Carbon-Monoxide Oxidation in Cell-Free Extracts of Clostridium pasteurianum

Cell-free extracts of NH4-grown and N2-grown Clostridium pasteurianum were found to catalyze the oxidation of carbon monoxide to CO2 under strictly anaerobic conditions. Free formate was not an intermediate. FAD, FMN, methylene blue and viologen dyes were effective as electron acceptors, while NAD+, NADP+ or oxidized ferredoxin were ineffective. The reaction was routinely followed by measuring the reduction of methyl viologen with carbon monoxide photometrically. The rate of carbon monoxide oxidation followed simple Michaelis-Menten kinetics. 0.1–0.2 μmol carbon monoxide was oxidized by methyl viologen per min and mg soluble protein at pH 8.7. Half-maximal velocities were obtained with 5 μM carbon monoxide and 0.4 mM methyl viologen. The rate increased with increasing pH with an inflection point near pH 8.4. The carbon monoxide oxidizing activity was acid-labile (pH 4.5), precipitable with acids at pH 5 and sensitive to molecular oxygen. It was relatively stabile to heat. At 90 °C only 50%, of the activity were destroyed within 15 min. Incubation of the extracts with cyanide (10 μM) or with methyl iodide (2.5 mM) resulted in a reversible loss of the carbon monoxide oxidizing activity. The rate and extent of inactivation by cyanide was decreased in the presence of carbon monoxide by a factor of 100 and 10, respectively. On the contrary, the rate of inactivation by methyl iodide was increased in the presence of carbon monoxide by a factor of 6. The activity of extracts inactivated by cyanide in the absence of carbon monoxide was partially restored by incubation of the extracts with carbon monoxide; the inactivation by methyl iodide was reversed by exposure of the extracts to the light of a projection lamp. These properties are taken to indicate that a vitamin B12 compound is probably involved in the catalysis of anaerobic carbon monoxide oxidation.