A ferredoxin from the thermohalophilic bacterium Rhodothermus marinus.
A [3Fe-4S](1+/0) ferredoxin was isolated from the thermohalophilic and strict aerobic bacterium Rhodothermus marinus. It is a small protein, with an apparent molecular mass of 9 kDa. Its N-terminal amino acid sequence reveals the capability of binding two tetranuclear clusters. However, upon purification, it contains a single [3Fe-4S](1+/0), with an unusually low reduction potential of -650 mV, determined by cyclic voltammetry at pH 7.6. [1H]NMR spectroscopy shows that the protein contains a single, homogeneous, trinuclear centre. When purified under anaerobic conditions, the EPR [3Fe-4S](1+/0) centre signal is also observed. However, it can now be reduced by dithionite and a new signal attributed to a [4Fe-4S](2+/1+) cluster develops. This can also be observed upon reconstitution of the prosthetic groups. The function of this ferredoxin in R. marinus is still unknown but it is very sensitive to oxygen, an unexpected characteristic for a protein from an aerobic organism. The thermodynamic stability of the R. marinus ferredoxin was also investigated and was shown to be high. Thermal and chemical unfolding reactions appear as single, cooperative transitions. The midpoint (T(m)) for thermally induced unfolding is 102+/-2 degrees C (pH 7). Unfolding induced by the chemical denaturant guanidine hydrochloride (GuHCl) shows a transition midpoint at 5.0 M GuHCl (pH 7.0, 20 degrees C). The iron-sulfur cluster degrades upon polypeptide unfolding, resulting in an irreversible denaturation process.
Pereira
Manuela M
MM
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Rua da Quinta, Grande 6, Apt. 127, 2780-156 Oeiras, Portugal.
Jones
Kathryn L
KL
Campos
Marta G
MG
Melo
Ana M P
AM
Saraiva
Lígia M
LM
Louro
Ricardo O
RO
Wittung-Stafshede
Pernilla
P
Teixeira
Miguel
M
D000595:Amino Acid Sequence; D001420:Bacteria, Aerobic / Q000378:metabolism*; D004563:Electrochemistry / Q000379:methods; D005288:Ferredoxins / Q000737:chemistry* / Q000302:isolation & purification; D006358:Hot Temperature; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000302:isolation & purification; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013816:Thermodynamics
D005288:Ferredoxins; D007506:Iron-Sulfur Proteins