CRYSTAL-STRUCTURE AND RESONANCE RAMAN STUDIES OF PROTOCATECHUATE 3,4-DIOXYGENASE COMPLEXED WITH 3,4-DIHYDROXYPHENYLACETATE

The crystal structure of the anaerobic complex of Pseudomonas putida p rotocatechuate 3,4-dioxygenase (3,4-PCD) bound with the alternative su bstrate, 3,4-dihydroxyphenylacetate (HPCA), is reported at 2.4 Angstro m resolution and refined to an R factor of 0.17. Formation of the acti ve site Fe(III).HPCA chelated complex causes the endogenous axial tyro sinate, Tyr447 (147 beta), to dissociate from the iron and rotate into an alternative orientation analogous to that previously observed in t he anaerobic 3,4-PCD.3,4-dihydroxybenzoate complex (3,4-PCD.PCA) [Orvi lle, A. M., Lipscomb, J. D., & Ohlendorf, D. H. (1997) Biochemistry 36 , 10052-10066]. Two orientations of the aromatic ring of HPCA related by an approximate 180 degrees rotation within the active site are cons istent with the electron density. Resonance Raman (rR) spectroscopic d ata from Brevibacterium fuscum 3,4-PCD.HPCA complex in solution reveal s low frequency rR vibrational bands between 500 and 650 cm(-1) as wel l as a band at similar to 1320 cm(-1) which are diagnostic of a HPCA.F e(III) chelate complex. O-18 labeling of HPCA at either the C4 or C3 h ydroxyl group unambiguously establishes the vibrational coupling modes associated with the five-membered chelate ring system. Analysis of th ese data suggests that the Fe(III)-HPCA(O4) bond is shorter than the F e(III)-HPCA(O3) bond. This consequently favors the model for the cryst al structure in which the C3 phenolic function occupies the Fe3+ ligan d site opposite the endogenous ligand Tyr408(O eta) (108 beta). This i s essentially the same binding orientation as proposed for PCA in the crystal structure of the anaerobic 3,4-PCD.PCA complex based solely on direct modeling of the 2\F-o\-\F-c\ electron density and suggests tha t this is the conformation required for catalysis.