Mössbauer and EPR spectroscopy on protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

Larry Que, John D Lipscomb, R. Zimmermann, E. Münck, N. R. Ormejohnson, W. H. Orme-Johnson

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Abstract

Protocatechuate 3,4-dioxygenase (EC 1.13.11.3) from Pseudomonas aeruginosa has been investigated by EPR and Mössbauer spectroscopy. Low temperature Mössbauer data on the native enzyme (Fe3+, S = 5 2) yields a hyperfine field Hsat = -525 kG at the nucleus. This observation is inconsistent with earlier suggestions, based on EPR data, of a rubredoxin-like ligand environment around the iron, i.e. a tetrahedral sulfur coordination. Likewise, the dithionite-reduced enzyme has Mössbauer parameters unlike those of reduced rubredoxin. We conclude that the iron atoms are in a previously unrecognized environment. The ternary complex of the enzyme with 3,4-dihydroxyphenylpropionate and O2 yields EPR signals at g = 6.7 and g = 5.3; these signals result from an excited state Kramers doublet. The kinetics of the disappearance of these signals parallels product formation and the decay of the ternary complex as observed in the optical spectrum. The Mössbauer and EPR data on the ternary complex establish the iron atoms to be in a high-spin ferric state characterized by a large and negative zero-field splitting, D = {reversed tilde equals} -2 cm-1.

Original languageEnglish (US)
Pages (from-to)320-334
Number of pages15
JournalBBA - Enzymology
Volume452
Issue number2
DOIs
StatePublished - Dec 8 1976

Bibliographical note

Funding Information:
We would like to express our gratitude to the Freshwater Biological Research Foundation for making the Institute and the research facilitiesa vailable. In particular, we wish to acknowledge the efforts of Richard Gray, Sr., a private citizen, without whose efforts the Freshwater Biological Institute would not exist. We thank Dr. J.M. Wood for many valuable discussions. We thank Mr. W. Hamilton for capable technical assistance with the EPR spectroscopy, Mr. R.L. Thrift for his valuable help in getting the computer facility.and the MSssbauer spectrometer running and Mr. Francis Engle for assistance with biological preparations. This work was supported by U.S. Public Health Service Grants GM22701 and GM17170, by a Research Career Development Award KO4-GM70683 (E.M.), by National Science Foundation Grants BMS 14980 and PCM-17318, and by the Graduate Research Committee and the College of Agricultural and Life Sciences of the University of Wisconsin.

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