Circular dichroism of holo- and apoprotocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa.
Circular dichroism studies have been carried out on both apo- and holoprotocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa, in the absence and presence of competitive inhibitors, protocatechualdehyde and 4-nitrocatechol. The apo- and holoenzyme showed identical spectra in the ultraviolet region between 200 and 250 nm (peptide back bone region), but the low intensity negative bands at 330 and 480 nm of the holoenzyme were completely absent in the apoenzyme. On the side chain region, the positive ellipticity peaks of the holoenzyme change into a lower intensity and broader band indicating the participation of aromatic amino acid residues in the primary binding of iron ion. Under anaerobic conditions, spectral changes were evident in the side chain region for the binary complexes of both the holo- and the apoenzyme with protocatechuate. The presence of iron in the holoenzyme results in an increase in positive ellipticity between 290 and 320 nm. Either with or without the iron, the enzyme protein binds protocatechuate and has a greater positive circular dichroism increase at 240-260 nm. CD difference spectra indicate that the modes of binding to form the binary complexes of holo- or apoenzyme with either substrates or competitive inhibitors are different. The bound iron ion stimulates binding. Spectral changes of the holoenzyme in the aromatic region were also observed in different pH environments of lower enzymatic activity. It is still not established whether these aromatic residues play an active or passive role in the binding of iron and/or substrates and inhibitors.
D001051:Apoenzymes; D001059:Apoproteins; D001547:Benzaldehydes; D001665:Binding Sites; D002021:Buffers; D002396:Catechols; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000032:analysis; D010105:Oxygenases; D011485:Protein Binding; D011487:Protein Conformation; D011520:Protocatechuate-3,4-Dioxygenase; D011550:Pseudomonas aeruginosa / Q000201:enzymology*
D001051:Apoenzymes; D001059:Apoproteins; D001547:Benzaldehydes; D002021:Buffers; D002396:Catechols; C005581:protocatechualdehyde; D007501:Iron; D010105:Oxygenases; D011520:Protocatechuate-3,4-Dioxygenase