Brevibacterium fuscum protocatechuate 3,4-dioxygenase. Purification, crystallization, and characterization.
A protocatechuate 3,4-dioxygenase with exceptionally sharp spectral features and a new subunit composition has been purified and crystallized from the Gram-positive organism Brevibacterium fuscum. EPR spectra show that the catalytically essential Fe3+ resides in a site of almost the maximal rhombicity (E/D = 0.333 +/- 0.003). The spectral line widths (1.4 milliTesla at g = 9.67) are the smallest reported for any biological high spin Fe3+ complex and suggest that the enzyme is quite homogeneous in the vicinity of the Fe site. The same conclusion is drawn from Mössbauer spectra measured with enzyme prepared from cells cultured in 57Fe-enriched media as well as from resonance Raman and optical spectra. In contrast, EPR and Mössbauer spectra of the anaerobic complex with protocatechuate (PCA) are complex and demonstrate that multiple species with markedly different electronic symmetries and both positive and negative zero field splittings are present. The Mr = 315,000 enzyme has a composition of (alpha beta Fe)5 (Mr(alpha) = 22,500; Mr (beta) = 40,000). Amino acid analysis shows that neither subunit contains cysteine, thus eliminating this amino acid as a possible Fe ligand. The general features of the structure, spectra, and catalyzed reaction of this enzyme appear to be very similar to those of protocatechuate 3,4-dioxygenase isolated from Gram-negative organisms. However, the kinetic parameters (Km(PCA) = 125 microM, Km(O2) = 800 microM, turnover number = 25,000 min-1 at infinite PCA and O2 concentrations) are 5- to 50-fold higher. The sharp spectra and the kinetic properties facilitate mechanistic studies described in this and the following reports.
D000596:Amino Acids / Q000032:analysis; D001951:Brevibacterium / Q000201:enzymology*; D003460:Crystallization; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010105:Oxygenases / Q000302:isolation & purification*; D011520:Protocatechuate-3,4-Dioxygenase / Q000302:isolation & purification* / Q000378:metabolism; D013053:Spectrophotometry; D013057:Spectrum Analysis
D000596:Amino Acids; D046911:Macromolecular Substances; D010105:Oxygenases; D011520:Protocatechuate-3,4-Dioxygenase