EPR spectroscopic and computational characterization of the hydroxyethylidene-thiamine pyrophosphate radical intermediate of pyruvate:ferredoxin oxidoreductase.
The radical intermediate of pyruvate:ferredoxin oxidoreductase (PFOR) from Moorella thermoacetica was characterized using electron paramagnetic resonance (EPR) spectroscopy at X-band and D-band microwave frequencies. EPR spectra, obtained with various combinations of isotopically labeled substrate (pyruvate) and coenzyme (thiamine pyrophosphate (TPP)), were analyzed by spectral simulations. Parameters obtained from the simulations were compared with those predicted from electronic structure calculations on various radical structures. The g-values and 14N/15N-hyperfine splittings obtained from the spectra are consistent with a planar, hydroxyethylidene-thiamine pyrophosphate (HE-TPP) pi-radical, in which spin is delocalized onto the thiazolium sulfur and nitrogen atoms. The 1H-hyperfine splittings from the methyl group of pyruvate and the 13C-hyperfine splittings from C2 of both pyruvate and TPP are consistent with a model in which the pyruvate-derived oxygen atom of the HE-TPP radical forms a hydrogen bond. The hyperfine splitting constants and g-values are not compatible with those predicted for a nonplanar, sigma/n-type cation radical.
Mansoorabadi
Steven O
SO
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53726-4087, USA.
Seravalli
Javier
J
Furdui
Cristina
C
Krymov
Vladimir
V
Gerfen
Gary J
GJ
Begley
Tadhg P
TP
Melnick
Jonathan
J
Ragsdale
Stephen W
SW
Reed
George H
GH
D004578:Electron Spin Resonance Spectroscopy; D008958:Models, Molecular; D050743:Pyruvate Synthase / Q000737:chemistry*; D013835:Thiamine Pyrophosphate / Q000031:analogs & derivatives* / Q000737:chemistry
C444428:hydroxyethylidene-thiamine pyrophosphate; D050743:Pyruvate Synthase; D013835:Thiamine Pyrophosphate