Journal
BIOCHEMISTRY
Volume 51, Issue 44, Pages 8755-8763Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi301115c
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Funding
- Biological Sciences Research Council [BB/H001905/1]
- National Institutes of Health [GM 24689]
- Biotechnology and Biological Sciences Research Council [BB/H001905/1] Funding Source: researchfish
- BBSRC [BB/H001905/1] Funding Source: UKRI
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Homoprotocatechuate 2,3-dioxygenase (FeHPCD) utilizes an active site Fe-II to activate O-2 in a reaction cycle that ultimately results in aromatic ring cleavage. Here, the roles of the conserved active site residue Tyr257 are investigated by solving the X-ray crystal structures of the Tyr257-to-Phe variant (Y257F) in complex with the substrate homoprotocatechuate (HPCA) and the alternative substrate 4-nitrocatechol (4NC). These are compared with structures of the analogous wild type enzyme complexes. In addition, the oxy intermediate of the reaction cycle of Y257F-4NC + O-2 is formed in crystallo and structurally characterized. It is shown that both substrates adopt a previously unrecognized, slightly nonplanar, strained conformation affecting the geometries of all aromatic ring carbons when bound in the FeHPCD active site. This global deviation from planarity is not observed for the Y257F variant. In the Y2S7F-4NC-oxy complex, the O-2 is bound side on to the Fe-II, while the 4NC is chelated in two adjacent sites. The ring of the 4NC in this complex is planar, in contrast to the equivalent FeHPCD intermediate, which exhibits substantial local distortion of the substrate hydroxyl moiety (C2-O-) that is hydrogen bonded to Tyr257. We propose that Tyr257 incluces the global and local distortions of the substrate ring in two different ways. First, van der Waals conflict between the Tyr257-OH substitueni and the substrate C2 carbon is relieved by adopting the globally strained structure. Second, Tyr257 stabilizes the localized out position of the C2-O- by forming a stronger hydrogen bond as the distortion increases. Both types of distortions favor transfer of one electron out of the substrate to form a reactive semiquinone radical. Then, the localized distortion at substrate C2 promotes formation of the key allcylperoxo intermediate of the cycle resulting from oxygen attack on the activated substrate at C2, which becomes sp(3) hybridized. The inability of Y257F to promote the distorted substrate structure may explain the observed 100-fold decrease in the rates of the O-2 activation and insertion steps of the reaction.
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