Fundamentally Divergent Strategies for Oxygen Activation by Fe2+ and Fe3+ Catecholic Dioxygenases

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We propose that the redox state of the active site iron of ring-cleaving catecholic dioxygenases determines both the site of ring cleavage and the mechanism of O2 activation. Spectroscopic and crystallographic studies show that substrates bind to the Fe2+ of extradiol dioxygenases as asymmetric chelates in which only one hydroxyl becomes ionized. Charge transfer onto the iron increases the affinity for small molecules such as NO and O2, which bind in another metal coordination site. Oxygen is activated by accepting electron density from the catechol via the iron, promoting nucleophilic attack of the resulting superoxide on the now electron-deficient catechol. In contrast, studies of intradiol Fe3+ ring-cleaving dioxygenases show that catechols bind as dianions. This provides a site for electrophilic attack by O2 at a hydroxyl-bearing carbon, which has been shown via model studies to lead to intradiol ring opening. Both dioxygenase classes shift from five-to six-coordinate iron sites during catalysis to allow oxygen binding. In the Fe2+ enzymes, this site is occupied before attack on the catechol, while in the Fe2+ case, it follows initial attack. In both cases, the expansion of the iron coordination allows the second oxygen required for dioxygenase stoichiometry to be retained for incorporation into the product during the final step.