Ferric reductase activity in Azotobacter vinelandii and its inhibition by Zn2+.
Ferric reductase activity was examined in Azotobacter vinelandii and was found to be located in the cytoplasm. The specific activities of soluble cell extracts were not affected by the iron concentration of the growth medium; however, activity was inhibited by the presence of Zn2+ during cell growth and also by the addition of Zn2+ to the enzyme assays. Intracellular Fe2+ levels were lower and siderophore production was increased in Zn2+-grown cells. The ferric reductase was active under aerobic conditions, had an optimal pH of approximately 7.5, and required flavin mononucleotide and Mg2+ for maximum activity. The enzyme utilized NADH to reduce iron supplied as a variety of iron chelates, including the ferrisiderophores of A. vinelandii. The enzyme was purified by conventional protein purification techniques, and the final preparation consisted of two major proteins with molecular weights of 44,600 and 69,000. The apparent Km values of the ferric reductase for Fe3+ (supplied as ferric citrate) and NADH were 10 and 15.8 microM, respectively, and the data for the enzyme reaction were consistent with Ping Pong Bi Bi kinetics. The approximate Ki values resulting from inhibition of the enzyme by Zn2+, which was a hyperbolic (partial) mixed-type inhibitor, were 25 microM with respect to iron and 1.7 microM with respect to NADH. These results suggested that ferric reductase activity may have a regulatory role in the processes of iron assimilation in A. vinelandii.
Huyer
M
M
Department of Microbiology, University of Alberta, Edmonton, Canada.
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D001395:Azotobacter / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D038181:FMN Reductase; D007502:Iron Chelating Agents / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D017262:Siderophores; D015032:Zinc / Q000494:pharmacology*
D001426:Bacterial Proteins; D007502:Iron Chelating Agents; D017262:Siderophores; D010088:Oxidoreductases; D038181:FMN Reductase; C023903:ferric citrate iron reductase; D015032:Zinc