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Antioxidant activities of 3-oxygenated and 3,4-dioxygenated carbazole alkaloids and their related carbazoles were comprehensively evaluated. In all assay systems, the 3,8-dihydroxycarbazoles carbazomadurin A (2) and B (3), and their synthetic precursors 2a and 3a exhibited higher antioxidant activities than the 3-monohydroxycarbazoles carazostatin (1), and the synthetic precursors 4a and 4b of carquinostatin A (4). In particular, 2a and 3a exhibited strong scavenging activities due to the reducing ability of formyl group at the C-5 position of carbazoles. The results suggest that these compounds could serve as useful clues for designing and developing novel antioxidants.

Dipeptidyl peptidase (DPP) III is a zinc-dependent exopeptidase that has a unique motif, “HELLGH,” as the zinc-binding site. In the present study, a three-dimensional (3D) model of rat DPP III was generated with the X-ray crystal structure of human DPP III (PDB: 3FVY [Dobrovetsky E. et al . (2009) SGC]) as a template. The replacement of the seven charged amino acid residues with a hydrophobic amino acid around the zinc ion did not cause any significant changes in K m values or in the substrate specificity. However, the k cat values of H568R and H568Y were remarkably reduced, by factors of 50 and 400, respectively. The His 568 residue of rat DPP III is essential for enzyme catalysis. The k cat values of the mutants E507A and E512A were 2.38 and 3.88 s − 1 toward Arg-Arg-NA, and 0.097 and 0.59 s − 1 toward Phe-Arg-NA, respectively. These values were markedly lower than those of the wild-type DPP III. Furthermore, the zinc contents of E507A and E512A were 0.29 and 0.08 atom per mol of protein, respectively, and those mutations caused remarkable increases in the dissociation constants of the zinc ions from DPP III by factors of 5 × 10 3 to 2 × 10 4 . The 3D model of the catalytic domain of rat DPP III showed that the carboxyl oxygen atoms of Glu 507 and Glu 512 form the hydrogen bonds to the nitrogen atoms of His 455 and His 450 . All of these results showed that Glu 507 or Glu 512 stabilizes the coordination bond between the zinc ion and His 455 or His 450 .

The electron-transfer reactions from Fe(II)(CN)6 4− to the cupric ion in human- or bovine-copper, zinc-superoxide dismutase were followed at various pHs from pH 5.0 to 8.0 by the micro-stopped-flow method. From pH 5.0 to 8.0, Fe(II)(CN)6 4− first forms an adduct through the charge interaction with Arg 143 or 141 of the active cavity; electron-transfer then occurs from Fe(II)(CN)6 4− to the cupric ion of the enzyme in human- or bovine-copper, zinc-superoxide dismutase. The formation-constant values of the adducts in human- and bovine-copper, zinc-superoxide dismutase are almost constant between pH 7.0 and 8.0, but increase with decreasing pH at pH values lower than 7.0. In contrast, the electron-transfer rate constants from Fe(II)(CN)6 4− to the cupric ion of the enzyme after adduct-formation in human- and bovine-copper, zinc-superoxide dismutase are nearly constant from pH 5.0 to 8.0 and give almost the same values in bovine- or human-copper, zinc-superoxide dismutase. The pH dependencies of adduct-formation-constants in human- and bovine superoxide dismutase indicate that the protonation of some residue with pKa value less than 5.0 accelerates the formation of adducts formed through the charge interaction between Arg 143 (human) or Arg 141 (bovine) and Fe(II)(CN)6 4−. X-ray diffraction analysis data show that the arrangement of Arg 143 and Glu 133 around the copper ion in the active cavity of human-copper, zinc-superoxide dismutase is very similar to that of bovine copper, zinc-superoxide dismutase. The low pKa value of the residue involved in adduct-formation suggests that the protonation group should be the carboxylate. Protonation of the carboxylate group of Glu 133 (Glu 131) results in neutralization of the minus charge of the carboxylate group and a decrease in the electronic repulsion for Fe(II)(CN)6 4−, so that the formation-constant of the adduct should increase with decreases in pH at lower pH values.

