Ferredoxins from the archaeon Acidianus ambivalens: Overexpression and characterization of the non-zinc-containing ferredoxin FdB

Two ferredoxin genes, fdA and fdB, from the extremely thermoacidophilic crenarchaeon Acidianus ambivalens have been sequenced; the sequences share 86% similarity. Whereas the deduced protein sequence of the ferredoxin FdA clearly contains a zinc-binding motif, the corresponding sequence of the FdB is devoid of this motif. Thus far, only the zinc-containing ferredoxin, FdA, from A. ambivalens has been chemically and functionally characterized from its native source. Using RT-PCR and Northern blot analysis, we show that both ferredoxins are expressed by A. ambivalens under either anaerobic or aerobic growth conditions. The zinc-free ferredoxin, FdB, was overexpressed in E. coli and purified to homogeneity. Using EPR spectroscopy, we could demonstrate that FdB contains one [3Fe-4S]^1+/0 and one [4Fe-4S]^2+/1+ cluster. The reduction potential of the [3Fe-4S]^1+/0 cluster was determined as -235±10 mV, at pH 6.5, by EPR-monitored redox titration. The high melting temperature of 108±2 °C of FdB determined by CD spectroscopy reveals that it is not the binding of the Zn²⁺ that induces the extreme thermostability of these ferredoxins.