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Structural basis for the role of tyrosine 257 of homoprotocatechuate 2,3-dioxygenase in substrate and oxygen activation

Authors :
Elena G. Kovaleva
John D. Lipscomb
Source :
Biochemistry. 51(44)
Publication Year :
2012

Abstract

Homoprotocatechuate 2,3-dioxygenase (FeHPCD) utilizes an active site FeII to activate O2 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 + O2 is formed in crystallo and structurally characterized. It is shown that both substrates adopt a previously unrecognized, slightly non-planar, 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 Y257F-4NC-oxy complex, the O2 is bound side-on to the FeII, 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 induces the global and local distortions of the substrate ring in two different ways. First, van der Waals conflict between the Tyr257-OH substituent and the substrate C2 carbon is relieved by adopting the globally strained structure. Second, Tyr257 stabilizes the localized out-of-plane position of the C2-O− by forming a stronger hydrogen bond as the distortion increases. Both types of distortion 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 alkylperoxo intermediate of the cycle resulting from oxygen attack on the activated substrate at C2, which becomes sp3 hybridized. The inability of Y257F to promote the distorted substrate structure may explain the observed 100-fold decrease in the rates of the O2 activation and insertion steps of the reaction. Homoprotocatechuate 2,3-dioxygenase (FeHPCD) from Brevibacterium fuscum catalyzes the fission of the O-O bond of O2 with incorporation of both atoms into the aromatic substrate (HPCA) resulting in ring cleavage as shown in Scheme 1.1–5 The mechanism shown in Scheme 1 begins with O2 and HPCA binding in adjacent coordination sites of the active site FeII.4,6–10 Electron transfer from HPCA to the O2 via the iron would give both substrates radical character, allowing facile recombination to form an alkylperoxo intermediate from which O-O bond fission and aromatic ring cleavage could ensue. Both the initial intermediate, in which each substrate has radical character, and the alkylperoxo intermediate resulting from oxygen attack have been structurally characterized by X-ray crystallography after a crystal of the enzyme soaked with the slow substrate 4-nitrocatechol (4NC) was exposed to low concentrations of O2. 3 While the chemical nature of the alkylperoxo species is apparent from the structure, the diradical character of the initial intermediate was surmised from the pronounced lack of planarity observed for the aromatic substrate. It was proposed that the non-planar ring results from a localized semiquinone radical (SQ•) on the ring C2 (See Scheme 1 for numbering system used here) where the attack by oxygen (at the level of O2•−) will occur. Accordingly, in the alkylperoxo intermediate, this carbon in observed to be fully sp3 hybridized and 4 coordinate. Scheme 1 Proposed reaction mechanism for extradiol dioxygenases. In the case of FeHPCD, R is –CH2COO− (optimal substrate HPCA) or –NO2 (alternative substrate 4NC), B(H) is His200. The ring carbon numbering system shown is adopted for both ... The structural studies from our laboratory and others have shown that there are several second sphere active site residues that may contribute to catalysis (e.g. Figure 1).3,5,9–11 Extensive investigations of an active site His (His200 in the case of FeHPCD) have shown that it plays many roles including stabilization of the Fe-bound O2 via hydrogen bonding and charge interaction, acid-base catalysis of the alkylperoxo intermediate formation and subsequent oxygen insertion chemistry, and possibly, stabilization of the bound oxygen in the side-on orientation properly aligned to attack the aromatic substrate.3,12–15 Intermediates trapped from solution reactions of FeHPCD and its variants at His200 show that iron converts transiently to the FeIII state in the absence of H200, but it is always observed in the FeII state in its presence. 13,14 Thus, when His200 is present, one or two electrons appear to pass from the aromatic substrate to the oxygen with either no change in iron oxidation state or a transient change that persists for much less than the freezing time for rapid freeze quench (RFQ) experiments (~10 ms).14 This suggests that there is a considerable driving force for electron transfer that is not apparent from the redox potentials of the isolated aromatic substrate and O2. Figure 1 Active site environment of FeHPCD in complex with the optimal substrate HPCA (PDB 4GHG, subunit C). Atom color code: gray, carbon (enzyme); yellow, carbon (substrate); blue, nitrogen; red, oxygen; bronze, iron. Red dashed lines show hydrogen bonds (A). ... Another second sphere amino acid, Tyr257, is hydrogen bonded to the deprotonated C2-O− of bound HPCA (Figure 1). The orientation of Tyr257 is such that this interaction might stabilize the non-planar structure of the substrate in the reaction cycle intermediate. In the accompanying report,16 it is shown that mutation of Tyr257 to Phe (Y257F) results in a significant decrease in the rate constants for many steps with reduction approaching 100-fold in the oxygen binding and insertion steps despite the presence of the acid-base catalyst His200. The results suggest that the putative HPCA SQ•-FeII-O2•− is either not formed or is unreactive, leading to formation of an observed HPCA quinone-FeII-(H)peroxo species that then slowly yields the correct ring cleaved product. Here, structural studies of the Y257F variant, its complexes with HPCA and 4NC, and the intermediate that results from exposure of the Y257F-4NC complex to O2 are reported. The results give insight into the structural basis for the observed decreases in rate constants and the change in the chemical nature of the reactive intermediate as well as the strategy used by aromatic dioxygenases to simultaneously maintain high rates and high fidelity in ring cleaving reactions.

Details

ISSN :
15204995
Volume :
51
Issue :
44
Database :
OpenAIRE
Journal :
Biochemistry
Accession number :
edsair.doi.dedup.....84fa720371661f9d9761aa8591745f8c