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 degrees 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.
Janssen
S
S
Institut für Biochemie, Medizinische Universität zu Lübeck, Germany.
Trincão
J
J
Teixeira
M
M
Schäfer
G
G
Anemüller
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003001:Cloning, Molecular; D019615:Crenarchaeota / Q000235:genetics / Q000378:metabolism*; D005288:Ferredoxins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D019848:Gene Expression Regulation, Archaeal; D008969:Molecular Sequence Data; D013057:Spectrum Analysis; D014158:Transcription, Genetic; D015032:Zinc / Q000378:metabolism*
D005288:Ferredoxins; D015032:Zinc