Properties of two clostridial flavodoxins

Abstract

1. 1. Flavodoxins have been purified from Clostridium pasteurianum and Clostridium MP and their properties examined. Clostridium MP flavodoxin, like flavodoxins from C. pasteurianum and Peptostreptococcus elsdenii, has a molecular weight of about 14 000, it contains 1 molecule of FMN, and it replaces ferredoxin as an electron carrier in the phosphoroclastic oxidation of pyruvate. The two clostridial proteins have different amino acid compositions.2. 2. The absorption spectra of flavodoxins from Clostridium MP and C. pasteurianum are very similar. Both proteins are reduced by irradiation with visible light in the presence of EDTA, NADPH in the presence of ferredoxin-NADP+ reductase (EC 1.6.99.4), hydrogen in the presence of hydrogenase (EC 1.12.1.1), and Na2S2O4. At half reduction, high yields of the blue neutral flavin semiquinone are obtained. Clostridium MP flavodoxin, like P. elsdenii flavodoxin, is completely reduced by 1 mole of Na2S2O4. C. pasteurianum flavodoxin is initially reduced by a small excess of Na2S2O4, but the reduced protein slowly reoxidizes to give a mixture of semiquinone and fully reduced flavodoxin. The extent of this reoxidation depends on the Na2S2O4 concentration and on pH. Extinction coefficients for the three oxidation-reduction states of the flavodoxins have been determined.3. 3. The oxidation-reduction potential (E1) for the couple flavodoxin semiquinone-fully reduced flavodoxin is -0.399 V and -0.419 V at pH 7 and 25[deg] for Clostridium MP and C. pasteurianum flavodoxins, respectively. The oxidation-reduction potential (E2) for the couple oxidized flavodoxin-flavodoxin semiquinone is -0.092 V and -0.132 V at pH 7 and 25[deg] for Clostridium MP and C. pasteurianum flavodoxins, respectively. The effects of pH on E1 suggest that an ionization occurs at about pH 6.7 in the fully reduced forms of these proteins. Potentiometric titrations with P. elsdenii flavodoxin have confirmed earlier results which indicated that this flavodoxin also is reduced in two 1-electron oxidation-reduction steps.4. 4. FMN is displaced from the flavodoxins by low concentrations of phenylmercuric acetate. The rate of this reaction increases with flavodoxins from C. pasteurianum, P. elsdenii and Clostridium MP, respectively, and a correlation is drawn between the observed rate and the cysteine content of the protein.