Mn(II)-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase (MndD) is an extradiol-cleaving catechol dioxygenase from Arthrobacter globiformis that has 82% sequence identity to and cleaves the same substrate (3,4-dihydroxyphenylacetic acid) as Fe(II)-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase (HPCD) from Brevibacterium fuscum. We have observed that MndD binds the chromophoric 4-nitrocatechol (4-NCH2) substrate as a dianion and. cleaves it extremely slowly, in contrast to the Fe(II)-dependent enzymes which bind 4-NCH2 mostly as a monoanion and cleave 4-NCH2 4-5 orders of magnitude faster. These results suggest that the monoanionic binding state of 4-NC is essential for extradiol cleavage. In order to address the differences in 4-NCH2 binding to these enzymes, we synthesized and characterized the first mononuclear monoanionic and dianionic Mn(II)-(4-NC) model complexes as well as their Fe(II)-(4-NC) analogs. The structures of [(6-Me2-bpmcn)Fe(II)(4-NCH)]+, [(6-Me3TPA)Mn(II)(DBCH)+, and [(6-Me2-bpmcn)Mn(II) (4-NCH)]+ reveal that the monoanionic catecholate is bound in an asymmetric fashion (Δrmetal-O(catecholate) = 0.25-0.35 Å), as found in the crystal structures of the E S complexes of extradiol-cleaving catechol dioxygenases. Acid-base titrations of [(L)M(II)(4-NCH)]+ complexes in aprotic solvents show that the pKa of the second catecholate proton of 4-NCH bound to the metal center is half a pKa unit higher for the Mn(II) complexes than for the Fe(II) complexes. These results are in line with the Lewis acidities of the two divalent metal ions but are the opposite of the trend observed for 4-NCH2 binding to the Mn(II)- and Fe(II)-catechol dioxygenases. These results suggest that the MndD active site decreases the second pKa of the bound 4-NCH2 relative to the HPCD active site.
- Catecholate model complexes
- Extradiol-cleaving catechol dioxygenase
- Manganese(II)-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase