In vivo self-hydroxylation of an iron-substituted manganese-dependent extradiol cleaving catechol dioxygenase.

The homoprotocatechuate 2,3-dioxygenase from Arthrobacter globiformis (MndD) catalyzes the oxidative ring cleavage reaction of its catechol substrate in an extradiol fashion. Although this reactivity is more typically associated with non-heme iron enzymes, MndD exhibits an unusual specificity for manganese(II). MndD is structurally very similar to the iron(II)-dependent homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum (HPCD), and we have previously shown that both MndD and HPCD are equally active towards substrate turnover with either iron(II) or manganese(II) (Emerson et al. in Proc. Natl. Acad. Sci. USA 105:7347-7352, 2008). However, expression of MndD in Escherichia coli under aerobic conditions in the presence of excess iron results in the isolation of inactive blue-green iron-substituted MndD. Spectroscopic studies indicate that this form of iron-substituted MndD contains an iron(III) center with a bound catecholate, which is presumably generated by in vivo self-hydroxylation of a second-sphere tyrosine residue, as found for other self-hydroxylated non-heme iron oxygenases. The absence of this modification in either the native manganese-containing MndD or iron-containing HPCD suggests that the metal center of iron-substituted MndD is able to bind and activate O(2) in the absence of its substrate, employing a high-valence oxoiron oxidant to carry out the observed self-hydroxylation chemistry. These results demonstrate that the active site metal in MndD can support two dramatically different O(2) activation pathways, further highlighting the catalytic flexibility of enzymes containing a 2-His-1-carboxylate facial triad metal binding motif.