Fig. 1.

Quantitative analyses of substrate consumption and product generation indicate nearly complete coupling of NADH/O₂ consumption to substrate O-demethylation for guaiacol and progressively more uncoupling for syringol and 3MC. NADH (200 µM) and guaiacol, syringol, or 3MC (100 or 200 µM) were incubated in air with 0.2 μM GcoA-F169A and GcoB (each with 25 mM Hepes, 50 mM NaCl, pH 7.5 at 25 °C and 210 µM O₂). Reactants and products were quantified when the UV/vis spectrum ceased changing and the reaction was deemed complete. The total NADH consumed is compared with the amounts of formaldehyde and demethylated aromatic compound produced. Pyrogallol, the O-demethylated product of 3MC, is unstable in air under the conditions used in the assay and was not detected. Error bars represent ±1 SD from three or more independent measurements. P values comparing NADH consumption and formaldehyde production were 0.035 for guaiacol, 0.20 for 100 µM syringol, 0.41 for 200 µM syringol, and 0.0035 for 3MC. For NADH consumption and aromatic product production, these P values were 0.011 for guaiacol, 0.00031 for 100 µM syringol, and 0.0084 for 200 µM syringol.