Purification and characterization of a 7Fe ferredoxin from Streptomyces griseus.
A ferredoxin has been purified from Streptomyces griseus grown in soybean flour-containing medium. The homogeneous protein has a molecular weight near 14,000 as determined by both PAGE and size exclusion chromatography. The iron and labile sulfide content is 6-7 atoms/mole protein. EPR spectroscopy of native S. griseus ferredoxin shows an isotropic signal at g = 2.01 which is typical of [3Fe-4S]1+ clusters and which quantitates to 0.9 spin/mole. Reduction of the ferredoxin by excess dithionite at pH 8.0 produces an EPR silent state with a small amount of a g = 1.95 type signal. Photoreduction in the presence of deazaflavin generates a signal typical of [4Fe-4S]1+ clusters at much higher yields (0.4-0.5 spin/mole) with major features at g-values of 2.06, 1.94, 1.90 and 1.88. This latter EPR signal is most similar to that seen for reduced 7Fe ferredoxins, which contain both a [3Fe-4S] and [4Fe-4S] cluster. In vitro reconstitution experiments demonstrate the ability of the S. griseus ferredoxin to couple electron transfer between spinach ferredoxin reductase and S. griseus cytochrome P-450soy for NADPH-dependent substrate oxidation. This represents a possible physiological function for the S. griseus ferredoxin, which if true, would be the first functional role demonstrated for a 7Fe ferredoxin.
Trower
M K
MK
E.I. du Pont de Nemours & Company Inc., Central Research and Development Department, Wilmington, DE 19880-0228.
Emptage
M H
MH
Sariaslani
F S
FS
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004227:Dithionite; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D005288:Ferredoxins / Q000302:isolation & purification* / Q000378:metabolism; D007501:Iron / Q000032:analysis*; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010777:Photochemistry; D013305:Streptomyces griseus / Q000032:analysis*; D013455:Sulfur / Q000032:analysis
D005288:Ferredoxins; D004227:Dithionite; D013455:Sulfur; D003577:Cytochrome P-450 Enzyme System; D007501:Iron