EPR Spectroscopic and Computational Characterization of the Hydroxyethylidene-Thiamine Pyrophosphate Radical Intermediate of Pyruvate:Ferredoxin Oxidoreductase
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- 作者：Steven O. Mansoorabadi ; Javier Seravalli ; Cristina Furdui ; Vladimir Krymov ; Gary J. Gerfen ; Tadhg P. Begley ; Jonathan Melnick ; Stephen W. Ragsdale ; and George H. Reed
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：June 13, 2006
- 年：2006
- 卷：45
- 期：23
- 页码：7122 - 7131
- 全文大小：221K
- 年卷期：v.45,no.23(June 13, 2006)
- ISSN：1520-4995
文摘
The radical intermediate of pyruvate:ferredoxin oxidoreductase (PFOR) from Moorellathermoacetica was characterized using electron paramagnetic resonance (EPR) spectroscopy at X-bandand D-band microwave frequencies. EPR spectra, obtained with various combinations of isotopically labeledsubstrate (pyruvate) and coenzyme (thiamine pyrophosphate (TPP)), were analyzed by spectral simulations.Parameters obtained from the simulations were compared with those predicted from electronic structurecalculations on various radical structures. The g-values and 14N/15N-hyperfine splittings obtained fromthe spectra are consistent with a planar, hydroxyethylidene-thiamine pyrophosphate (HE-TPP) 
hars/pi.gif" BORDER=0 >-radical,in which spin is delocalized onto the thiazolium sulfur and nitrogen atoms. The 1H-hyperfine splittingsfrom the methyl group of pyruvate and the 13C-hyperfine splittings from C2 of both pyruvate and TPP areconsistent with a model in which the pyruvate-derived oxygen atom of the HE-TPP radical forms a hydrogenbond. The hyperfine splitting constants and g-values are not compatible with those predicted for a nonplanar,hars/sigma.gif" BORDER=0 >/n-type cation radical.
hars/pi.gif" BORDER=0 >-radical,in which spin is delocalized onto the thiazolium sulfur and nitrogen atoms. The 1H-hyperfine splittingsfrom the methyl group of pyruvate and the 13C-hyperfine splittings from C2 of both pyruvate and TPP areconsistent with a model in which the pyruvate-derived oxygen atom of the HE-TPP radical forms a hydrogenbond. The hyperfine splitting constants and g-values are not compatible with those predicted for a nonplanar,hars/sigma.gif" BORDER=0 >/n-type cation radical.