Metal ion requirements and regulation by redox

The best characterized members of the class of metallophosphatases are the purple acid phosphatases. Besides the Fe +-Zn + metal cluster in the kidney bean enzyme. 

PAPase FGLIGDL.GQSFDSNTTLSHYELSPKKGQT.VLFVGDLSYADR.YPNHDNVRWDTWGRFTERSVAYQPWIW.TAGHHEIEFAPE 

The oxidation state of the Fe ion of calcineurin is still the subject of some debate. In a recent study, Klee and colleagues proposed that the functional redox state was the reduced Fe -Zn form [48]. This proposal was based on data which showed that a portion of calcineurin activity in crude brain extract was susceptible to inactivation following incubation with Ca + and calmodulin and that inactivation could be prevented by the addition of superoxide dismutase. Inactivation was more pronounced imder aerobic than in anaerobic conditions. Additionally, incubation with and calmodulin in the presence of Snm ascorbate protected calcineurin from inactivation while reactivation could be achieved by addition of 0.5 mM Fe(NH4)2(S04)2 in the presence of 5mM dithiothreitol and 5mM ascorbate. These data were taken to propose that the active redox state was Fe +-Zn and that inactivation was due to oxidation of the cluster to the Fe +-Zn + state. Direct confirmation of the oxidation states was not provided. 

A similar conclusion was alluded to in studies which found that activation of PPl could be achieved in the presence of FeCh and ZnCl2 [49] or FeS04 and ascorbic add [50]. In these studies, the exact nature of the metal cluster and ultimate oxidation state of the Fe ion was not determined. Since incubations were carried out with an excess of both FeCl2 and ZaCh, a heterogeneous mixture of species was possible (e.g. Fe-Fe and Fe-Zn clusters). Furthermore, aerobic conditions will result in significant oxidation of Fe + to Fe making it difficult to assign Fe oxidation states without definitive spectroscopic analysis. 

The active form of the cluster being Fe +-Zn + is inconsistent with data obtained by Yu et al. which followed the phosphatase activity of either the Fe-Zn or Fe-Fe forms of calcineurin imder different redox states [14, 35]. In those studies, the pure enzyme was utilized and the redox state of the cluster could be followed by EPR 

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