Hydrodynamic characterization of the SufBC and SufCD complexes and their interaction with fluorescent adenosine nucleotides.
Bacteria, as well as the plastid organelles of algae and higher plants, utilize proteins of the suf operon. These are involved in Fe-S cluster assembly, particularly under conditions of iron limitation or oxidative stress. Genetic experiments in some organisms found that the ATPase SufC is essential, though its role in Fe-S biogenesis remains unclear. To ascertain how interactions with other individual Suf proteins affect the activity of SufC we coexpressed it with either SufB or SufD from Thermotoga maritima and purified the resulting SufBC and SufCD complexes. Analytical ultracentrifuge and multiangle light-scattering measurements showed that the SufBC complex exists in solution as the tetrameric SufB(2)C(2) species, whereas SufCD exists as an equilibrium mixture of SufCD and SufC(2)D(2). Transient kinetic studies of the complexes were made using fluorescent 2'(3')-O-(N-methylanthraniloyl-(mant) analogues of ATP and ADP. Both SufBC and SufCD bound mantATP and mantADP much more tightly than does SufC alone. Compared to the cleavage step of the mantATPase of SufC alone, that of SufBC was accelerated 180-fold and that of SufCD only fivefold. Given that SufB and SufD have 20% sequence identity and similar predicted secondary structures, the different hydrodynamic properties and kinetic mechanisms of the two complexes are discussed.
Petrovic
Arsen
A
MRC National Institute for Medical Research, London NW7 1AA, United Kingdom.
Davis
Colin T
CT
Rangachari
Kaveri
K
Clough
Barbara
B
Wilson
R J M Iain
RJ
Eccleston
John F
JF
D000244:Adenosine Diphosphate / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D007700:Kinetics; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate