Pulse Q-band EPR and ENDOR spectroscopies of the photochemically generated monoprotonated benzosemiquinone radical in frozen alcoholic solution

Abstract

Quinones are essential cofactors in many physiological processes, among them proton-coupled electron transfer (PCET) in photosynthesis and respiration. A key intermediate in PCET is the monoprotonated semiquinone radical. In this work we produced the monoprotonated benzosemiquinone (BQH(•)) by UV illumination of BQ dissolved in 2-propanol at cryogenic temperatures and investigated the electronic and geometric structures of BQH(•) in the solid state (80 K) using EPR and ENDOR techniques at 34 GHz. The g-tensor of BQH(•) was found to be similar to that of the anionic semiquinone species (BQ(•-)) in frozen solution. The peaks present in the ENDOR spectrum of BQH(•) were identified and assigned by (1)H/(2)H substitutions. The experiments reconfirmed that the hydroxyl proton (O-H) on BQH(•), which is abstracted from a solvent molecule, mainly originates from the central CH group of 2-propanol. They also showed that the protonation has a strong impact on the electron spin distribution over the quinone. This is reflected in the hyperfine couplings (hfc's) of the ring protons, which dramatically changed with respect to those typically observed for BQ(•-). The hfc tensor of the O-H proton was determined by a detailed orientation-selection ENDOR study and found to be rhombic, resembling those of protons covalently bound to carbon atoms in a π-system (i.e., α-protons). It was found that the O-H bond lies in the quinone plane and is oriented along the direction of the quinone oxygen lone pair orbital. DFT calculations were performed on different structures of BQH(•) coordinated by four, three, or zero 2-propanol molecules. The O-H bond length was found to be around 1.0 Å, typical for a single covalent O-H bond. Good agreement between experimental and DFT results were found. This study provides a detailed picture of the electronic and geometric structures of BQH(•) and should be applicable to other naturally occurring quinones.

Figure 1

Molecular structure of the monoprotonated benzosemiquinone radical (BQH^•). The principal axes of the g–tensor are expected to be approximately along the molecular axes x, y, z (see DFT section). The principal axes of the hfc tensor corresponding to the O−H proton are along the axes x′, y′, z′, which are related to the g–tensor axes by rotations: (A) around z (angle ϕ) followed by consecutive rotations (B) around y′ (angle θ) and (C) around z′ (angle ψ). The first two Euler angles, ϕ and θ, define the direction of the O−H bond (B). The rotation around z′ (angle ψ) is shown for the case in which the O−H proton lies in the plane of the quinone (θ = 90°) (C).

Figure 2

Experimental (solid line) and simulated (dashed line) Q-band EPR powder spectra of BQH^•−d₄ in (CH₃)₂CHOD. ENDOR spectra were taken at the magnetic field positions indicated by arrows including those corresponding to g_x, g_y and g_z. The linewidth values of ΔB_x, ΔB_y, and ΔB_z obtained from the simulation are 0.46 mT, 0.36 mT, and 0.61 mT. Experimental conditions: T = 80 K, microwave (MW) frequency = 33.9 GHz, spectrum obtained by pseudo-modulating the field-swept FID-detected EPR spectrum with 0.15 mT, 1 scan, 76 s per scan.

Figure 3

¹H Davies Q-band ENDOR spectra of benzosemiquinone radicals prepared under different conditions. Samples were prepared: (a) by UV illumination in (CH₃)₂CHOH, (b) using an alkaline solution of (CD₃)₂CDOD forming the anionic semiquinone radical and (c) by UV illumination in (CD₃)₂CDOD. All spectra were taken along the magnetic field position corresponding to g_z (B₁₁ in Fig. 2) yielding single-crystal type spectra. ENDOR lines corresponding to five different proton species are clearly observed. The respective low and high frequency peaks belonging to hyperfine coupling are labeled (L₁/L₁´, L₂/L₂´, L₃/L₃´, L₄/L₄´ and L₅/L₅´). Experimental conditions: T = 80 K, microwave (MW) frequency = 33.9 GHz. Number of scans: 235 (a), 2093 (b) and 1885 (c). Scan time: 8 s (a), 20 s (b) and 8 s (c).

Figure 4

¹H Davies Q-band ENDOR spectra of fully deuterated benzosemiquinone radicals prepared by UV illumination (BQH^•−d₄) in differently deuterated 2-propanol: (a) (CH₃)₂CHOH, (b) (CH₃)₂CHOD and (c) (CH₃)₂CDOH. Spectra were taken along the magnetic field position corresponding to g_z (B₁₁ in Fig. 2). ENDOR lines corresponding to three different proton species are clearly observed. The peaks are labeled as in Fig. 3. Experimental conditions: T = 80 K, microwave (MW) frequency = 33.9 GHz. Number of scans: 55 (a), 630 (b) and 200 (c). Scan time: 103 s (a), 8 s (b) and 62 s (c).

Figure 5

Experimental (solid lines) and simulated (dotted lines) ¹H Davies Q-band ENDOR spectra of BQH^•−d₄ in (CH₃)₂CHOD at 10 different magnetic field positions of the EPR spectrum (see arrows in Fig. 2). In the fitting of the spin Hamiltonian to the data the frequency range between 49.5 MHz and 53.5 MHz was excluded. In addition, the smallest hfc of O−H (~6 MHz), which is clearly resolved only in the spectra recorded at magnetic fields B₃, B₄ and B₅, was fitted using the positions (frequencies) of the respective ENDOR peaks (see Supporting Information). The hfc splitting A_z is shown by an arrow in (B₁₁). Experimental conditions: T = 80 K, microwave (MW) frequency = 33.89 GHz. Number of scans per position: 420. Scan time: 8 s.

Figure 6

Presentation of the single occupied molecular orbital (SOMO) (A) and the spin density distribution of the monoprotonated benzosemiquinone radical BQH^• (B) coordinated by four 2-propanol molecule (oxygen = red, carbon = gray, hydrogen = white). The non-protonated carbonyl oxygen (O₁, right) is hydrogen bonded to two 2-propanol molecules, and the protonated carbonyl oxygen (O₄, left) to only one 2-propanol molecule. Furthermore, the O−H proton is hydrogen bonded to the oxygen of a fourth 2-propanol molecule. In all calculations, in-plane hydrogen bonding was found.

Figure 7

π-spin density distribution of the benzoquinone radical anion, and the monoprotonated benzosemiquinone neutral radical ligated by four 2-propanol solvent molecules (dotted lines indicate H−bonds) calculated using DFT. The spin density is proportional to the area of the circles (blue = positive, yellow = negative). For values see Table 2 and ref. .