Protocatechuate 2,3-dioxygenase (2,3-PCD) from Bacillus macerans JJlb 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 02. 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 Mrs of 35,500, each containing one iron atom. Mossbauer spectroscopy of "7Fe-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, AEQ, and isomer shift, 8, of the Mossbauer spectrum changed from AEQ = 2.57 mm/s and 8 = 1.29 mm/s to AEQ = 2.73 mm/s and 8 = 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 H202 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 02. 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.Protocatechuate (PCA) is one of a relatively small number of single-ring aromatic compounds that are found at the points of confluence of bacterial pathways for metabolism of complex aromatics (12,17,28,37). The aromatic ring of PCA is opened in reactions catalyzed by dioxygenase enzymes that result in the incorporation of both atoms of 02 into the open chain products (14,19,25,46,65). Two of these enzymes, protocatechuate 3,4-dioxygenase (3,4-PCD) (10, 21, 25, 59, 65, 70) and 4,5-PCD (5,18,19,53) were among the first of this class of enzyme to be recognized. They continue to serve as prototypical enzymes for the two major subclasses of catecholic dioxygenases termed intradiol and extradiol on the basis of the site of ring clea...

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