The electron transfer reactions from Fe II (CN) 4− 6 and W IV (CN) 4− 8 to the cupric ions in human copper, zinc superoxide dismutase were followed by the micro-stopped-flow method. The kinetic rate data clearly indicate that Fe II (CN) 4− 6 or W IV (CN) 4− 8 first forms an adduct with the enzyme through the interaction with Arg143 of the active cavity and then an electron from Fe II (CN) 4− 6 or W IV (CN) 4− 8 of the adduct transfers to the cupric ion in the enzyme. The dissociation constants of the adducts of Fe II (CN) 4− 6 and W IV (CN) 4− 8 were 4.0(±0.3) × 10 −3 and 2.2(±0.3) × 10 −3 M, respectively. In spite of the difference between the standard redox potentials of Fe III (CN) 3− 6 /Fe II (CN) 4− 6 (468 mV) and W V (CN) 3− 8 /W IV (CN) 4− 8 (556 mV), the electron transfer rate constant (0.148(±0.005) s −1 ) of Fe II (CN) 4− 6 at 25°C is very similar to that of W IV (CN) 4− 8 (0.072(±0.011) s −1 ). The entropy values of the adduct formations and the activation energies of the electron transfer rates were determined by the temperature dependence of the dissociation constants of the adducts and the electron transfer rates. The enthalpy values of the formation of adducts are almost zero, so that the driving forces to form the adducts are mainly derived from the entropy. The activation energy of the electron transfer rate of Fe II (CN) 4− 6 is very similar to that of W IV (CN) 4− 8 . The formation of the adduct between Fe II (CN) 4− 6 and the enzyme was inhibited by the presence of various anions (ClO − 4 , SO 2− 4 , SCN − , and N − 3 ). The bulky anions SO 2− 4 and ClO − 4 behave as competitive inhibitors for Fe II (CN) 4− 6 ; these anions should interact mainly with Arg143, as it has a positive charge at the entrance of the active cavity. The competitive inhibition constants of ClO − 4 , SO 2− 4 , and SCN − were 0.010, 0.012, and 0.008 M. The azide ion, which is smaller than SO 2− 4 or ClO − 4 , shows mixed inhibition, because N − 3 can interact with Arg143 (competitive inhibition) and also directly binds to the cupric ion in h-SOD (noncompetitive inhibition). The competitive and noncompetitive inhibition constants of N − 3 were 0.004 and 0.016 M, respectively.

Bilirubin oxidase obtained from Trachyderma tsunodae K-2593 is a multi-copper enzyme which has type 1:type 2:type 3 copper atoms in the ratio 1:1:2. Anion binding properties of bilirubin oxidase were investigated to determine the structural and functional properties of bilirubin oxidase. The anions N3−, SCN−, F−, Cl−, and Br− were non-competitive inhibitors against the substrate, bilirubin ditaurate, and the inhibition constants of the anions were in the following order: N3−

The behavior of complexing agents for the copper removal reaction was studied by the equilibrium dialysis method. In the copper removal reaction, complexing agents are divided into two types: those that are reducing agents and those that are not. Sodium cyanide and sodium thiosulfate are of the first type, and 8-hydroxyquinoline-5-sulfonic acid, 2,2′-bipyridyl, and picolinic acid are of the second type. From equilibrium dialysis with the first type of complexing agent, the apparent binding constant (pH 6.0) between cuprous ions and apotyrosinase was calculated to be 10 15 m −1 . Similarly, the apparent binding constant (pH 6.0) between cupric ions and apo-tyrosinase was about 10 13 m −1 , which was calculated from equilibrium dialysis with the second type of complexing agent. The apparent binding constant between cuprous ions and apo-tyrosinase was larger than that between cupric ions and apo-tyrosinase.

For the removal of the cobalt ion from cobalt-bovine carbonic anhydrase with 5-methyl-1,10-phenanthroline, it was proved spectrophotometrically that the substitution mechanism proceeded through the ternary complex in which the central metal ion bound with the protein and the chelating agent. The spectrum of the ternary complex had low molar absorption coefficient in visible region, so that it was assumed that the ternary complex had a five- or six-coordination geometry.

Interaction of cobalt(II) bovine carbonic anhydrase with 3- and 4-pyridinecarboxylates, 2-pyridinecarboxylate, and 2,6-pyridinedicarboxylate has been investigated by the spectrophotometric method. The apparent formation constant of the ternary complex ( ligand : cobalt ion : apoenzyme = 1 : 1 : 1 ) was determined from spectral data. The spectroscopic data of the ternary complex indicate that the 3- or 4-pyridinecarboxylate adduct has a five-coordination geometry through three donor atoms of the protein part of the enzyme, the carboxyl group of 3- or 4-pyridinecarboxylate, and a water molecule. 3- or 4-Pyridinecarboxylate behaves as a monodentate ligand. The spectrum of the ternary complex of 2-pyridinecarboxylate was very different from that of 3- or 4-pyridinecarboxylate. The spectra data indicate that 2-pyridinecarboxylate adduct has a five-coordination geometry and that it behaves as a bidentate ligand. The ternary complex of 2,6-pyridinedicarboxylate was so unstable that the spectrum of the ternary complex was determined by the indirect method. The spectrum of 2,6-pyridinedicarboxylate adduct shows lower molar absorption than that of 2-pyridinecarboxylate adduct. This result indicates that 2,6-pyridinedicarboxylate behaves possibly as a tridentate ligand.