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Crystal structures of substrate & substrate analog complexes of protocatechuate 3,4-dioxygenase yield mechanistic insights
NON-HEME IRON PROTEINS 367
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CRYSTAL STRUCTURES OF SUBSTRATE & SUBSTRATE ANALOG COMPLEXES OF PROTOCATECHUATE 3,4-DIOXYGENASE YIELD MECHANISTIC INSIGHTS. Allen M. Orville, Natesan Elango, Douglas H. Ohlendorf, & John D. Lipscomb Dept. of Biochemistry, ivied. School, Univ. of Minnesota, Minneapolis, MN 55455 USA Protocatechuate 3,4-dioxygenase (3,4-PCD) utilizes mononuclear Fe ~ to catalyze the cleavage of 02 and the intradiol ring fission of 3,4-(OH)2-benzoate (PCA) without undergoing an observable redox change. Crystallographic analysis to 2.1 A of 3,4-PCD as isolated reveals 1 exogenous solvent and 4 endogenous (2 Tyr & 2 His) ligands with distorted trigonal bipyramidal coordination geometry of the Fe ~ [ 1]. Docking PCA into the active site of the native structure suggests that only its C4-OH approaches the Fe3+. However, previous spectroscopic studies [2] have suggested a 5- coordinate, chelated substrate complex with coincident conformational change at the metal cofactor. The proposed reaction mechanism therefore, includes two salient features: 0 PCA chelation of the Fe3+ that displaces an endogenous iron ligand and the exogenous solvent; i0 PCA activation for electrophilic attack by 02. Here we present novel results from [ I an X-ray crystallographic analysis of 13 binary and/or ternary ligand complexes of 3,4-PCD intended to mimic the enzyme in conformations likely encountered during catalysis. Sc~..=v3,4-PCD rnonohydrc,~:y aromatic inhibitor complexes reveal several possible organic ligand binding orientations within the active site that include a) no Fe3+ ligation, b) monodentate, five coordinate Fe3+ binding, and c) mondentate, six coordinate Fe 3+ complexes. Two anaerobic substrate complexes and two complexes formed with N-oxide analogs of PCA (Fig. 1), yield 5- or 6-coordinate bidentate Fe3+ ligation in which the axial Tyr447 is displaced from the Fe3+. The structures highlight residues that may influence PCA activation and reaction cycle intermediate stabilization. Ternary N-oxide-3,4-PCD.CN- complexes yield octahedral ligand spheres in which the Fe3+ bound CN- protrudes into a cavity adjacent to the N-oxides suggesting the locus of O 2 attack on PCA. The structures indicate that endogenous Fe3+ligand displacement with subsequent expansion to an octahedral Fe3+ coordination sphere may be critical for 3,4-PCD function. Moreover, the role of dynamic ligand exchange and coordination number flexibility illustrated in this study may provide insights into metalloenzyme catalytic function in general. 1. D.H.Ohlendorf, A.M. Orville, & J.D. Lipscomb, J.Mol. Biol. 244, 586 (1994). 2. J.D. Lipscomb, & A.M. Orville, Metallons in Biol. Systems, 28, 243 (1992).
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CRYSTAL STRUCTURES OF SUBSTRATE & SUBSTRATE ANALOG COMPLEXES OF PROTOCATECHUATE 3,4-DIOXYGENASE YIELD MECHANISTIC INSIGHTS. Allen M. Orville, Natesan Elango, Douglas H. Ohlendorf, & John D. Lipscomb Dept. of Biochemistry, ivied. School, Univ. of Minnesota, Minneapolis, MN 55455 USA Protocatechuate 3,4-dioxygenase (3,4-PCD) utilizes mononuclear Fe ~ to catalyze the cleavage of 02 and the intradiol ring fission of 3,4-(OH)2-benzoate (PCA) without undergoing an observable redox change. Crystallographic analysis to 2.1 A of 3,4-PCD as isolated reveals 1 exogenous solvent and 4 endogenous (2 Tyr & 2 His) ligands with distorted trigonal bipyramidal coordination geometry of the Fe ~ [ 1]. Docking PCA into the active site of the native structure suggests that only its C4-OH approaches the Fe3+. However, previous spectroscopic studies [2] have suggested a 5- coordinate, chelated substrate complex with coincident conformational change at the metal cofactor. The proposed reaction mechanism therefore, includes two salient features: 0 PCA chelation of the Fe3+ that displaces an endogenous iron ligand and the exogenous solvent; i0 PCA activation for electrophilic attack by 02. Here we present novel results from [ I an X-ray crystallographic analysis of 13 binary and/or ternary ligand complexes of 3,4-PCD intended to mimic the enzyme in conformations likely encountered during catalysis. Sc~..=v3,4-PCD rnonohydrc,~:y aromatic inhibitor complexes reveal several possible organic ligand binding orientations within the active site that include a) no Fe3+ ligation, b) monodentate, five coordinate Fe3+ binding, and c) mondentate, six coordinate Fe 3+ complexes. Two anaerobic substrate complexes and two complexes formed with N-oxide analogs of PCA (Fig. 1), yield 5- or 6-coordinate bidentate Fe3+ ligation in which the axial Tyr447 is displaced from the Fe3+. The structures highlight residues that may influence PCA activation and reaction cycle intermediate stabilization. Ternary N-oxide-3,4-PCD.CN- complexes yield octahedral ligand spheres in which the Fe3+ bound CN- protrudes into a cavity adjacent to the N-oxides suggesting the locus of O 2 attack on PCA. The structures indicate that endogenous Fe3+ligand displacement with subsequent expansion to an octahedral Fe3+ coordination sphere may be critical for 3,4-PCD function. Moreover, the role of dynamic ligand exchange and coordination number flexibility illustrated in this study may provide insights into metalloenzyme catalytic function in general. 1. D.H.Ohlendorf, A.M. Orville, & J.D. Lipscomb, J.Mol. Biol. 244, 586 (1994). 2. J.D. Lipscomb, & A.M. Orville, Metallons in Biol. Systems, 28, 243 (1992).