Mansoorabadi Steven O, Seravalli Javier, Furdui Cristina, Krymov Vladimir, Gerfen Gary J, Begley Tadhg P, Melnick Jonathan, Ragsdale Stephen W, Reed George H
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53726-4087, USA.
Biochemistry. 2006 Jun 13;45(23):7122-31. doi: 10.1021/bi0602516.
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.
利用X波段和D波段微波频率的电子顺磁共振（EPR）光谱对来自嗜热栖热菌的丙酮酸：铁氧化还原蛋白氧化还原酶（PFOR）的自由基中间体进行了表征。通过光谱模拟分析了用同位素标记的底物（丙酮酸）和辅酶（硫胺焦磷酸（TPP））的各种组合获得的EPR光谱。将从模拟中获得的参数与从各种自由基结构的电子结构计算预测的参数进行了比较。从光谱中获得的g值和14N/15N超精细分裂与平面羟基亚乙基硫胺焦磷酸（HE-TPP）π自由基一致，其中自旋离域到噻唑鎓硫和氮原子上。来自丙酮酸甲基的1H超精细分裂以及来自丙酮酸和TPP的C2的13C超精细分裂与一个模型一致，在该模型中，HE-TPP自由基的丙酮酸衍生氧原子形成氢键。超精细分裂常数和g值与非平面σ/n型阳离子自由基预测的不兼容。
