Characterization of BphF, a Rieske-type ferredoxin with a low reduction potential.
BphF is a small, soluble, Rieske-type ferredoxin involved in the microbial degradation of biphenyl. The rapid, anaerobic purification of a heterologously expressed, his-tagged BphF yielded 15 mg of highly homogeneous recombinant protein, rcBphF, per liter of cell culture. The reduction potential of rcBphF, determined using a highly oriented pyrolytic graphite (HOPG) electrode, was -157+/- 2 mV vs the standard hydrogen electrode (SHE) (20 mM MOPS, 80 mM KCl, and 1 mM dithiothreitol, pH 7.0, 22 degrees C). The electron paramagnetic resonance spectrum of the reduced rcBphF is typical of a Rieske cluster while the close similarity of the circular dichroic (CD) spectra of rcBphF and BedB, a homologous protein from the benzene dioxygenase system, indicates that the environment of the cluster is highly conserved in these two proteins. The reduction potential and CD spectra of rcBphF were relatively independent of pH between 5 and 10, indicating that the pK(a)s of the cluster's histidinyl ligands are not within this range. Gel filtration studies demonstrated that rcBphF readily oligomerizes in solution. Crystals of rcBphF were obtained using sodium formate or poly(ethylene glycol) (PEG) as the major precipitant. Analysis of the intermolecular contacts in the crystal revealed a head-to-tail interaction that occludes the cluster, but is very unlikely to be found in solution. Oligomerization of rcBphF in solution was reversed by the addition of dithiothreitol and is unrelated to the noncovalent crystallographic interactions. Moreover, the oligomerization state of rcBphF did not influence the latter's reduction potential. These results indicate that the 450 mV spread in reduction potential of Rieske clusters of dioxygenase-associated ferredoxins and mitochondrial bc(1) complexes is not due to significant differences in their solvent exposure.
Couture
M M
MM
Department of Biochemistry, Université Laval, Québec, Canada.
Colbert
C L
CL
Babini
E
E
Rosell
F I
FI
Mauk
A G
AG
Bolin
J T
JT
Eltis
L D
LD
D019117:Burkholderia / Q000737:chemistry / Q000235:genetics; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D014450:Electron Transport Complex III; D005288:Ferredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000138:chemical synthesis; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012996:Solutions; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D005288:Ferredoxins; D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; C037989:Rieske iron-sulfur protein; D012996:Solutions; D006867:Hydrolases; C062907:2,6-dioxo-6-phenylhexa-3-enoate hydrolase; D014450:Electron Transport Complex III