Purification and properties of cytochrome P-450 from Moraxella sp

• Published in: Biochimie Vol. 70; no. 10; pp. 1385 - 1395
• Main Authors: Sauret-Ignazi, Ginette, Dardas, Anastase, Pelmont, Jean
• Format: Journal Article
• Language: English
• Published: Paris Elsevier Masson SAS 01.10.1988; Elsevier
• Subjects: alkoxyphenols; Analytical, structural and metabolic biochemistry; bacteria; Biological and medical sciences; Chromatography, Gel; cytochrome P-450; Cytochrome P-450 Enzyme System - isolation & purification; Cytochrome P-450 Enzyme System - metabolism; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; guaiacol; Guaiacol - metabolism; Hemoproteins; Metalloproteins; Molecular Weight; Moraxella; Moraxella - enzymology; Oxidation-Reduction; Proteins; Spectrum Analysis; alkoxyphenols; guaiacol; cytochrome P-450; bacteria; Moraxella; Spectral data; Hemoprotein; Purification; Cytochrome P450; Neisseriaceae; Bacteria; Micrococcales
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/S0300-9084(88)80001-4

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. 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 om in the P-450 + ligand minus P-450 spectrum was 65 mM −1·cm 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 μM (guaiacol) to 24 μM {2-butoxyphenol). The dissociation constants were 1μM for anisole, and over 90μM 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 μM in the absence of any added ligand and disappeared after dilution. The reduction of p4re P-450 by dithionite was immediate, but became very slow, and was complete after 10 min or more at 23°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-4S0 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 WQS obtained from the substrate, most likely a consequence of the potent catechol 1,2-dioxygenase activity present in the non-purified protein fractions used.