Relaxation Filtered Hyperfine (REFINE) Spectroscopy: A Novel Tool for Studying Overlapping Biological Electron Paramagnetic Resonance Signals Applied to Mitochondrial Complex I
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- 作者：Thorsten Maly ; Fraser MacMillan ; Klaus Zwicker ; Noushin Kashani-Poor ; Ulrich Brandt ; and Thomas F. Prisner
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：April 6, 2004
- 年：2004
- 卷：43
- 期：13
- 页码：3969 - 3978
- 全文大小：122K
- 年卷期：v.43,no.13(April 6, 2004)
- ISSN：1520-4995
文摘
A simple strategy to separate overlapping electron paramagnetic resonance (EPR) signals inbiological systems is presented. Pulsed EPR methods (inversion- and saturation-recovery) allow thedetermination of the T1 spin-lattice relaxation times of paramagnetic centers. T1 may vary by severalorders of magnitude depending on the species under investigation. These variations can be employed tostudy selectively individual species from a spectrum that results from an overlap of two species using aninversion-recovery filtered (IRf) pulsed EPR technique. The feasibility of such an IRf field-swept techniqueis demonstrated on model compounds (
,
-bisphenylene-
-phenylallyl-benzolate, BDPA, and 2,2,6,6-tetramethyl-piperidine-1-oxyl, TEMPO) and a simple strategy for the successful analysis of such mixturesis presented. Complex I is a multisubunit membrane protein of the respiratory chain containing severaliron-sulfur (FeS) centers, which are observable with EPR spectroscopy. It is not possible to investigatethe functionally important FeS cluster N2 separately because this EPR signal always overlaps with theother FeS signals. This cluster can be studied selectively using the IRf field-swept technique and its EPRspectrum is in excellent agreement with previous cw-EPR data from the literature. In addition, the possibilityto separate the hyperfine spectra of two spectrally overlapping paramagnetic species is demonstrated byapplying this relaxation filter together with hyperfine spectroscopy (REFINE). For the first time, theapplication of this filter to a three-pulse electron spin-echo envelope modulation (ESEEM) pulse sequenceis 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.
,-bisphenylene--phenylallyl-benzolate, BDPA, and 2,2,6,6-tetramethyl-piperidine-1-oxyl, TEMPO) and a simple strategy for the successful analysis of such mixturesis presented. Complex I is a multisubunit membrane protein of the respiratory chain containing severaliron-sulfur (FeS) centers, which are observable with EPR spectroscopy. It is not possible to investigatethe functionally important FeS cluster N2 separately because this EPR signal always overlaps with theother FeS signals. This cluster can be studied selectively using the IRf field-swept technique and its EPRspectrum is in excellent agreement with previous cw-EPR data from the literature. In addition, the possibilityto separate the hyperfine spectra of two spectrally overlapping paramagnetic species is demonstrated byapplying this relaxation filter together with hyperfine spectroscopy (REFINE). For the first time, theapplication of this filter to a three-pulse electron spin-echo envelope modulation (ESEEM) pulse sequenceis 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.