Abstract

The X-ray absorption spectra of rubredoxin from Peptococcus aerogenes and of the model iron sulfur components prepared by Holm [Fe(S 2- O- xyl) 2] have been obtained in the oxidized and reduced forms using X-rays from synchrotron radiation at the Stanford Synchrotron Radiation Project. The spectra were measured both in transmission and by fluorescence, with the latter giving a several-fold higher signal-to-noise ratio for the protein. The edge spectra of the protein and the model compounds were extremely similar and both showed similar changes upon reduction. The best resolved change of the edge spectra upon reduction was a shift of the 1s-3d transition by ≈0.7 eV to lower energies. The EXAFS region of the spectra were converted to k-space and the data were fitted in order to determine first the average Fe-S distances, R ave, and second their spread which, for rubredoxin, was described by a model wherein three Fe-S bonds had length R 3 and one R 1. Three different methods have been tried consisting essentially of fitting (1) both phase and amplitude simultaneously to the R 3, R 1 model, (2) phase alone to an R ave model and using the phase to determine the spread of distance, and (3) using phase and amplitude to fit an R ave model and then using the amplitudes alone to determine the spread of distances. The values of R ave obtained for the oxidized state of rubredoxin were R ave = 2.265(13) A ̊ for the powder and 2.256(16) Å in solution. Upon reduction the value increased to 2.32(2) å. The spread of the distances was determined most accurately by the third method and was ¦R 3 — R 1¦= 0.04 +0.06 −0.04 A ̊ for the oxidized powder and ¦R 3 — R 1¦= 0.08 for the reduced form. These values agree within combined experimental errors with the values found by extended X-ray absorption fine structure in the oxidized and reduced forms of Fe(S 2- O- xyl) 2, which also agreed with the previously published X-ray diffraction results. The agreement between the average distances in rubredoxin and the model compounds and the small spread allowable in the rubredoxin distances lead to the conclusion that any strain energy in the iron — sulfur bonds of Rub ox is appreciably less than thermal energy. Hence the redox potential is not being regulated by strain in the iron — sulfur bond lengths.

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