Very Low-Frequency Broadband Electron Paramagnetic Resonance Spectroscopy of Metalloproteins

Abstract

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 c 3 , 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.

Low-frequency EPR of ferrimyoglobin fluoride and ferrimyoglobin cyanide: a case study on the applicability of broadband analysis to high-spin hemoproteins and to HALS hemoproteins

“…Quadrupole splitting from the nitrogen ligands is also ignored. Furthermore, dipolar broadening from intermolecular interaction is expected to be present as previously observed in broadband EPR of low-spin hemoproteins [11]. As can be seen in Fig.…”

“…10A. The g-strain simulator has been described before [11,18]. Lowering the g x to 0.2 (B x = 33,631; S g = 15.3) gives an almost identical (in fact slightly better) fit to the experimental spectrum.…”

“…At frequencies below some 1 GHz spectra of lowspin hemoproteins begin to broaden due to intermolecular dipole-dipole interactions [11], and since this is a (11) S g = g 2…”

“…As a competitive alternative to the single low-frequency spectrometer, I have developed a new type of machine called a broadband EPR spectrometer as it can be tuned to resonance at many microwave frequencies over a range of up to some seven octaves from above X-band down to well into the sub-gigahertz range [9][10][11]. Applicability to dilute randomly oriented samples has been illustrated with analysis of site symmetry in a low-symmetry copper complex [10] and recently with analysis of intra-and inter-molecular dipolar heme-heme interaction in low spin ferric cytochromes (S = 1/2) [11]. This exploration of usefulness is now extended to the class of high-spin ferric hemoproteins (S = 5/2), which pose a very different problem than the previous examples since they exhibit strong zero-field interaction as well as a complex linewidth behavior.…”

X-Band Parallel-Mode and Multifrequency Electron Paramagnetic Resonance Spectroscopy of S = 1/2 Bismuth Centers

“…In conclusion, the application of parallel-mode EPR to an S = 1/2 spin system is an unconventional utilization of the technique and warrants further considerations in other complex S = 1/2 systems. One may envision the development of a new or the modification of current low-frequency and broadband EPR spectrometers 35 to perform parallel-mode EPR spectroscopy for S = 1/2 with large hyperfine couplings.…”