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S. Janssen, J. Trincão, M. Teixeira, G. Schäfer, S. Anemüller
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 zincbinding motif, the corresponding sequence of the FdB is devoid of this motif. Thus far, only the zinccontaining ferredoxin, FdA, from A. ambivalens has been chemically and functionally characterized from its native source. Using RTPCR and Northern blot analysis, we show that both ferredoxins are expressed by A. ambivalens under either anaerobic or aerobic growth conditions. The zincfree 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 EPRmonitored 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.
Biological Chemistry, Walter de Gruyter
Print ISSN: 1431-6730
Volume: 382, 10/2001
Pages: 1501 - 1507