Relaxation Filtered Hyperfine (REFINE) Spectroscopy:  A Novel Tool for Studying Overlapping Biological Electron Paramagnetic Resonance Signals Applied to Mitochondrial Complex I

Abstract

A simple strategy to separate overlapping electron paramagnetic resonance (EPR) signals in biological systems is presented. Pulsed EPR methods (inversion- and saturation-recovery) allow the determination of the T(1) spin-lattice relaxation times of paramagnetic centers. T(1) may vary by several orders of magnitude depending on the species under investigation. These variations can be employed to study selectively individual species from a spectrum that results from an overlap of two species using an inversion-recovery filtered (IRf) pulsed EPR technique. The feasibility of such an IRf field-swept technique is demonstrated on model compounds (alpha,gamma-bisphenylene-beta-phenylallyl-benzolate, BDPA, and 2,2,6,6-tetramethyl-piperidine-1-oxyl, TEMPO) and a simple strategy for the successful analysis of such mixtures is presented. Complex I is a multisubunit membrane protein of the respiratory chain containing several iron-sulfur (FeS) centers, which are observable with EPR spectroscopy. It is not possible to investigate the functionally important FeS cluster N2 separately because this EPR signal always overlaps with the other FeS signals. This cluster can be studied selectively using the IRf field-swept technique and its EPR spectrum is in excellent agreement with previous cw-EPR data from the literature. In addition, the possibility to separate the hyperfine spectra of two spectrally overlapping paramagnetic species is demonstrated by applying this relaxation filter together with hyperfine spectroscopy (REFINE). For the first time, the application of this filter to a three-pulse electron spin-echo envelope modulation (ESEEM) pulse sequence is demonstrated to selectively observe hyperfine spectra on a system containing two paramagnetic species. Finally, REFINE is used to assign the observed nitrogen modulation in complex I to an individual iron-sulfur cluster.

Resolving the EPR Spectra in the Cytochrome bc 1 Complex from Saccharomyces cerevisiae

“…2b) at such times, then only one of the two species is detectable, at a reduced intensity. These so-called filter times (T F ) can be experimentally determined or calculated [28,30]. Alternatively a two-dimensional experiment can be performed (Fig.…”

“…Three-pulse ESEEM and ESEEM-REFINE experiments were performed as described in Refs. [28,34]. Figure 2a shows the X-band cw-EPR spectra of the reduced cytochrome bc 1 complex from S. cerevisiae at 9 K. A broad signal from the reduced Rieske center is clearly visible, as well as an intense narrow signal situated at g * 2.0042 typical of a semiquinone [35].…”

“…The inversion-recovery field-swept spectra were obtained as described in Ref. [28] by integrating the area of the echo after detection of the three-pulse sequence (p -T F -p/2 -t -p). Three-pulse ESEEM and ESEEM-REFINE experiments were performed as described in Refs.…”

“…Recently, a technique of relaxation filtered hyperfine (REFINE) spectroscopy was introduced, through which the characterization of the important FeS center N2 in complex I was performed [28]. It was not possible to study this FeS center individually in complex I using pulsed EPR, and this technique was thus successfully developed to address the question of the amino acid coordination of this center in the protein.…”

“…In the experiment a pulse sequence was used which includes an inversion-recovery pulse applied together with a standard Hahn-echo detection sequence [29], which allows the separation of different paramagnetic species by differences in their longitudinal relaxation times (T 1e ). The time between the inversion-recovery pulse and the detection sequence is varied, and depending on this so-called filter time (T F ), it is possible to selectively suppress one of the two overlapping EPR species and thus record the individual EPR spectrum of a second species [28,30].…”