TY - JOUR
T1 - Trapping and spectroscopic characterization of an FeIII-superoxo intermediate from a nonheme mononuclear iron-containing enzyme
AU - Mbughuni, Michael M.
AU - Chakrabarti, Mrinmoy
AU - Hayden, Joshua A.
AU - Bominaar, Emile L.
AU - Hendrich, Michael P.
AU - Münck, Eckard
AU - Lipscomb, John D.
PY - 2010/9/28
Y1 - 2010/9/28
N2 - FeIII-O2•- intermediates are well known in heme enzymes, but none have been characterized in the nonheme mononuclear FeII enzyme family. Many steps in the O2 activation and reaction cycle of FeII-containing homoprotocatechuate 2,3-dioxygenase are made detectable by using the alternative substrate 4-nitrocatechol (4NC) and mutation of the active site His200 to Asn (H200N). Here, the first intermediate (Int-1) observed after adding O2 to the H200N-4NC complex is trapped and characterized using EPR and Mössbauer (MB) spectroscopies. Int-1 is a high-spin (S1 = 5/2) FeIII antiferromagnetically (AF) coupled to an S2 = 1/2 radical (J ≈ 6 cm-1 in H = JS1•S2). It exhibits parallel-mode EPR signals at g = 8.17 from the S = 2 multiplet, and g = 8.8 and 11.6 from the S = 3 multiplet. These signals are broadened significantly by 17O2 hyperfine interactions (A17O ≈ 180 MHz). Thus, Int-1 is an AF-coupled FeIII-O2 •- species. The experimental observations are supported by density functional theory calculations that show nearly complete transfer of spin density to the bound O2. Int-1 decays to form a second intermediate (Int-2). MB spectra show that it is also an AF-coupled Fe III-radical complex. Int-2 exhibits an EPR signal at g = 8.05 arising from an S = 2 state. The signal is only slightly broadened by 17O2 (<3% spin delocalization), suggesting that Int-2 is a peroxo-FeIII-4NC semiquinone radical species. Our results demonstrate facile electron transfer between FeII, O2, and the organic ligand, thereby supporting the proposed wild-type enzyme mechanism.
AB - FeIII-O2•- intermediates are well known in heme enzymes, but none have been characterized in the nonheme mononuclear FeII enzyme family. Many steps in the O2 activation and reaction cycle of FeII-containing homoprotocatechuate 2,3-dioxygenase are made detectable by using the alternative substrate 4-nitrocatechol (4NC) and mutation of the active site His200 to Asn (H200N). Here, the first intermediate (Int-1) observed after adding O2 to the H200N-4NC complex is trapped and characterized using EPR and Mössbauer (MB) spectroscopies. Int-1 is a high-spin (S1 = 5/2) FeIII antiferromagnetically (AF) coupled to an S2 = 1/2 radical (J ≈ 6 cm-1 in H = JS1•S2). It exhibits parallel-mode EPR signals at g = 8.17 from the S = 2 multiplet, and g = 8.8 and 11.6 from the S = 3 multiplet. These signals are broadened significantly by 17O2 hyperfine interactions (A17O ≈ 180 MHz). Thus, Int-1 is an AF-coupled FeIII-O2 •- species. The experimental observations are supported by density functional theory calculations that show nearly complete transfer of spin density to the bound O2. Int-1 decays to form a second intermediate (Int-2). MB spectra show that it is also an AF-coupled Fe III-radical complex. Int-2 exhibits an EPR signal at g = 8.05 arising from an S = 2 state. The signal is only slightly broadened by 17O2 (<3% spin delocalization), suggesting that Int-2 is a peroxo-FeIII-4NC semiquinone radical species. Our results demonstrate facile electron transfer between FeII, O2, and the organic ligand, thereby supporting the proposed wild-type enzyme mechanism.
KW - Oxygen activation
KW - Oxygenase
KW - Spectroscopy
KW - Superoxide
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U2 - 10.1073/pnas.1010015107
DO - 10.1073/pnas.1010015107
M3 - Article
C2 - 20837547
AN - SCOPUS:78049259568
VL - 107
SP - 16788
EP - 16793
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 39
ER -