Valley Michael P, Brown C Kent, Burk David L, Vetting Matthew W, Ohlendorf Douglas H, Lipscomb John D
Department of Biochemistry, Molecular Biology, and Biophysics and Center for Metals in Biocatalysis, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Biochemistry. 2005 Aug 23;44(33):11024-39. doi: 10.1021/bi050902i.
The active site Fe(III) of protocatechuate 3,4-dioxygenase (3,4-PCD) from Pseudomonas putida is ligated axially by Tyr447 and His462 and equatorially by Tyr408, His460, and OH(-). Tyr447 and OH(-) are displaced as protocatechuate (3,4-dihydroxybenzoate, PCA) chelates the iron and appear to serve as in situ bases to promote this process. The role(s) of Tyr408 is (are) explored here using mutant enzymes that exhibit less than 0.1% wild-type activity. The X-ray crystal structures of the mutants and their PCA complexes show that the new shorter residues in the 408 position cannot ligate the iron and instead interact with the iron through solvents. Moreover, PCA binds as a monodentate rather than a bidentate ligand, and Tyr447 fails to dissociate. Although the new residues at position 408 do not directly bind to the iron, large changes in the spectroscopic and catalytic properties are noted among the mutant enzymes. Resonance Raman features show that the Fe-O bond of the monodentate 4-hydroxybenzoate (4HB) inhibitor complex is significantly stronger in the mutants than in wild-type 3,4-PCD. Transient kinetic studies show that PCA and 4HB bind to 3,4-PCD in a fast, reversible step followed by a step in which coordination to the metal occurs; the latter process is at least 50-fold slower in the mutant enzymes. It is proposed that, in wild-type 3,4-PCD, the Lewis base strength of Tyr408 lowers the Lewis acidity of the iron to foster the rapid exchange of anionic ligands during the catalytic cycle. Accordingly, the increase in Lewis acidity of the iron caused by substitution of this residue by solvent tends to make the iron substitution inert. Tyr447 cannot be released to allow formation of the usual dianionic PCA chelate complex with the active site iron, and the rate of electrophilic attack by O(2) becomes rate limiting overall. The structures of the PCA complexes of these mutant enzymes show that hydrogen-bonding interactions between the new solvent ligand and the new second-sphere residue in position 408 allow this residue to significantly influence the spectroscopic and kinetic properties of the enzymes.
恶臭假单胞菌原儿茶酸3,4 -双加氧酶（3,4 - PCD）的活性位点铁（III）轴向由Tyr447和His462配位，赤道面由Tyr408、His460和OH(-)配位。当原儿茶酸（3,4 -二羟基苯甲酸，PCA）螯合铁时，Tyr447和OH(-)会发生位移，它们似乎作为原位碱来促进这一过程。本文利用活性低于野生型0.1%的突变酶探究了Tyr408的作用。突变体及其PCA复合物的X射线晶体结构表明，408位上新的较短残基无法与铁配位，而是通过溶剂与铁相互作用。此外，PCA以单齿而非双齿配体的形式结合，并且Tyr447不会解离。尽管408位上的新残基不直接与铁结合，但突变酶的光谱和催化性质发生了很大变化。共振拉曼特征表明，单齿4 -羟基苯甲酸（4HB）抑制剂复合物的Fe - O键在突变体中比在野生型3,4 - PCD中显著更强。瞬态动力学研究表明，PCA和4HB以快速、可逆的步骤与3,4 - PCD结合，随后是与金属配位的步骤；在突变酶中，后一过程至少慢50倍。有人提出，在野生型3,4 - PCD中，Tyr408的路易斯碱强度降低了铁原子的路易斯酸度，以促进催化循环中阴离子配体的快速交换。因此，该残基被溶剂取代导致铁原子路易斯酸度增加，这往往使铁原子的取代变得惰性。Tyr447无法释放，从而无法与活性位点的铁形成通常的双阴离子PCA螯合复合物，并且O(2)的亲电攻击速率总体上成为限速步骤。这些突变酶的PCA复合物结构表明，新的溶剂配体与408位上新的第二配位层残基之间的氢键相互作用使该残基能够显著影响酶的光谱和动力学性质。
