Relaxation-induced Dipolar Modulation Enhancement Research Articles

High-Field Dipolar Electron Paramagnetic Resonance (EPR) Spectroscopy of Nitroxide Biradicals for Determining Three-Dimensional Structures of Biomacromolecules in Disordered Solids

We consider the state-of-the-art capabilities and future perspectives of electron-spin triangulation by high-field/high-frequency dipolar electron paramagnetic resonance (EPR) techniques designed for determining the three-dimensional structure of large supra-molecular complexes dissolved in disordered solids. These techniques combine double site-directed spin labeling (SDSL) with orientation-resolving pulsed electron-electron double resonance (PELDOR) spectroscopy. In particular, we appraise the prospects of angular triangulation, which extends the more familiar distance triangulation. As a model case for spin-labeled proteins, the three-dimensional structures of two nitroxide biradicals with rather stiff bridging blocks and deuterated nitroxide headgroups have been derived. To this end we applied 95 GHz high-field electron dipolar EPR spectroscopy with the microwave pulse-sequence configurations for PELDOR and relaxation-induced dipolar modulation enhancement (RIDME). Various specific spectroscopic strategies are discussed to overcome the problems of overlapping spectra of the chemically identical nitroxide labels when attached to macromolecular systems. We conclude that due to the high detection sensitivity and spectral resolution the combination of SDSL with high-field RIDME/PELDOR stands out as an extremely powerful tool for 3D structure determination of large disordered systems. The approach compares favorably with other structure-determining magnetic-resonance methods. This holds true both for stable and transient radical-pair states. Angular constraints are provided in addition to distance constraints obtained for the same sample. Thereby, the number of necessary distance constraints is strongly reduced. Since each measurement of a distance constraint requires an additional doubly spin-labeled sample, the reduction of necessary distance constraints is another appealing aspect of orientation-resolving EPR spin triangulation for protein structure determination.

Use of Additional Fast-Relaxing Paramagnetic Species for Improvement of RIDME Performance

A method of improving relaxation-induced dipolar modulation enhancement (RIDME) performance by an artificial increase of electron spin–lattice relaxation rate is introduced and tested on a model nitroxide biradical. A substantial increase of its electron spin–lattice relaxation rate is achieved by the addition of a fast-relaxing paramagnetic holmium complex to the sample solution. The electron spin–spin relaxation rate of the nitroxide does not change dramatically upon this addition. The suggested method allows obtaining a deep dipolar modulation in the RIDME experiment, which is free from distortions caused by spectral diffusion processes.

Pulsed EPR Determination of the Distance between Heme Iron and FMN Centers in a Human Inducible Nitric Oxide Synthase

Mammalian nitric oxide synthase (NOS) is a homodimeric flavo-hemoprotein that catalyzes the oxidation of L-arginine to nitric oxide (NO). Regulation of NO biosynthesis by NOS is primarily through control of interdomain electron transfer (IET) processes in NOS catalysis. The IET from the flavin mononucleotide (FMN) to heme domains is essential in the delivery of electrons required for O(2) activation in the heme domain and the subsequent NO synthesis by NOS. The NOS output state for NO production is an IET-competent complex of the FMN-binding domain and heme domain, and thereby it facilitates the IET from the FMN to the catalytic heme site. The structure of the functional output state has not yet been determined. In the absence of crystal structure data for NOS holoenzyme, it is important to experimentally determine the Fe...FMN distance to provide a key calibration for computational docking studies and for the IET kinetics studies. Here we used the relaxation-induced dipolar modulation enhancement (RIDME) technique to measure the electron spin echo envelope modulation caused by the dipole interactions between paramagnetic FMN and heme iron centers in the [Fe(III)][FMNH(*)] (FMNH(*): FMN semiquinone) form of a human inducible NOS (iNOS) bidomain oxygenase/FMN construct. The FMNH(*)...Fe distance has been directly determined from the RIDME spectrum. This distance (18.8 +/- 0.1 A) is in excellent agreement with the IET rate constant measured by laser flash photolysis [Feng, C. J.; Dupont, A.; Nahm, N.; Spratt, D.; Hazzard, J. T.; Weinberg, J.; Guillemette, J.; Tollin, G.; Ghosh, D. K. J. Biol. Inorg. Chem. 2009, 14, 133-142].

Orientation-Resolving Pulsed Electron Dipolar High-Field EPR Spectroscopy on Disordered Solids: I. Structure of Spin-Correlated Radical Pairs in Bacterial Photosynthetic Reaction Centers

Distance and relative orientation of functional groups within protein domains and their changes during chemical reactions determine the efficiency of biological processes. In this work on disordered solid-state electron-transfer proteins, it is demonstrated that the combination of pulsed high-field EPR spectroscopy at the W band (95 GHz, 3.4 T) with its extensions to PELDOR (pulsed electron-electron double resonance) and RIDME (relaxation-induced dipolar modulation enhancement) offers a powerful tool for obtaining not only information on the electronic structure of the redox partners but also on the three-dimensional structure of radical-pair systems with large interspin distances (up to about 5 nm). Strategies are discussed both in terms of data collection and data analysis to extract unique solutions for the full radical-pair structure with only a minimum of additional independent structural information. By this novel approach, the three-dimensional structure of laser-flash-induced transient radical pairs P(865)(*+)Q(A)(*-) in frozen-solution reaction centers (RCs) from the photosynthetic bacterium Rhodobacter (Rb.) sphaeroides is solved. The measured positions and relative orientations of the weakly coupled ion radicals P(865)(*+) and Q(A)(*-) are compared with those of the precursor cofactors P865 and QA known from X-ray crystallography. A small but significant reorientation of the reduced ubiquinone QA is revealed and interpreted as being due to the photosynthetic electron transfer. In contrast to the large conformational change of Q(B)(*-) upon light illumination of the RCs, the small light-induced reorientation of Q(A)(*-) had escaped previous attempts to detect structural changes of photosynthetic cofactors upon charge separation. Although small, they still may be of functional importance for optimizing the electronic coupling of the redox partners in bacterial photosynthesis both for the charge-separation and charge-recombination processes.

Electron dipole–dipole interaction in ESEEM of nitroxide biradicals

Fourier transform of the standard primary electron spin echo envelope modulation (ESEEM) of nitroxide biradicals in organic glass shows a peak ascribed to electron–electron dipole–dipole interaction. The dipolar couplings obtained in imidazoline biradical and double spin-labeled peptide Trichogin GA IV are in good agreement with pulsed electron–electron double resonance (PELDOR) data. The intensity of this peak is increased substantially in a new three-pulse relaxation-induced dipolar modulation enhancement (RIDME) experiment. This experiment involves the non-resonant partner spin in the formation of ESEEM via a relaxation-induced change of the local dipolar field experienced by the resonant spin.