Spin Distribution and the Location of Protons in Paramagnetic Proteins

Abstract

Two current frontiers in EPR research are high-field ( nu0 > 70 GHz, B0 > 2.5 T ) electron paramagnetic resonance (EPR) and high-field electron-nuclear double resonance (ENDOR). This review focuses on recent advances in high-field ENDOR and its applications to the study of proteins containing native paramagnetic sites. It concentrates on two aspects; the first concerns the determination of the location of protons and is related to the site geometry, and the second focuses on the spin density distribution within the site, which is inherent to the electronic structure. Both spin density and proton locations can be derived from ligand hyperfine couplings determined by ENDOR measurements. A brief description of the experimental methods is presented along with a discussion of the advantages and disadvantages of high-field ENDOR compared with conventional X-band (~ 9.5 GHz) experiments. Specific examples of both protein single crystals and frozen solutions are then presented. These include the determination of the coordinates of water ligand protons in the Mn(II) site of concanavalin A, the detection of hydrogen bonds in a quinone radical in the bacterial photosynthetic reaction center as well as in the tyrosyl radical in ribonuclease reductase, and the study of the spin distribution in copper proteins. The copper proteins discussed are the type I copper of azurin and the binuclear CuA center in a number of proteins. The last part of the review presents a brief discussion of the interpretation of hyperfine couplings using quantum chemical calculations, primarily density functional theory (DFT) methods. Such methods are becoming an integral part of the data analysis tools, as they can facilitate signal assignment and provide the ultimate relation between the experimental hyperfine couplings and the electronic wave function.

Cross-polarisation edited ENDOR

“…here [i-j] equal [1][2][3] and [2][3][4] for the {e-n} spin system and [1][2][3][4][5], [2][3][4][5][6], [3][4][5][6][7] and [4][5][6][7][8] for the {e-n 1 -n 2 } spin system. Angle ϕ [i−j ] corresponds to the projection of the effective field in the [i-j] electron manifold on the x-y plane.…”

“…This technique at high magnetic fields provides strongly improved spectral resolution [5,6]. Here, not only orientational selectivity but also increased resolution in the nuclear Larmor frequencies and suppression of second-order effects substantially simplify analysis and interpretation of hyperfine spectra.…”

Exploring hyperfine interactions in spin-correlated radical pairs from photosynthetic proteins: High-frequency ENDOR and quantum beat oscillations

“…ENDOR is sensitive only to specific nuclei that are interacting with a specific electron spin. This feature establishes the main application of ENDOR spectroscopy as a tool to probe the electronic structure, wave-function delocalization, and magnetic interactions with the surroundings of paramagnetic centers [2][3][4]. Although the techo.G.…”

“…nique was initially developed for applications in solid-state physics, particularly for the study of the defects in semiconductor and dielectric materials [5,6], ENDOR soon became a crucial tool for biophysical research, such as studies of photosynthesis [3,7]. Thus, the identification of the structure of the primary electron donor of the purple photosynthetic bacterial reaction center (RC) protein asa dimer of bacteriochlorophyll molecules was unequivocally established with ENDOR spectroscopy [8,9].…”

Spin-correlated radical pairs: the differential effect in high-field ENDOR spectra

“…These approaches yield unique information on dynamics, structure and electron transfer pathways of the photosynthetic proteins [1][2][3]. Concomitantly, the associated HF method of electron-nuclear double resonance (ENDOR), which measures electron-nuclear hyperfine interactions (HFI), has the potential to directly probe the structure of cofactors and their local protein environments [1,2,[11][12][13].…”