An EXAFS study of the interaction of substrate with the ferric active site of protocatechuate 3,4-dioxygenase

Abstract

X-ray crystallographic studies of the intradiol cleaving protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa have shown that the enzyme has a trigonal bipyramidal ferric active site with two histidines, two tyrosines, and a solvent molecule as ligands [Ohlendorf, D.H., Lipscomb, J.D., & Weber, P.C. (1988) Nature 336, 403-405]. Fe K-edge EXAFS studies of the spectroscopically similar protocatechuate 3,4-dioxygenase from Brevibacterium fuscum are consistent with a pentacoordinate geometry of the iron active site with 3 O/N ligands at 1.90 A and 2 O/N ligands at 2.08 A. The 2.08-A bonds are assigned to the two histidines, while the 1.90-A bonds are associated with the two tyrosines and the coordinated solvent. The short Fe-O distance for the solvent suggests that it coordinates as hydroxide rather than water. When the inhibitor terephthalate is bound to the enzyme, the XANES data indicate that the ferric site becomes 6-coordinate and the EXAFS data show a beat pattern which can only be simulated with an additional Fe-O/N interaction at 2.46 A. Together, the data suggest that the oxygens of the carboxylate group in terephthalate displace the hydroxide and chelate to the ferric site but in an asymmetric fashion. In contrast, protocatechuate 3,4-dioxygenase remains 5-coordinate upon the addition of the slow substrate homoprotocatechuic acid (HPCA). Previous EPR data have indicated that HPCA forms an iron chelate via the two hydroxyl functions.(ABSTRACT TRUNCATED AT 250 WORDS)

Substrate activation for O 2 reactions by oxidized metal centers in biology

“…These enzymes catalyze the cleavage of molecular oxygen accompanied by insertion of both oxygen atoms between the vicinal hydroxyl groups of the catecholic substrate, resulting in ring cleavage to yield muconic acid derivatives (3-5, 18, 19). The resting state of intradiol dioxygenases contains a highspin ferric center in a distorted trigonal bipyramidal geometry, with Tyr and His as the axial ligands and Tyr, His, and a hydroxide ligand defining the equatorial plane (20)(21)(22). Upon anaerobic substrate binding, the active site shifts to a square pyramidal geometry in which the axial Tyr and equatorial OH Ϫ are displaced by the substrate, which binds bidentate in its doubly deprotonated form (23,24).…”

Absence of Mn-Centered Oxidation in the S2→ S3Transition: Implications for the Mechanism of Photosynthetic Water Oxidation

“…This formally dipole-forbidden transition contains information about the oxidation state and geometry 42 of Mn in the OEC. Analysis of these features in mononuclear metalloproteins with Fe at the active site 47,[82][83][84][85][86][87] and Fe model complexes 88,89 has proven useful in determining the coordination number and oxidation state of Fe. The pre-edge region in the inset of Figure 2A shows changes with S-state; the most prominent change occurs between the S 0 and S 1 states.…”

Ring-Cleaving Dioxygenases with a Cupin Fold

“…Within this family, protocatechuate 3,4-dioxygenase has been studied most extensively. Crystal structures and X-ray absorption data revealed a ferric center in a trigonal bipyramidal geometry, with a hydroxide ligand completing the coordination sphere (31,93,94,123). The substrate binds as a dianion, donating both its protons to the displaced hydroxide and tyrosyl ligands (36,60,96).…”