Structural roles of the active site iron(III) ions in catechol 1,2-dioxygenases and differential secondary structure changes in isoenzymes A and B from Acinetobacter radioresistens S13.
The reversible active site metal ion removal process for two catechol 1,2-dioxygenase isoenzymes (IsoA and IsoB) isolated from Acinetobacter radioresistens S13 has been monitored using circular dichroism and fluorescence spectroscopic techniques. IsoA and IsoB are homodimers, containing one iron(III) ion per subunit. Their amino acid sequence identity is 48.4%. Previous experiments suggested that structural diversities could be responsible for the differential thermal and pH stabilities of the two isoenzymes and of their distinct demetallation kinetics. The far-UV CD spectra of IsoA and IsoB catechol 1,2-dioxygenases from A. radioresistens S13 provide information on their secondary structures. IsoB appears to have a content of alpha-helices higher than IsoA. Upon metal ion removal, both proteins reversibly lose part of their secondary structure following distinct pathways. CD spectra simulations allowed us to estimate the content of alpha-helices, beta-sheets, and turns for each isoenzyme and to monitor the secondary structure rearrangements. The metal ion withdrawal has large influence on the secondary structure: in particular a significant reduction of alpha-helices content is observed for both isoenzymes. Intrinsic fluorescence emission spectra clearly support such results, adding information on the local environment changes of the tryptophan residues. The positioning of Trp250 in IsoB has been shown to be of particular interest for monitoring the local structure changes occurring upon metal ion removal. For the first time these studies allow to underline the role of active site iron ions on dioxygenases folding and stability, further evidencing the differences in structural assembling between the two isoenzymes from A. radioresistens S13.
Dipartimento di Biologia Animale, Università di Torino,Via A. Albertina 13, 10123, Turin, Italy.
Giovanna
G
Di Nardo
Silvia
S
Tilli
Enrica
E
Pessione
Maria
M
Cavaletto
Carlo
C
Giunta
Fabrizio
F
Briganti
D000150:Acinetobacter / Q000201:enzymology*; D050548:Catechol 1,2-Dioxygenase; D002942:Circular Dichroism; D049308:Dioxygenases / Q000737:chemistry / Q000378:metabolism*; D007501:Iron / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence
D007527:Isoenzymes; D007501:Iron; D049308:Dioxygenases; D050548:Catechol 1,2-Dioxygenase