Extracts of aerobically, CO-autotrophically grown cells of P. carboxydovorans catalyzed the oxidation of CO to CO2 in the presence of methylene blue, pyocyanine, thionine, phenazine methosulfate or toluylene blue under strictly anaerobic conditions. Viologen dyes and NAD(P)+ were ineffective as electron acceptors. The same extracts catalyzed the oxidation of formate and of H2; the spectrum of electron acceptors was identical for the 3 substrates, CO, formate and H2. The CO- and the formate-oxidizing activities were soluble enzymes, whereas hydrogenase was membrane bound exclusively. The rates of oxidation of CO, formate and H2 were measured spectrophotometrically following the reduction of methylene blue. The rate of CO oxidation followed simple Michaelis-Menten kinetics; the apparent Km for CO was 45 .mu.M. The reaction rate was maximal at pH 7.0, and the temperature dependence followed the Arrhenius equation with an activation energy (.DELTA.Ho) of 35.9 kJ/mol (8.6 kcal/mol). Neither free formate nor H3 is an intermediate of the CO oxidation reaction. This conclusion is based on the differential sensitivity of the activities of formate dehydrogenase, hydrogenase and CO dehydrogenase to heat, hypophosphite, chlorate, cyanide, azide and fluoride and on the failure to trap free formate or H3 in coupled optical assays. These results support the following equation for CO oxidation in P. carboxydovorans: CO + H2O .fwdarw. CO2 + 2H+ 2e-. The CO-oxidizing activity of P. carboxydovorans differed from that of Clostridium pasteurianum by not reducing viologen dyes and by a pH optimum curve that did not show an inflection point.
