Applications of pulsed EPR spectroscopy to the study of mitochondrial iron-sulphur clusters and Escherichia coli fumarate reductase

Abstract

Pulsed methods of electron paramagnetic resonance (EPR) spectroscopy open up new possibilities for studying the structure and properties of paramagnetic centres in biological systems 11 I . The electron spin-echo envelope modulation (ESEEM) technique offers improved resolution of hyperfine interactions between an EPR-detectable species and nuclei of ligand atoms. I t has been shown to be particularly sensitive to weak interactions with quadrupolar nuclei, and is therefore suited for the detection of nuclei, such as 14N, in the vicinity of a paramagnetic centre 121. The spin-echo method allows the accurate determination of time-domain properties, such as the hptn-lattice (T,) and the spin-spin (Tz) relaxation times. Spinspin interactions between the various mitochondrial centres may be observed, and the distances between the centres can then, in principle, be determined. These pulsed methods have so far mainly been applied to the study of transition metal complexes and purified metalloproteins, such as ferredoxins. Our experiments were undertaken to assess the feasibility of pulsed EPR measurements on the more complex proteins of unpurified electron-transferring membranes. The spectrometer used was a Bruker ESP380, fitted with a liquid helium flow cryostat (Oxford Instruments, CS935). Preliminary experiments were done on the, previously studied [3], soluble, 2-subunit form of fumarate reductase (FRD) from Escherichia coli , a membrane protein analogous to the mitochondrial succinate dehydrogenase (SDH). In mitochondria the major EPR-detectable species at low temperatures are the iron-sulphur clusters 141 o f Complex I (Centres Nl-N4), Complex I1 (Centres S 1 S 3 ) and Complex 111 (the Rieske iron-sulphur protein). Submitochondrial particles (SMP) were prepared by sonication of bovine heart mitochondria. The high protein concentration required for pulsed measurements was achieved by centrifuging the SMP suspension inside quartz EPR tubes. Two samples were prepared, and NADH was then added to one tube. Continuous-wave (c.w) EPR spectra were recorded at ISK, with a Bruker ESP300 spectrometer, and verified that each tube contained the expected spectral species; that is, the sample as prepared (oxidized) gave a signal at g=2.01, typical of SDH (a subunit of Complex 11) centre S3, and the NADHreduced sample gave rise to various signals typical of centres N 1 , N2, N3 and N4 of Complex 1. Note that centres S1 and S2 of SDH are probably superimposed on the g=1.94 signal from centre N 1. ESEEM data were collected at 4K (by the Bruker Pulsespel