PYRUVATE, FERREDOXIN OXIDOREDUCTASE FROM THE SULFATE-REDUCING ARCHAEOGLOBUS-FULGIDUS - MOLECULAR COMPOSITION, CATALYTIC PROPERTIES, AND SEQUENCE ALIGNMENTS

Archaeoglobus fulgidus is a hyperthermophilic sulfate-reducing archaeo n. In this communication we de scribe the purification and properties of pyruvate:ferredoxin oxidoreductase from this organism. The cataboli c enzyme was purified 250-fold to apparent homogeneity with a yield of 16%. The native enzyme had an apparent molecular mass of 120 kDa and was composed of four different subunits of apparent molecular masses o f 45, 33, 25, and 13 kDa, indicating an alpha beta gamma delta structu re. Per mel, the enzyme contained 0.8 mol thiamine pyrophosphate, 9 mo l non-heme iron, and 8 mol acid-labile sulfur. FAD, FMN, lipoic acid, and copper were not found. The purified enzyme showed an apparent K-m for coenzyme A of 0.02 mM, for pyruvate of 0.3 mM, and for clostridial ferredoxin of 0.01 mM, an apparent V-max of 64 U/mg (at 65 degrees C) with a pH optimum near 7.5 and an Arrhenius activation energy of 75 k J/mol (between 30 and 70 degrees C). The temperature optimum was above 90 degrees C. At 90 degrees C, the enzyme lost 50% activity within 60 min in the presence of 2 M KCl. The enzyme did not catalyze the oxida tion of 2-oxoglutarate, indolepyruvate, phenylpyruvate, glyoxylate, an d hydroxypyruvate. The N-terminal amino acid sequences of the four sub units were determined. The sequence of the alpha-subunit had similarit ies to the N-terminal amino acid sequence of the alpha-subunit of the heterotetrameric pyruvate:ferredoxin oxidoreductase from Pyrococcus fu riosus and from Thermotoga maritima, and unexpectedly, to the N-termin al amino acid sequence of the homodimeric pyruvate:ferredoxin oxidored uctase from proteobacteria and from cyanobacteria. No sequence similar ities were found, however, between the alpha-subunits of the enzyme fr om A. fulgidus and the heterodimeric pyruvate:ferredoxin oxidoreductas e from Halobacterium halobium.