title: Thermodynamics of substrate binding to the metal site in homoprotocatechuate 2,3-dioxygenase: Using ITC under anaerobic conditions to study enzyme-substrate interactions.
abstract: BACKGROUND
  Extradiol dioxygenases are a family of nonheme iron (and sometimes manganese) enzymes that catalyze an O2-dependent ring-opening reaction in a biodegradation pathway of aromatic compounds. Here we characterize the thermodynamics of two substrates binding in homoprotocatechuate 2,3-dioxygenase (HPCD) prior to the O2 activation step.
  
  
  METHODS
  This study uses microcalorimetry under an inert atmosphere to measure thermodynamic parameters associated with catechol binding to nonheme metal centers in HPCD. Several stopped-flow rapid mixing experiments were used to support the calorimetry experiments.
  
  
  RESULTS
  The equilibria constant for 4-nitrocatechol and homoprotocatechuate binding to the iron(II) and manganese(II) forms of HPCD range from 2×10(4) to 1×10(6), suggesting there are distinctive differences in how the enzyme-substrate complexes are stabilized. Further experiments in multiple buffers allowed us to correct the experimental ΔH for substrate ionization and to fully derive the pH and buffer independent thermodynamic parameters for substrate binding to HPCD. Fewer protons are released from the iron(II) dependent processes than their manganese(II) counterparts.
  
  
  CONCLUSIONS
  Condition independent thermodynamic parameters for 4-nitrocatechol and homoprotocatechuate binding to HPCD are highly consistent with each other, suggesting these enzyme-substrate complexes are more similar than once thought, and the ionization state of metal coordinated waters may be playing a role in tuning redox potential and in governing reactivity.
  
  
  CONCLUSIONS
  Substrate binding to HPCD is a complex set of equilibria that includes ionization of substrate and water release, yet it is also the key step in O2 activation.
authors: Department of Chemistry, Mississippi State University, 310 President's Circle, Mississippi State, MS 39762 USA.
authors: Kate L
authors: KL
authors: Henderson
authors: Department of Chemistry, Mississippi State University, 310 President's Circle, Mississippi State, MS 39762 USA.
authors: Danielle H
authors: DH
authors: Francis
authors: Department of Chemistry, Mississippi State University, 310 President's Circle, Mississippi State, MS 39762 USA. Electronic address: elewis@ichemsitry.msstate.edu.
authors: Edwin A
authors: EA
authors: Lewis
authors: Department of Chemistry, Mississippi State University, 310 President's Circle, Mississippi State, MS 39762 USA. Electronic address: jemerson@chemistry.msstate.edu.
authors: Joseph P
authors: JP
authors: Emerson
mesh_terms: D015102:3,4-Dihydroxyphenylacetic Acid / Q000737:chemistry*; D000693:Anaerobiosis; D002151:Calorimetry; D020134:Catalytic Domain; D002396:Catechols / Q000737:chemistry*; D002413:Cations, Divalent; D049308:Dioxygenases / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry*; D007700:Kinetics; D008024:Ligands; D008345:Manganese / Q000737:chemistry*; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000737:chemistry; D012996:Solutions; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D002396:Catechols; D002413:Cations, Divalent; D008024:Ligands; D011994:Recombinant Proteins; D012996:Solutions; D015102:3,4-Dihydroxyphenylacetic Acid; D008345:Manganese; D007501:Iron; D049308:Dioxygenases; C020055:3,4-dihydroxyphenylacetate 2,3-dioxygenase; C001833:4-nitrocatechol
keywords: 4-nitrocatechol; Extradiol dioxygenase; Homoprotocatechuate; Manganese(II); Nonheme iron(II)