Very Low-Frequency Broadband Electron Paramagnetic Resonance Spectroscopy of Metalloproteins

A previously developed
spectrometer for broadband electron paramagnetic
resonance (EPR) spectroscopy of dilute randomly oriented systems has
been considerably modified to extend the frequency reach down to the
hundred MHz range and to boost concentration sensitivity by 1 to 2
orders of magnitude. The instrument is now suitable for the study
of biological systems in particular metalloproteins. As a proof of
concept, examples from the class of low-spin ferric hemoproteins are
studied in terms of frequency-dependent changes in their EPR spectra.
Mono-heme cytochrome c EPR is determined by g-strain
over a wide frequency range, whereas a combination of unresolved ligand
hyperfine interaction and concentration-dependent intermolecular dipolar
interaction becomes dominant at very low frequencies. In the four
heme containing cytochrome c3, g-strain
combines with intramolecular dipolar interaction over the full-studied
frequency range of 0.23–12.0 GHz. It is concluded that the
point-dipole approach is inappropriate to describe magnetic interactions
between low-spin ferric heme systems and that a body of literature
on redox interactions in multi-heme proteins will be affected by this
conclusion.

此前研制的一台适用于稀薄随机取向系统的宽带电子顺磁共振（EPR）光谱仪，已经过大幅改进，以拓展其频率范围至百兆赫兹以下，并显著提升其浓度灵敏度达一个至两个数量级。该仪器现适用于研究生物系统，尤其是金属蛋白。作为验证概念的实例，对低自旋铁血红素蛋白类别的频率依赖性EPR光谱变化进行了研究。在单血红素细胞色素c的EPR研究中，g-应变在宽频率范围内被确定，而在极低频率下，未解决的配体超精细相互作用和浓度依赖性的分子间偶极相互作用变得突出。在含四个血红素的细胞色素c3中，g-应变与分子内偶极相互作用在整个0.23–12.0 GHz的研究频率范围内结合。据此得出结论，点偶极方法不适用于描述低自旋铁血红素系统之间的磁相互作用，因此，关于多血红素蛋白中氧化还原相互作用的文献体系将受到此结论的影响。