Pyrocatechases

On the other hand, Hayaishi and his associates found that the two atoms of oxygen incorporated into catechol by the action of pyrocatechase were both derived from molecular oxygen3 [Eq. (2)]. 

As in the cases of phenolase and pyrocatechase, respectively, oxygenases catalyze the incorporation of either one or two atoms of molecular oxygen into their substrates. Therefore, they are classified into two major groups, monooxygenases and dioxygenases1S). 

Similar but not identical pyrocatechases have been purified to homogeneity from various microorganisms including Brevibacterium fuseum P-13112,113 and... 

Spectral Properties. The absorption spectra of these three pyrocatechase are very similar to each other. A concentrated solution of the enzyme shows a distinct red color with a broad absorption between 390 and 650 nm. The peak is at about 440 nm and the molecular absorbance at 440 nm is estimated to be 4,670s5. The trivalent iron bound to the enzyme is responsible for the visible absorption since the apo-enzyme shows neither significant absorption in the visible region nor enzyme activity which is restored upon reconstitution56. The visible absorption also de-... 

Table 4. Substrate specificity of pyrocatechases from Pseudomonas, Brevibacterium and Acinetobactor...

Circular Dichroism. The circular dichroism (CD) bands of Pseudomonas pyrocatechase can be classified into three groups ... 

Serological Properties. Antisera prepared against purified preparation of Acineto-bactor pyrocatechase cross-react and inhibit the enzyme activity in the crude extracts prepared from other strains of Acinetobactor calcoaceticus, but fail to cross-react and inhibit isofunctional enzymes prepared from strains of Pseudomonas, Nocardia, and Alcaligenes114. ... 

A few [VO(OMe)(/8-diketonato)]2 and [VO(L)]2 complexes (L = polydentate O-coordinating ligands) also have a high catalytic activity for the oxygenation of catechols (namely 3,5-di-f-butyl-1,2-dihy droxybenzene)554 and this reaction was considered similar to the one catalyzed by pyrocatechase. 

Tsuji and coworkers reported that copper(I) chloride in the presence of pyridine, methanol and dioxygen promotes the stoichiometric oxidation of pyrocatechol to methyl muconate.606 Labeling lg02 studies have shown that only one atom of the dioxygen molecule is incorporated in the substrate, while the other one is transformed into water as in enzymic monooxygenases (equation 275)607 (and not as in dioxygenases, viz. pyrocatechase). This reaction has been shown by Rogic et al. to proceed via two steps (equation 276).580,58 ... 

Both of the oxygen atoms incorporated into catechol have been shown639 by labeling studies to be derived from molecular oxygen. Pyrocatechase and other related oxygenases are known to require either copper or iron for maximum activity. An efficient, nonenzymatic oxidation of catechol to s,os-muconate with molecular oxygen activated by cuprous chloride has been recently presented as a model reaction for pyrocatechase.649... 

Pyrocatechase (PC) is representative of a class of proteins that facilitates the transfer of both atoms of O2 to a substrate molecule. With this particular dioxygenase, pyrocatechol is transformed to mnconic acid (eqnation 29). 

Scheme 8 Proposed mechanism for the activation of O2 by pyrocatechase (PC) for the dioxygenation of catechols...

Fe (non-heme) Pyrocatechase, ferredoxin, hemerythrin, transferrin, aconitase, nitrogenase... 

These enzymes are red in color and contain ferric ion as the sole cofactor. Two types of enzymes have been isolated from various bacteria — pyrocatechases which catalyze catechol cleavage and protocatechuate 3,4-dioxygenases which cleave 3,4-dihydroxybenzoate (protocatechuate). The pyrocatechases have molecular weights in the 60.000-90.000 dalton range9-11) the enzyme from Pseudomonas arvilla (MW 63.000) has two subunits with the molecular form a0Fe9), while the enzyme from Acinetobacter calcoaceticus (MW 85.000) is a dimer of the form (aFe)210). 

Fig. 2. Visible spectra of native pyrocatechase and the pyrocatechase-catechol complex (SO mM Tris-acetate buffer pH 8)...

Fig. 4. A-D. Resonance Raman spectra of pyrocatechase complexed with o-fluorophenol (A) o-chlorophenol (B) o-bromophenol (C) o-chlorophenol-4,6-d2 (D). Conditions 647.1 nm excitation, 150-200 mW power, 4 cm-1 slit width, 40-50 mg/mL of enzyme in Tris-acetate pH 8.5 buffer, 4 °C sample temperature. SO - was used as internal standard...

We have also found carboxylates to be weak inhibitors (Kj 5 mM) of these dioxygenases44) upon binding, the carboxylates cause a red shift in the absorption maximum. For example, the addition of benzoate to pyrocatechase shifts from 458 nm to 5.05 nm. Only dicarboxylates such as succinate and glutarate effect this spectral change for protocatechuate 3,4-dioxygenase ( ,ax 530 nm). Presumably,... 

Fig. S. A and B. Visible spectra of (A) 4-nitrocatechol at pH 8.S and the pyrocatechase-4-nitro-catechol complex (50 mM Tris-acetate pH 8) (B) 4-nitrocatechol at pH 13 and the protocatechuate 3,4-dioxygenase-4-nitrocatechol complex (50 mM Tris-acetate pH 8)...

Recently, Ques ) reported that when o-aminophenol was added to pyrocatechase from Pseudomonas arvilla, ring cleavage occurred between C-l and C-6 yielding an intermediate which cyclized rapidly to picolinic acid. There was some hydrolysis of the intermediate but no cleavage between C-l and C-2 was observed. 

A series of Fe(salen)X complexes have been synthesized58) and their visible spectral maxima have been compared with the corresponding pyrocatechase complexes (Table 7). These synthetic complexes are clearly imperfect analogues of the enzyme complexes, since their spectral maxima are consistently blue-shifted relative to the corresponding enzyme complexes. Nevertheless, it is encouraging to note the similar ligand dependence on the position of the absorption maxima. 

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