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Addition to the end of the initial paragraph:
In most instances, the exact motivation for these transfer processes remains to be determined. The electron transfer self- exchange rate constant for rubredoxin, as isolated from Clostridium pasteurianum, is quite rapid at kese= 3.1 x 105 M-1·s-1 [1]
Addition to Structure section:
It is found that upon changing from the oxidized (Fe3+) to reduced (Fe2+) form, the Fe-S bond lengths consistently increase by about 0.10 Å. Rubredoxin is shown to maintain Fe-S distances at intermediate lengths between those ideal for the oxidized and reduced forms, allowing for more rapid electron transfer. There is also an observed decrease in the bond distances (0.08 Å) of the five NH-S hydrogen bonds, which are present in both forms of rubredoxin. This is thought to aid in the stabilization of the negative charge introduced to most of the sulfur atoms. [2]
There is a proposed mechanism which facilitates the reduction of rubredoxin involving the Leu 41 side chain located at the protein’s surface and beside the Cys 42 ligand. Leu 41 possesses a closed conformation in the oxidized state, which then switches to an open conformation upon the reduction of rubredoxin. This conformational change provides exposure of the Fe(S-Cys)4 complex to a more polar environment, which encourages H bonding with the Cys(9)-S- donor. Therefore, this increased hydration is thought to aid in the reduction of rubredoxin, which is more effectively stabilized in a polar environment. [2]
Initial Isolation[edit]
Rubredoxin was discovered by Lovenburg and Sobel in 1965, as a red protein in Clostridium pasteurianum, during the isolation of another Fe-S cluster, ferredoxin. To summarize this initial isolation, C. pasteurianum cells were suspended in water, and following centrifugation, the supernatant was processed on a DEAE cellulose column. After subsequent washings were performed, ferredoxin and rubredoxin were eluted with 0.15M Tris-HCl pH 7.3 with 0.65M NaCl. After salt was removed from the eluent, the concentration was adjusted to 0.05M with the Cl- buffer, and the solution was 60% saturated using ammonium sulfate. Subsequently, 90% saturation was achieved, at which point most of the ferredoxin was contained within the precipitate, while the supernatant is comprised mostly of rubredoxin. The supernatant was then subjected to a similar DEAE cellulose column elution, desalting and saturation procedure as just performed. The precipitate containing the remainder of the ferredoxin was discarded and the supernatant fraction was readsorbed and eluted once more using the same purification procedure, to ultimately yield the final purified rubredoxin. Recrystallization of rubredoxin was achieved with ammonium sulfate. [3]
References[edit]
- ^ Kummerle, R.; et al. (1997). "Site-Directed Mutagenesis of Rubredoxin Reveals the Molecular Basis of Its Electron Transfer Properties". Biochemistry. 36: 15983–15991. doi:10.1021/bi971636e.
{{cite journal}}: Explicit use of et al. in:|last2=(help) - ^ a b Min, T.; et al. (2001). "Leucine 41 is a gate for water entry in the reduction of Clostridium pasteurianum rubredoxin". Protein Science. 10: 613–621. doi:10.1110/gad.34501.
{{cite journal}}: Explicit use of et al. in:|last2=(help) - ^ Lovenburg, W.; Sorbel, B.E. (July 15, 1965). "Rubredoxin: A New Electron Transfer Protein From Clostridium pasteurianum". Proceedings of the National Academy of Sciences of the United States of America. 54: 193–199.