Oxy Intermediates of Homoprotocatechuate 2,3-Dioxygenase: Facile Electron Transfer between Substrates

• Published in: Biochemistry (Easton) 2011-11, Vol.50 (47), p.10262-10274
• Main Authors: Mbughuni, Michael M, Chakrabarti, Mrinmoy, Hayden, Joshua A, Meier, Katlyn K, Dalluge, Joseph J, Hendrich, Michael P, Münck, Eckard, Lipscomb, John D
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites; Brevibacterium flavum - chemistry; Brevibacterium flavum - enzymology; Brevibacterium flavum - genetics; Dioxygenases - chemistry; Dioxygenases - genetics; Dioxygenases - metabolism; Electron Transport; Ferrous Compounds - chemistry; Ferrous Compounds - metabolism; Kinetics; Models, Molecular; Oxygen - chemistry; Oxygen - metabolism; Protein Binding
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi201436n

Substrates homoprotocatechuate (HPCA) and O2 bind to the FeII of homoprotocatechuate 2,3-dioxygenase (FeHPCD) in adjacent coordination sites. Transfer of an electron(s) from HPCA to O2 via the iron is proposed to activate the substrates for reaction with each other to initiate aromatic ring cleavage. Here, rapid-freeze-quench methods are used to trap and spectroscopically characterize intermediates in the reactions of the HPCA complexes of FeHPCD and the variant His200Asn (FeHPCD–HPCA and H200N–HPCA, respectively) with O2. A blue intermediate forms within 20 ms of mixing of O2 with H200N–HPCA (H200NInt1 HPCA). Parallel mode electron paramagnetic resonance and Mössbauer spectroscopies show that this intermediate contains high-spin FeIII (S = 5/2) antiferromagnetically coupled to a radical (S R = 1/2) to yield an S = 2 state. Together, optical and Mössbauer spectra of the intermediate support assignment of the radical as an HPCA semiquinone, implying that oxygen is bound as a (hydro)peroxo ligand. H200NInt1 HPCA decays over the next 2 s, possibly through an FeII intermediate (H200NInt2 HPCA), to yield the product and the resting FeII enzyme. Reaction of FeHPCD–HPCA with O2 results in rapid formation of a colorless FeII intermediate (FeHPCDInt1 HPCA). This species decays within 1 s to yield the product and the resting enzyme. The absence of a chromophore from a semiquinone or evidence of a spin-coupled species in FeHPCDInt1 HPCA suggests it is an intermediate occurring after O2 activation and attack. The similar Mössbauer parameters for FeHPCDInt1 HPCA and H200NInt2 HPCA suggest these are similar intermediates. The results show that transfer of an electron from the substrate to the O2 via the iron does occur, leading to aromatic ring cleavage.