Low activation barriers characterize intramolecular electron transfer in ascorbate oxidase.

Abstract

Anaerobic reduction kinetics of the zucchini squash ascorbate oxidase (AO; L-ascorbate:oxygen oxidoreductase, EC 1.10. x 109 M-1-s-1] was followed by its subsequent reoxidation in three distinct phases, all found to be unimolecular processes with the respective specific rates of 201 ± 8, 20 ± 4, and 2.3 ± 0.2 s"' at pH 5.5 and 298 K. While at this pH no direct bimolecular reduction was resolved in the 330-nm band, at pH 7.0 such a direct process was observed [(6.5 are similar to values reported for the limiting-rate constants of AO reduction by excess substrate, suggesting that internal electron transfer is the rate-determining step ofAO activity. The temperature dependence of the intramolecular electron transfer rate constants was measured from 275 to 308 K at pH 5.5 and, from the Eyring plots, low activation enthalpies were calculat--namely, 9.1 ± 1.1 and 6.8 ± 1.0kJ mol' for the fastest and slowest phases, respectively. The activation entropies observed for these respective phases were -170 ± 9 and -215 ± 16 J-K'1-mol'1. The exceptionally low enthalpy barriers imply the involvement of highiy optimized electron transfer pathways for internal electron transfer.The blue copper oxidases are widespread redox enzymes that catalyze specific one-electron substrate oxidation by dioxygen, which in turn is reduced to two water molecules (1-3). Their minimal catalytic unit contains four copper ions, which are bound to distinct sites classified according to unique spectroscopic properties (3). The type 1 (Ti) site confers on the bound Cu(II) ions an intense absorption band in the visible region and a rather narrow hyperfine g1l value in the electron paramagnetic resonance (EPR) spectrum. The pair of Cu(II) ions bound to the type 3 (T3) site is characterized by a strong absorbance in the near UV region and by the absence of an EPR signal. The type 2 (T2) Cu(II) site has a rather weak optical absorption spectrum and exhibits a typical hyperfine EPR signal.Ascorbate oxidase (AO; L-ascorbate:oxygen oxidoreductase, EC 1.10.3.3) was found to be a 140-kDa protein containing eight copper ions per molecule, which can be classified into the above T1, T2, and T3 by their spectroscopic properties (4).The reduction potentials ofT1 and T3 are identical at 298 K (E' = 350 mV; pH 7.0) (5). The recently determined highresolution three-dimensional model of AO provides unique insights into the detailed structure and spatial relationship among the copper binding centers (6, 7). Thus, AO has been shown to exist as a dimer of identical 70-kDa subunits, each containing one T1, one T2, and one T3 site. These subunits are folded into three interacting domains, all of similar (-barreltype structure, distantly related to the small, blue single copper proteins (6, 7). The T1 site, highly conserved among all blue copper proteins, is within one domain. The T2 and T3 sites were found to be proximal, thus creating a trinuclear center at a distance of 1.23 nm between the T3 copper pair and the T1 site. Ligands coordinated to the T2 and T3 coppe...

The intramolecular electron transfer between copper sites of nitrite reductase: a comparison with ascorbate oxidase

“…1, the amplitude of the increase in absorbance associated with the reoxidation of the T1 Cu(I) centre during the intramolecular ET from T1 Cu(I) to T2 Cu(II) does not correspond to the complete reoxidation of the T1 site. A similar observation was made in the earlier pulse radiolysis studies [7,12,13]. Since the midpoint potential of T1 Cu(II)/Cu(I) for AxNiR has been reported to be 240 mV at 298 K [12] one may calculate a midpoint potential of T2 Cu(II)/Cu(I) from the above ET equilibrium to be 230 mV.…”

“…In both enzymes the distance between the two centres is 12.5 A î . The activation parameters for the faster phase of intramolecular ET in AO are: vH g = 9.1 þ 1.1 kJ mol 3I and vS g = 3170 þ 9 J K 3I mol 3I [7]. It is of considerable interest to compare these values with those determined in the present study for the forward ET process in NiR: vH g = 22.7 þ 3.4 kJ mol 3I and vS g = 3126 þ 11 J K 3I mol 3I .…”

Crystal structure and electron transfer kinetics of CueO, a multicopper oxidase required for copper homeostasis in Escherichia coli

“…These kinetic results are in sharp contrast to those obtained from the more extensively studied MCO, AO from zucchini squash (20,(30)(31)(32)(33)(34). Upon reduction of AO by laser flash photolysis, a rapid bimolecular reaction with the T1 Cu occurs with a rate constant of 5 ϫ 10 20), about 10-fold larger than that for CueO.…”

“…This second kinetic process results in an Ϸ50% return of the absorbance at 610 nm caused by Cu 2ϩ regeneration at the T1 site, implying that the two copper centers have similar reduction potentials, an interpretation further supported for AO by using pulse radiolytic reduction and monitoring of absorption by the trinuclear cluster at 330 nm (31,32). The rate constant for this intramolecular electron transfer reaction in AO, which is the sum of the forward and reverse rate constants, has a value of Ϸ160 s Ϫ1 (20,33), 16-fold larger than that for CueO (Ϸ10 s…”

Human Ceruloplasmin

“…Ascorbate oxidase is a homodimer consisting of two subunits both containing the four copper arrangement. Here, two intramolecular ET processes are observed between T1[Cu(I)] and T3[Cu(II)], one with a rate constant of 200 s Ϫ1 and a slower one with a rate constant of 2.3 s Ϫ1 (9). The difference between the two rates could be explained by differences in activation entropies, which were attributed to differences in electronic coupling between the electron donor and acceptor in the two ascorbate oxidase monomers.…”