Kinetic, spectroscopic, and mutagenesis studies of homoprotocatechuate 2,3-dioxygenase.

Abstract

Homoprotocatechuate 2,3-Dioxygenase (2,3-HPCD) from Brevibacterium fuscum catalyzes the O2-dependent extradiol ring-opening reaction of its catecholic substrate homoprotocatechuate (HPCA) to yield a α-hydroxy-δ-carboxymethyl cis-muconic semialdehyde. In this process, both O2 and HPCA are first coordinated to a mononuclear Fe(II) in the active site. The Fe(II) cofactor is postulated to act as an electronic conduit for facile electron transfer between the bound substrates, working in concert with second sphere amino acids to guide the formation of a reactive •HPCA-Fe(II)-O2•- intermediate. The work presented in this thesis focuses on developing tools for solution trapping of reaction intermediates from 2,3-HPCD for spectroscopic studies, in particular mutagenesis of key active site residues and use of a novel rapid-freeze-quench apparatus (RFQ). 2,3-HPCD is found to catalyze the oxygen activation and radical recombination reactions on the sub-millisecond time scale at 4 °C, forming the Fe(II)-alkylperoxo (or lactone) intermediate in the dead time of the RFQ apparatus. The reaction was slowed to reveal intermediates in this important phase of the catalytic cycle by mutating the postulated second sphere acid/base catalyst His200 to Asn200 (H200N). This results in formation of a •HPCA-Fe(III)-(H)peroxo intermediate that decays slowly through an Fe(II) intermediate to form ring-cleaved product. When H200N reacts with the electron deficient 4-nitrocatechol (4NC) substrate, a 4NC-Fe(III)-O2•- intermediate is formed. Both this species and the Fe(II)-alkylperoxo species described above represent the first time such species have been trapped from the solution reaction of a mononuclear non-heme iron enzyme. The 4NC-Fe(III)-O2•- intermediate decays to form an •4NC-Fe(III)-(Hydro)peroxo species and then releases 4NC quinone without ring cleavage. These studies demonstrate facile electron transfer between substrates and reveal important roles for His200 in promoting efficient O2 binding and rapid reaction between the O2 and organic substrate. Mutation of Tyr257 to Phe257 (Y257F) results in an enzyme that forms a HPCA-Fe(II)-O2 intermediate. This initial oxygenated complex decays to form a Quinone-Fe(II)-(Hydro)peroxo intermediate which slowly forms ring-cleaved product. Tyr257 hydrogen bonds to the HPCA C3-O- coordinated to the iron. This may stabilize both the substrate semiquinone state and the Fe(II)-alkylperoxo state because the HPCA C3-O- would move closer to Tyr257 in both intermediates. Consequently, in the absence of Y257, both electron transfer and radical recombination are slowed, leading to the accumulation of the observed intermediates. The six new types of trapped reaction intermediates described in this thesis reveal the range of catalytic strategies used by 2,3-HPCD. However, the very rapid reactions of the native enzyme using its natural substrate suggest that the enzyme has evolved to prevent accumulation of any of these intermediates, thereby ensuring high flux and specificity.