.The interaction of the Cu(II) complex with calf thymus DNA (CT-DNA) was investigated by UV-visible and fluorescence emission spectrometry, as well as agarose gel electrophoresis. The apparent binding constant (K app ) values of 2.47 Â 10 4 M À1 for 1 and 3.04 Â 10 4 M À1 for 2 suggest moderate intercalative binding mode between the complexes and DNA. Both complexes displayed efficient oxidative cleavage of supercoiled DNA in the presence of external agents. In addition, fluorescence spectrometry of bovine serum albumin (BSA) with the complexes showed that the quenching mechanism of 1 might be a dynamic procedure, while 2 showed a combined dynamic and static quenching mechanism. For both 1 and 2, the number of binding sites was about one for BSA. Moreover, synchronous fluorescence spectral experiments revealed that 1 and 2 affect the microenvironment of tryptophan residues of BSA.

The directed assembly of N,N‐bis(benzimidazol‐2‐yl‐methyl)amine (BMA), 4,4′‐bipyridine (4,4′‐bipy) and NiII or MnII salts resulted in the formation of  the novel compounds [{Ni(BMA)(SCN)2}2]·(μ‐4,4′‐bipy)(DMSO)2 (1) and [{Mn(BMA)(SCN)2}2]·(μ‐4,4′‐bipy) (2), which were characterized by single‐crystal X‐ray diffraction, elemental analysis, IR spectroscopy, UV/Vis spectroscopy and magnetic measurement. The X‐ray single‐crystal analysis reveals that 1 and 2 are discrete dinuclear compounds bridged by 4,4′‐bipy, further arranged in multidimensional supramolecular networks which are stabilized by different modes of hydrogen bonding and π–π interactions. Variable‐temperature magnetic measurements (2–300 K) of 1 and 2 were modeled with magnetic coupling constants J = –0.16 and –0.022 cm–1, respectively showing the weak antiferromagnetic coupling between the two metal ions in each dinuclear complex, which is also observed for similar 4,4′‐bipyridine bridging complexes.