Purification and characterization of protocatechuate 2,3-dioxygenase from Bacillus macerans: A new extradiol catecholic dioxygenase

Protocatechuate 2,3-dioxygenase (2,3-PCD) from Bacillus macerans JJ1b has been purified to homogeneity for the first time. The enzyme catalyzes proximal extradiol ring cleavage of protocatechuate (PCA) with the attendant incorporation of both atoms of oxygen from O₂. The holoenzyme has a mass of 143 ± 7 kDa as determined by ultracentrifugation and other techniques. It is composed of four apparently identical subunits with M(r)s of 35,500, each containing one iron atom. Mossbauer spectroscopy of ⁵⁷Fe-enriched enzyme showed that the irons are indistinguishable and are high spin (S = 2) Fe²⁺ in both the uncomplexed and substrate-bound enzyme. However, the quadrupole splitting, ΔE(Q), and isomer shift, δ, of the Mossbauer spectrum changed from ΔE(Q) = 2.57 mm/s and δ = 1.29 mm/s to ΔE(Q) = 2.73 mm/s and δ = 1.19 mm/s upon PCA binding to the enzyme, showing that the iron environment is altered when substrate is present. The enzyme was also found to bind variable and substoichiometric amounts of Mn²⁺, but this metal could be removed without loss of activity or stability. The inherently electron paramagnetic resonance (EPR)-silent Fe²⁺ of the enzyme reversibly bound nitric oxide to produce an EPR-active species (g = 4.11, 3.95; S = 3/2). The specific activity of the enzyme was found to be correlated with the amount of the S = 3/2 species formed, showing that activity is dependent on Fe²⁺. Anaerobic addition of substrates to the enzyme-nitric oxide complex significantly altered the EPR spectrum, suggesting that substrates bind to or near the iron. The enzyme was inactivated by reagents that oxidize the Fe²⁺, such as H₂O₂ and K₃Fe(CN)₆; full activity was restored after reduction of the iron by ascorbate. Steady-state kinetic data were found to be consistent with an ordered bi-uni mechanism in which the organic substrate must add to 2,3-PCD before O₂. The enzyme has the broadest substrate range of any of the well-studied catecholic dioxygenases. All substrates have vicinal hydroxyl groups on the aromatic ring except 4-NH₂-3-hydroxybenzoate. This is the first substrate lacking vicinal hydroxyl groups reported for catecholic extradiol dioxygenases. 2,3-PCD is the final member of the PCA dioxygenase family to be purified. It is compared with other members of this family as well as other catecholic dioxygenases.