Ferric reductase activity in Azotobacter vinelandii and its inhibition by Zn2+

Abstract

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 ,uM, respectively, and the data for the enzyme reaction were consistent with Ping Pong Bi Bi kinetics. The approximate K. values resulting from inhibition of the enzyme by Zn2+, which was a hyperbolic (partial) mixed-type inhibitor, were 25 ,uM with respect to iron and 1.7 ,uM 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.The presence of iron in many enzymes such as nitrogenase and ribonucleotide reductase, and in many cellular structures such as cytochromes and ferredoxins, make it an essential element for almost all forms of life (26). The extreme usefulness of this particular metal is related to its ability to exist in two stable redox states, Fe3" and Fe2 .

Growth and siderophores production in Alcaligenes faecalis is regulated by metal ions

“…2+ in the intracellular control of siderophores synthesis and are responsible for repressing siderophore synthesis [26]. Huyer and Page [27][28], and Page [29] have also reported the inhibition of azotobactin, azotochelin and aminochelin synthesis in vinelandii at elevated levels of Mn 2+ and Zn 2+ . Maximum growth with good siderophoregenesis was obtained even at high concentration (50 μM) of (CH 3 COO) 2 Pb (Table 1).…”

Role of Molybdate and Other Transition Metals in the Accumulation of Protochelin by Azotobacter vinelandii

“…At these concentrations, the metals were not detrimental to A. vinelandii growth. In addition, strain RP40, which is defective in both high-and low-affinity molybdate transport, was found to form protochelin in response to different molybdate concentrations (18,28). A. vinelandii cells grown in medium containing 1 M molybdate and 3 M ferric citrate were used to prepare CX.…”

The haemoglobin‐like protein (HMP) of Escherichia coli has ferrisiderophore reductase activity and its C‐terminal domain shares homology with ferredoxin NADP+ reductases

“…FSR activity has been detected in a variety of bacteria and in some cases the enzymes have been isolated [2][3][4]. However, these enzymes have not been characterised in detai!, their role in the cell has not been established, and in most cases the corresponding genes have not been cloned and sequenced.…”