Purification and properties of cytochrome P-450 from Moraxella sp.
A cytochrome P-450 has been purified to homogeneity from a Moraxella species that is able to grow on guaiacol as the sole source of carbon and energy. The pure cytochrome was a monomeric protein of about 52 kDa, with no catalytic activity towards guaiacol. The difference in mM extinction coefficients between 450 and 490 nm in the CO-difference spectrum was 89.5 mM-1.cm-1. The typical shift of the Soret band from 415 to 390 nm that is attributed to the high-spin state of the cytochrome was observed in the presence of guaiacol and other 2-alkoxyphenols with up to 5 carbons in the side chain. It was also obtained with anisole. The maximum difference in mM extinction coefficients between 390 and 420 nm in the P-450 + ligand minus P-450 spectrum was 65 mM-1.cm-1 in all instances. The dissociation constants of the complexes formed between the pure protein and various O-alkoxyphenols were measured, and ranged from 0.1 microM (guaiacol) to 24 microM (2-butoxyphenol). The dissociation constants were 1 microM for anisole, and over 90 microM for phenol. Catechol induced no spectral change in cytochrome P-450 and appeared to be a weak inhibitor of guaiacol binding. The same spectral shift as induced by guaiacol was observed at high P-450 concentration over 1 microM in the absence of any added ligand and disappeared after dilution. The reduction of pure P-450 by dithionite was immediate, but became very slow, and was complete after 10 min or more at 25 degrees C in the presence of guaiacol. This effect was also obtained with the 2 isomers, 3- and 4-methoxyphenols, and with metyrapone, an inhibitor of guaiacol binding that induced the low-spin state. Preliminary experiments using the crude cell lysate or a reconstructed system with purified P-450 and a protein fraction indicated NADH-dependent guaiacol degradation. This was in agreement with the former hypothesis of Moraxella P-450 acting as a monooxygenase in the demethylation of guaiacol. However, cis, cis-muconate rather than catechol was obtained from the substrate, most likely a consequence of the potent catechol 1,2-dioxygenase activity present in the non-purified protein fractions used.
Sauret-Ignazi
G
G
Laboratoire de Biochimie des Micro-organismes, Université Grenoble 1, Saint-Martin-d'Hères, France.
Dardas
A
A
Pelmont
J
J
D002850:Chromatography, Gel; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006139:Guaiacol / Q000378:metabolism; D008970:Molecular Weight; D009016:Moraxella / Q000201:enzymology*; D010084:Oxidation-Reduction; D013057:Spectrum Analysis
D006139:Guaiacol; D003577:Cytochrome P-450 Enzyme System