The role of histidine 200 in MndD, the Mn(II)-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase from Arthrobacter globiformis CM-2, a site-directed mutagenesis study

Joseph P. Emerson, Michelle L. Wagner, Mark F. Reynolds, Lawrence Que, Michael J. Sadowsky, Lawrence P. Wackett

Research output: Contribution to journalArticlepeer-review

27 Scopus citations

Abstract

The manganese-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase (MndD) from Arthrobacter globiformis CM-2 is an extradiol-cleaving catechol dioxygenase that catalyzes aromatic ring cleavage of 3,4-dihydroxyphenylacetate (DHPA). Based on the recent crystal structure of the MndD-DHPA complex, a series of site-directed mutations were made at a conserved second-sphere residue, histidine 200, to gain insight into and clarify the role this residue plays in the Mn(II)-dependent catalytic mechanism. In this study, we report the activities and spectroscopic data of these H200 variants and their DHPA and 4-nitrocatechol (4-NC) complexes. The data collected from wild-type and mutant MndDs are consistent with a role for H200 interacting with a manganese-bound dioxygen moiety and are inconsistent with other previously proposed roles involving proton transfer. Spectroscopic observations, including unique low-field EPR signals found when DHPA and 4-NC are bound to the Mn(II) center of MndD, are discussed and their relationship to dioxygen activation catalyzed in MndD is explored.

Original languageEnglish (US)
Pages (from-to)751-760
Number of pages10
JournalJournal of Biological Inorganic Chemistry
Volume10
Issue number7
DOIs
StatePublished - Nov 2005

Keywords

  • Dioxygen activation
  • Electron paramagnetic resonance
  • Extradiol-cleaving catechol dioxygenase
  • Mononuclear nonheme manganese active center

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