Protocatechuate 3,4-dioxygenase. Resonance Raman studies of the oxygenated intermediate

Keyes, W.E.; Loehr, T.M.; Taylor, M.L.; Loehr, J.S.

Biochemical and Biophysical Research Communications 89(2): 420-427

1979


ISSN/ISBN: 0006-291X
PMID: 114174
DOI: 10.1016/0006-291x(79)90646-6
Accession: 068521013

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Abstract
Resonance Raman spectra of protocatechuate 3,4-dioxygenase from P. aeruginosa were investigated during the reaction of the enzyme with substrate and O2. The spectrum of the turned-over enzyme is indistinguishable from that of the resting enzyme in the absence of substrate, and is characterized by resonance-enhanced tyrosinate ring vibrational modes at 1263 and 1174 cm-1. In the ternary ESO2 complex, the tyrosinate vibrational modes are shifted to 1252 and 1165 cm-1, respectively. There is no evidence for any O2 vibrations in the spectra of ESO2 complexes prepared with 16O2, 18O2 and 16O18O in the region between 1300-200 cm-1. Apparently O2 is attached to the substrate (but not Fe) in the stable intermediate, and the concomitant rearrangement at C4 of the substrate induces a substantial change in geometry of the tyrosine residues associated with the Fe complex. The optical spectrum of the ESO2 complex (.lambda.max = 520 nm) is dominated by tyrosinate .fwdarw. Fe(III) charge transfer and contains little or no peroxide .fwdarw. Fe(III) charge transfer. These results invalidate the previously advanced analogy in spectral properties between this enzyme and the respiratory protein, oxyhemerythrin.