Ascorbate oxidase spectra

Many multiple copper containing proteins (e.g., laccase, ascorbate oxidase, hemo-cyanin, tyrosinase) contain so-called type III copper centers, which is a historical name (cf. Section 5.8 for type I and type II copper) for strongly exchange-coupled Cu(II) dimers. In sharp contrast to the ease with which 5=1 spectra from copper acetate are obtained, half a century of EPR studies on biological type III copper has not produced a single triplet spectrum. Why all type III centers have thus far remained EPR silent is not understood. 

Mavicyanin (Mj = 18,000) is obtained from green squash (Cucurbito pepo medullosa), where it occurs alongside ascorbate oxidase [64]. It has a peak at 600 nm (e 5000 M cm and reduction potential of 285 mV. Further studies on this and the mung bean and rice bran proteins [65, 66] would be of interest. All the above type 1 Cu proteins have an intense blue color and characteristic narrow hyperfine EPR spectrum for the Cu(II) state. Table 3 summarizes the properties of those most studied. There is some variation in reduction potential and position of the main visible absorbance peak. In the case of azurin, for example, the latter is shifted from 597 to 625 nm. Stellacyanin has no methionine and the identity of the fourth ligand is therefore different [75]. The possibility that this is the 0(amide) of Gln97 has been suggested [63b]. It now seems unlikely that the disulfide is involved in coordination. Stellacyanin has 107 amino acids, with carbohydrate attached at three points giving a 40% contribution to the M, of 20,000 [75]. 

Fig. 5-14. The visible absorption spectrum of ascorbate oxidase (curve 1) and in the presence of a substrate AH2 (curve 2). Reproduced with permission from Dawson (1960).

The reoxidation studies on laccase and ascorbate oxidase are listed in Table IX. The reoxidation of the type-1 copper and of the trinuclear copper site occurs at a rate of 5 x 10 M" sec" both for tree laccase 134) and for ascorbate oxidase 135). During reoxidation with H2O2, an 02 " intermediate is formed in several minutes, which is documented for tree laccase by changes in the CD spectrum 136) and for ascorbate oxidase in the formation of an absorption band at 350 nm... 

Thereafter, crystals were brought back to the aerobic 25% MPD solution, buffered with 50 mAf sodium phosphate, pH 5.5. This procedure is based on Avigliano et al. s (157) method of preparing T2D ascorbate oxidase in solution and was modified by Merli et al. (159) for use with ascorbate oxidase crystals. The 2.5-A-resolution X-ray structure analysis by difference-Fourier techniques and crystallographic refinement shows that about 1.3 copper ions per ascorbate oxidase monomer are removed. The copper is lost from all three copper sites of the trinuclear copper species, whereby the EPR-active type-2 copper is the most depleted (see Fig. 10). Type-1 copper is not affected. The EPR spectra from polycrystalline samples of the respective native and T2D ascorbate oxidase were recorded. The native spectrum exhibits the type-1 and type-2 EPR signals in a ratio of about 1 1, as expected from the crystal structure. The T2D spectrum reveals the characteristic resonances of the type-1 copper center, also observed for T2D ascorbate oxidase in frozen solution, and the complete disappearance of the spectroscopic type-2 copper. This observation indicates preferential formation of a Cu-depleted form with the holes equally distributed over all three copper sites. Each of these Cu-depleted species may represent an anti-ferromagnetically coupled copper pair that is EPR-silent and that could explain the disappearance of the type-2 EPR signal. 

The first successful observation and characterization of the ascorbate free radical was carried out with ESR (14,15). A 1.7-G ESR doublet was reported and it was correctly concluded that the observed spectrum represented the anionic form (A ) of the radical. These measurements (14,15) showed that the enzyme-generated radical (horseradish peroxidase-hydrogen peroxide-ascorbate) was present as a free radical and decayed by second-order kinetics (see Figure 2). Recent experiments (16,17) have shown that ascorbate oxidase and dopamine-monooxygenase also generate unbound ascorbate radicals. 

Double integration of the area under the first derivative reveals that 48 =i= 2% of the chemically determined copper (IS) is EPR detectable in frozen solution. Table I summarizes the experimental g and A values measured from the recorded spectra. A best fit of the EPR spectrum of oxidized ascorbate oxidase is obtained by computer simulation, using the high-frequency measurements at 35 GHz (28). The ratio of type 1 to type 2 copper is estimated by double integration of the first low-field line, which arises from the type 2 copper, at approximately 0.270 T (IS). Roughly 25% of the EPR-detectable copper in ascorbate oxidase is type 2, whereas 75% is blue type 1 copper. This ratio is confirmed by computer analysis (IS) and agrees with earlier results (28) (Figure 4). 

Figure 9. EPR spectrum of the substrate radical formed during the rapid initial phase of the anaerobic reduction of ascorbate oxidase by reductate. Conditions as in Figure 6. The spectrum was recorded at 77 K, 9.4 GHz, 100 kHz modulation frequency, 0.05 mT modulation amplitude.

Deinum et al. 225) have shown that the EPR spectrum of ascorbate oxidase from green zuchini squash is very similar to that from cucumber peel shown in Fig. 9. They have carefully recorded and integrated the EPR spectrum of ascorbate oxidase finding that 47 3% of the Cu present was detectable by this method. Of this 47 % only 25 zh 5 % corresponded to T)q)e 2 Cu2+. With this information in hand, the authors quite accurately simulated the observed EPR spectrum assuming the presence of three EPR identical Type 1 Cu2+ and one T5q>e 2 Cu2+. On the basis of total Cu present and the above observations Deinum et al. 225) conclude that ascorbate oxidase contains three Type 1 Cu2+, one T q)e 2 and four... 

Fig. 9. The EPR spectrum of ascorbate oxidase from Cucumber. Native form (solid) and reduced with ascorbate (dashed). [Taken from Nakamura, Makino and Ogura , Ref. (797)]...

Figure 1. Difference spectrum (reduced vs oxidized) of beef heart mitochondria. Peak 1 represents cytochrome oxidaser peak 2 cytochrome b, peak 3 cytochromes c and ci. Average concentrations calculated according to Williams . Cytochrome oxidase 26 8 pN, cytochrome b 20.3 pM, cytochrome ci 3.1 pM and cytochrome c 5.1 pM. Protein 13.4 mg ml i. Reductants A. Ascorbate +...

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