Ferric center

Pseudomonas sp. strain P.J. 874 grown with tyrosine carried out dioxygenation of 4-hydroxyphenylpyruvate to 2,5-dihydroxyphenylacetate accompanied by an NIH shift (Lindstedt et al. 1977). The involvement of a high-spin ferric center coordinated with tyrosine is conclusively revealed in the primary structure of the enzyme (Riietschi et al. 1992). 

When, under identical conditions, ascorbic acid was used instead of mercaptoethanol, the reaction gave products with 3°/2° carbon reactivity of 0.28-0.42, suggestive of an autoxidation process (12). Furthermore, the kinetics of the reaction are biphasic for 2-mercaptoethanol and monophasic for ascorbic acid. These kinetics are consistent with the generation of a new catalytic system by the coordination of the thiol to the ferric center(s). For either reductant, bleaching of the complex was observed within minutes in the absence of substrate. 

Kitajima et al. (21) also reported a more efficient catalyst through a serendipitous modification of complex 2. They observed that an increase in the catalytic activity (by a factor of 1.5) occurred when hexafluo-roacetyl acetone (hfacac) was used instead of acetic acid. The reaction between 2 and hfacac produced [ Fe(HBpz3)(hfacac) 20], 4 in 50-60% yield that was structurally characterized to reveal a ferric dimer with a single n-oxo bridge (21). Each iron center is in a six-coordinate N303 environment. The two octahedral units are more distorted in this coordination environment than that of 2 the FeOFe unit is bent with an angle of 169.4°. Each ferric center has one hfacac ligand bound in a bidentate mode. 

The second question is about how the the high-valent oxo intermediate forms in both enzymes. For catalase and peroxidase, the evidence indicates that hydrogen peroxide binds to the ferric center and then undergoes heterolysis at the... 

Mossbauer spectra of native RRB2 reveals two quadrupole doublets of equal intensity with 8 values of 0.5 mm s", which are typical of high-spin ferric centers and AEq values of 1.65 and 2.45 mm s" (26). These large quadrupole splittings are expected of (p,-oxo)diiron(III) complexes. The appearance of two iron centers with distinct AEq values was difficult to rationalize in the RRB2 model proposed by Reichard and co-workers... 

Two intermediates have been detected in the reaction of catechol 1,2-dioxygenase with pyrogallol and dioxygen [88]. The active site in this enzyme has been studied by NMR spectroscopy a high-spin ferric center gives rise to paramagnetically shifted resonances [89]. 

Figure 6 shows the NMR spectra of the model complexes and 1,2-CTD after coordination of catechol and phenols [68-70]. In the case of model monodentate catecholate complexes, the methyl resonance appears at 100 ppm downfield if the 0(1) oxygen is coordinated to the iron and at -30 ppm upfield if the 0(2) oxygen is coordinated. The same shifts are observed for methyl-substituted phenols. For chelated catecholates, the methyl resonance appears at 50 ppm downfield. In the case of CTD, 4-methylcatecholate species exhibit a peak at 100 ppm downfield, indicating that 4-methylcatechol coordinates to the ferric center solely through the 0(1) oxygen, as 6 rather than 7 in Fig. 5. On the other hand, no methyl resonance has been observed with 3-methylcatechol. Considering that 3,6-dimethylcatechol exhibits only one peak near at -25 ppm, the coordination through 0(2) as 8 is suggested for 3-methylcatecholatoiron...

The character of the coordinated catechols has been studied by Mossbauer spectroscopy [38]. Since the value of A is a measure of the hyperfine interaction of the iron electron with Fe, it reflects the covalency of the metal-ligand bands. The value of A/gnPn for the catechol complex is -18.9 T, that is the smallest observed for a dioxygenase complex -20.8 T for the phenol complex and -20.0 T for the thiophenol complex. The lowest values are explained by a greater delocalization of unpaired spin density away from the ferric center onto the catechol, generating a radical character of the catechol. The unpaired spin density delocalization is consistent with the paramagnetic shifts of protons of the catecholate ligand observed by NMR. 

In the case of 3,4-PCD, formation of a binary ES complex has also been noticed by the optical [71, 72] and EPR [72] spectroscopic changes. The decrease in the peak intensity of the characteristic EPR signal at g = 4.31 upon addition of PCA to 3,4-PCD has been explained by the ligand field modification of the ferric center rather than the valence change from the ferric to ferrous state [72]. This has been confirmed by further EPR studies [40, 56, 60, 73, 74]. 

---