Cheryl E. Cote

CHARACTERIZATION OF BOVINE CERULOPLASMIN

Ceruloplasmin (ferroxidase, iron II: oxygen oxidoreductase, EC 1.16.3.1) has been extensively studied, not only because it is a member of the blue copper oxidases, but also because of its clinical importance and possible role(s) in iron and copper metabolism and transport [ 1-3]. Human and porcine ceruloplasrains have been well characterized [1-3] . Both contain three spectroscopically distinguishable types of copper sites (types 1,2 and 3) whose properties have been extensively discussed [1-5]. The copper stoichiometry of ceruloplasmin is still somewhat uncertain. Previous experiments indicated that porcine and human ceruloplasmin have identical copper contents • [2]. Recent measurements of the molecular mass and copper content of the human protein are consistent with 6 7 intrinsic coppers [6]. One copper ion can Be removed from a human ceruloplasmin preparation containing 8 coppers/enzyme molecule without affecting the catalytic activity [7 ]. In addition, human ceruloplasmin can bind up to 10 additional cupric ions [8]. Human, pig, horse, and rabbit ceruloplasmins are each composed of a single polypeptide chain with nearly the same molecular mass [6,9-11]. Further, the SDS gel electrophoretic patterns of multicomponent preparations of human ceruloplasmin have been qualitatively reproduced via limited proteolysis [ 12]. In comparison, bovine ceruloplasmin has been poorly characterized. We have purified it by a standard method and determined its enzymatic activity, copper content, and some of its spectroscopic properties.

Methylamine oxidase from Arthrobacter P1 as a prototype of eukaryotic plasma amine oxidase and diamine oxidase

Methylamine oxidase (MAOx) from Gram-positive soil bacterium Arthrobacter P1 catalyzes the oxidation of CH3NH2 to H2C = O and NH4+ via reduction of O2 to H2O2. Past work indicates that MAOx is similar to mammalian plasma amine oxidase (PAO) and diamine oxidase (DAO), plant DAO, and yeast peroxisomal amine oxidase (YAO). All have Mr congruent to 170,000 and are composed of 2 identical subunits, each of which contains 1 atom of Cu(II) and one molecule of quinonoid cofactor. Herein, we report further evidence as to the striking similarity of these enzymes, and describe properties of MAOx which offer insights into understanding the eukaryotic oxidases. It is our belief that the structure of the quinone cofactor, and the Cu(II) site in MAOx are identical to these sites in PAO and DAO.

Copper-PQQ Interactions in Amine Oxidases

Interactions between copper and PQQ in several amine oxidases have been probed by a variety of physical methods. A Cu(I)-semiquinone (PQQ●) state can be generated by substrate-reduction in the presence of ligands that stabilize Cu(I). This state has now been characterized in detail for amine oxidases from bovine plasma, porcine kidney, pea seedlings, and Arthrobacter P1. The properties of the semiquinone are independent of the enzyme, substrate, or copper ligand. It is possible that a Cu(I)-PQQ● state may be a catalytic intermediate in the oxidation of the substrate-reduced enzyme by O2. Additional substrate-reduced forms are observed for some amine oxidases; the distribution among these forms is sensitive to copper ligation. Resonance Raman spectroscopy has been used to characterize both native and metal-depleted amine oxidases, which have been derivatized by chromophoric carbonyl reagents. 2-Hydrazinopyridine reacts with all the amine oxidases (and PQQ) to form a hydrazone derivative with λmax ≅ 415 nm; subsequently this converts, in a copper-dependent reaction, to a form with an absorption band at ≃ 520 nm.

Active Site Structures of Copper-Containing Oxidases

Pig plasma amine oxidase (PPAO; EC 1.4.3.6) has been shown to contain PQQ as well as copper. Experiment are describer to quantitate and characterize the functional characteristics of the PQQ centres in PPAO. The Results of spectroscopic studies on PQQ are reviewed and a plausible model for the active site copper proposed. There is no evidence from the spectroscopic Studies for or against PQQ coordination to the copper. Galactose oxidase (EC 1.1.3.9) has structural and functional similarity to amine oxidase. Experiments to test for the presence of PQQ in galactose oxidase are discussed together with other structural properties of this enzyme.

Inhibition of amine oxidases by Cu(II) complexes and anions: Mechanistic implications

Abstract Copper-containing amine oxidases catalyze the oxidative deamination of primary amines by the following mechanism [1]: Our interests center on the role(s) of copper in reactions (1) and (2) and on the activation and utilization of O 2 by this enzymes. An important mechanistic question in this regard is whether then oxidation of E red proceeds via sequential one-electron steps or via a single, two-electron step. One-electron oxidation would generate O 2 − as an intermediate, whereas a two-electron oxidation [2] would not. Accordingly we investigated several Cu(II) complexes, previously shown to be superoxide dismutase active, as potential inhibitors of bovine plasma and pig kidney amine oxidase [3, 4]. Cu(II) complexes of 1, 10-phenanthroline (phen) and 2,2′-bipyridine (bipy) strongly inhibited both amine oxidases. Lysine, tyrosine, and salicylate complexes, as well as Cu 2+ (aq.), inhibited the plasma but not the kidney amine oxidase. No inhibition by the free ligands, by Cu(EDTA) 2− , or bovine erythrocyte SOD was observed. Cu(phen) 2 (NO 3 ) 2 , Cu 2+ (aq.), and Cu(salicylate) 2 displayed linear noncompetitive inhibition against amine substrate at saturating O 2 concentrations with either pig kidney or beef plasma amine oxidase. Since neither the free ligands nor Cu 2+ (aq.) inhibit the kidney enzyme at concentrations where Cu(phen) 2 (NO 3 ) 2 and Cl 2 are effective, the complexes. Two plausible inhibition mechanism are (1) dismutation of O 2 − , which is an intermediate in the reoxidation reaction; or (2) direct oxidation E red by the Cu(II) complexes. Steric requirements for the second should be mechanism should be less restrictive than for mechanism (1) as outer-sphere electron transfer is possible over substantial distances, but O 2 − dismutation is an inner-sphere process that requires the Cu(II) complex to penetrate to the O 2 reduction site. Thus if O 2 − is tightly bound within the dioxygen-reductions site, its accessibility to Cu(II) complexes of varying size and charge would be mechanically important. On this basis the reactivity pattern we have observed can be rationalized: only those complexes with hydrophobic ligands and a net positive charge inhibited pig kidney amine oxidase, but the active site in the plasma enzyme may be much more accessible to external reagents. N 3 − and SCN − are Cu(II)-specific inhibitors of several amine oxidases [1, 5–7]. Ligand-to-metal charge-transfer bands are evident at ∼365 nm (ϵ ∼3,000 M −1 cm −1 and ∼400 nm (ϵ ∼6,000 M −1 cm −1 when SCN − and N 3 − , respectively, are added to beef plasma or pig kidney amine oxidase. The intensities and energies establish that the anions are equitorially coordinated. Changes in the Cu(II) ligand field, as judged by absorption, circular dichroism and EPR spectroscopy, are consistent with a simple ligand substitution reaction. Preliminary NMR evidence indicates that H 2 O is the leaving group, as previously demonstrated for the pig plasma enzyme [6]. Depending on the amine concentration both the anions are linear noncompetitive or uncompetitive inhibitors of beef plasma amine oxidase with O 2 saturating; under the same conditions these anions were strictly linear uncompetitive inhibitors of the pig kidney enzyme. By determining in detail the inhibition mechanism of anions and the Cu(II) complexes, we should be able to learn much about the role(s) of copper in the amine oxidases.