The effect of azide on the spectral and catalytic properties of ascorbate oxidase

Abstract

(1) 45% of the total copper of green zucchini ascorbate oxidase is EPR-detectable. At least two species of copper are present, one with a small A parallel (Type 1) and one with a large A parallel (Type 2). Computer simulated spectra indicated 50% contribution by each type of copper. (2) Azide inhibited ascorbate oxidase activity by an uncompetitive mechanism. EPR and optical spectra performed on titration of ascorbate oxidase with azide indicated the formation of a copper-azide complex. The Type 2 copper appears to be the binding site of azide. The involvement of the EPR non-detectable copper as an anion binding site with high affinity toward azide can not be excluded.

Coordination environment and fluoride binding of type 2 copper in the blue copper protein ascorbate oxidase

“…1. Similar spectra have been reported by several laboratories (9)(10)(11)(12)28 Exposure of native ascorbate oxidase to nitric oxide under anaerobic conditions led to a substantially simplified spectrum (Fig. 2).…”

“…In addition, because the inhibition studies by Mondovi et al (28) and by Strothkamp and Dawson (29) have implicated type 2 copper as an anion binding site in ascorbate oxidase, we have also examined the effect of added fluoride on the EPR spectra of the native enzyme and the NO-treated derivative.…”

Ascorbate Oxidase from Cucurbita pepo medullosa

“…3 -6 Cu/molecule depending on the preparation. In the case of the material investigated by Mondovi et al [8], which contained 6-8 Cu/l40000 M,, computer simulations of the EPR spectra indicated an equal amount of type-1 and type-2 copper. These results are in contrast to the ratio of 3 to 1 as found by Deinum et al [13] in a sample of ascorbate oxidase prepared by one of us (A. M.) in 1973.…”

“…Double integration of the area under the first derivative reveals 48 f 2 % of the total chemically determined copper to be EPR detectable in frozen solution. Table 3 summarizes the experimental g and A values, taken from the recorded EPR spectrum, in comparison to the EPR parameter given by Nakamura et al [12], Mondovi et al [8,14], and Lee and Dawson [2]. A best fit of the EPR spectrum of oxidised ascorbate oxidase can be obtained by computer simulation on the basis of high-frequency measurements at 35 GHz [13].…”

“…A close examination of the EPR spectra shown by Lee and Dawson [2] clearly indicates the presence of so-called non-specific copper with large hyperfine splittings in the gll region similar to the type-2 copper in laccase or ceruloplasmin. Furthermore the absorption maximum at 880 nm [ 2 ] , which so far has not been detected in the preparations of Mondovi et al [8], Nakamura et al [12] or Deinum et al [13], and which has not been detected in laccase or ceruloplasmin either (see Table 1 in [2]), might result from another copper protein or from ascorbate oxidase with slightly modified copper sites. It should be noted that in the case of tree laccase the low-molecular-weight copper protein stellacyanin has been isolated from the same crude extract [I 81.…”