Structural aspects of the dye-linked alcohol dehydrogenase of Rhodopseudomonas acidophila

Abstract

A dye-linked alcohol dehydrogenase was purified 60-fold from extracts of R. acidophila 10050 grown aerobically on ethanol. The properties of this enzyme were identical with those of the alcohol dehydrogenase synthesized by this organism during growth on methanol anaerobically in the light, and they are judged to be the same enzyme. The enzyme gave a single protein band, coincident with alcohol dehydrogenase activity, during electrophoresis on polyacrylamide gel. The amino acid composition, isoelectric point, UV and visible absorption spectra of the enzyme were determined and compared with those of other similar enzymes. The presence of 0.7-1.0 g-atom of non-heme, acid-labile Fe/mol of enzyme was shown by atomic absorption spectrophotometry and colorimetric assay. The Fe could not be dissociated from the enzyme by dialysis against chelating agents. EPR spectroscopy of the enzyme did not indicate any redox function for the iron during alcohol dehydrogenation, but showed a signal at g=2.00 consistent with the presence of a protein-bound organic free radical. Antisera were raised against alcohol (methanol) dehydrogenases purified from R. acidophila, Paracoccus denitrificans and Methylophilus methylotrophus. The antiserum to the R. acidophila enzyme cross-reacted with neither of the 2 other antisera, nor with crude extracts of methanol-grown Hyphomicrobium X and Pseudomonas AM1, thus emphasizing its singular biochemical properties.