Methanosarcina barkeri (strain Fusaro) was grown on pyruvate as methanogenic substrate [Bock, A. K., Prieger-Kraft, A. & Schonheit, P. (1994) Arch. Microbiol. 161, 33-46]. The first enzyme of pyruvate catabolism, pyruvate oxidoreductase, which catalyzes oxidation of pyruvate to acetyl-CoA was purified about 90-fold to apparent electrophoretic homogeneity. The purified enzyme catalyzed the CoA-dependent oxidation of pyruvate with ferredoxin as an electron acceptor which defines the enzyme as a pyruvate:ferredoxin oxidoreductase. The deazaflavin, coenzyme F,,,,, which has been proposed to be the physiological electron acceptor of pyruvate oxidoreductase in methanogens, was not reduced by the purified enzyme. In addition to ferredoxin and viologen dyes, flavin nucleotides served as electron acceptors. Pyruvate : ferredoxin oxidoreductase also catalyzed the oxidation of 2-oxobutyrate but not the oxidation of 2-oxoglutarate, indolepyruvate, phenylpyruvate, glyoxylate, 3-hydroxypyruvate and oxaloacetate. The apparent K,,, values of pyruvate:ferredoxin oxidoreductase were 70 pM for pyruvate, 6 pM for CoA and 30 pM for clostridial ferredoxin. The apparent V,,,, with ferredoxin was about 30 U/mg (at 37°C) with a pH optimum of approximately 7. The temperature optimum was approximately 60°C and the Arrhenius activation energy was 40 kJ/mol (between 30°C and 60°C). The enzyme was extremely oxygen sensitive, losing 90% of its activity upon exposure to air for 1 h at 0°C. Sodium nitrite inhibited the enzyme with a K, of about 10 mM. The native enzyme had an apparent molecular mass of approximately 130 kDa and was composed of four different subunits with apparent molecular masses of 48, 30, 25, and 15 kDa which indicates that the enzyme has an a y 6 structure. The enzyme contained 1 mol/mol thiamine diphosphate, and about 12 mol/mol each of non-heme iron and acid-labile sulfur. FAD, FMN and lipoic acid were not found. The N-terminal amino acid sequences of the four subunits were determined. The sequence of the a-subunit was similar to the N-terminal amino acid sequence of the a-subunit of the heterotetrameric pyruvate : ferredoxin oxidoreductases of the hyperthermophiles Archaeoglobus fulgidus, Pyrococcus furiosus and Thermotoga maritima and of the mesophile Helicobacter pylori, and to the N-terminal amino acid sequence of the homodimeric pyruvate: ferredoxin oxidoreductase from proteobacteria and from cyanobacteria. No sequence similarities were found, however, between the a-subunit of the M. barkeri enzyme and the heterodimeric pyruvate: ferredoxin oxidoreductase of the archaeon Halobacter~~m halobiurn.