Two-dimensional pulsed electron spin resonance characterization of 15N-labeled archaeal Rieske-type ferredoxin

AbstractTwo-dimensional electron spin-echo envelope modulation (ESEEM) analysis of the uniformly 15N-labeled archaeal Rieske-type [2Fe–2S] ferredoxin (ARF) from Sulfolobus solfataricus P1 has been conducted in comparison with the previously characterized high-potential protein homologs. Major differences among these proteins were found in the hyperfine sublevel correlation (HYSCORE) lineshapes and intensities of the signals in the (++) quadrant, which are contributed from weakly coupled (non-coordinated) peptide nitrogens near the reduced clusters. They are less pronounced in the HYSCORE spectra of ARF than those of the high-potential protein homologs, and may account for the tuning of Rieske-type clusters in various redox systems

One and Two Dimensional Pulsed Electron Paramagnetic Resonance Studies of in vivo Vanadyl Coordination in Rat Kidney

The biological fate of a chelated vanadium source is investigated by/n vivo spectroscopic methods to elucidate the chemical form in which the metal ion is accumulated. A pulsed electron paramagnetic resonance study of vanadyl ions in kidney tissue, taken from rats previously treated with bis(ethylmaltolato)oxovanadium(IV) (BEOV) in drinking water, is presented. A combined approach using stimulated echo (3-pulse) electron spin echo envelope modulation (ESEEM) and the two dimensional 4-pulse hyperfine sublevel correlation (HYSCORE) spectroscopies has shown that at least some of the VO2+ ions are involved in the coordination with nitrogen-containing ligands. From the experimental spectra, a 4N hyperfine coupling constant of 4.9 MHz and a quadrupole coupling constant of 0.6 + 0.04 MHz were determined, consistent with amine coordination of the vanadyl ions. Study of VO-histidine model complexes allowed for a determination of the percentage of nitrogen-coordinated VO2+ ions in the tissue sample that is found nitrogen-coordinated. By taking into account the bidentate nature of histidine coordination to VO2+ ions, a more accurate determination of this value is reported. The biological fate of chelated versus free (i.e. salts) vanadyl ion sources has been deduced by comparison to earlier reports. In contrast to its superior pharmacological efficacy over VOSO4, BEOV shares a remarkably similar biological fate after uptake into kidney tissue

Two-Dimensional Pulsed EPR Resolves Hyperfine Coupling Strain in Nitrogen Hydrogen Bond Donors of Semiquinone Intermediates

Hydrogen bonding between semiquinone (SQ) intermediates and side-chain or backbone nitrogens in protein quinone processing sites (Q-sites) is a common motif. Previous studies on SQs from multiple protein environments have reported specific features in the ¹⁵N HYSCORE spectra not reproducible by a theory based on fixed hyperfine parameters, and the source of these lineshape distortions remained unknown. In this work, using the spectra of the SQ in the Q-sites of wild-type and mutant D75H cytochrome <i>bo</i>₃ ubiquinol oxidase from Escherichia coli, we have explained the observed additional features as originating from <i>a</i>-strain of the isotropic hyperfine coupling. In two-dimensional spectra, the <i>a</i>-strain manifests as well-resolved lineshape distortions of the basic cross-ridges and accompanying lines of low intensity in the opposite quadrant that allow its direct analysis. We have shown that their appearance is regulated by the relative values of the strain width, Δ<i>a</i>, and parameter, δ = |2<i>a</i> + <i>T</i>| – 4ν_15N. α-strain provides a direct measure of the structural dynamics and heterogeneity of the O···H···N bond in the SQ systems

Nuclear hyperfine and quadrupole tensor characterization of the nitrogen hydrogen bond donors to the semiquinone of the QB site in bacterial reaction centers: A combined X- and S-band 14,15N ESEEM and DFT study

[Image: see text] The secondary quinone anion radical Q(B)(–) (SQ(B)) in reaction centers of Rhodobacter sphaeroides interacts with N(δ) of His-L190 and N(p) (peptide nitrogen) of Gly-L225 involved in hydrogen bonds to the Q(B) carbonyls. In this work, S-band (∼3.6 GHz) ESEEM was used with the aim of obtaining a complete characterization of the nuclear quadrupole interaction (nqi) tensors for both nitrogens by approaching the cancelation condition between the isotropic hyperfine coupling and (14)N Zeeman frequency at lower microwave frequencies than traditional X-band (9.5 GHz). By performing measurements at S-band, we found a dominating contribution of N(δ) in the form of a zero-field nqi triplet at 0.55, 0.92, and 1.47 MHz, defining the quadrupole coupling constant K = e(2)qQ/4h = 0.4 MHz and associated asymmetry parameter η = 0.69. Estimates of the hyperfine interaction (hfi) tensors for N(δ) and N(p) were obtained from simulations of 1D and 2D (14,15)N X-band and three-pulse (14)N S-band spectra with all nuclear tensors defined in the SQ(B) g-tensor coordinate system. From simulations, we conclude that the contribution of N(p) to the S-band spectrum is suppressed by its strong nqi and weak isotropic hfi comparable to the level of hyperfine anisotropy, despite the near-cancelation condition for N(p) at S-band. The excellent agreement between our EPR simulations and DFT calculations of the nitrogen hfi and nqi tensors to SQ(B) is promising for the future application of powder ESEEM to full tensor characterizations

Conformational differences between the methoxy groups of QA and QB site ubisemiquinones in bacterial reaction centers: A key role for methoxy group orientation in modulating ubiquinone redox potential

Ubiquinone is an almost universal, membrane-associated redox mediator. Its ability to accept either one or two electrons allows it to function in critical roles in biological electron transport. The redox properties of ubiquinone in vivo are determined by its environment in the binding sites of proteins and by the dihedral angle of each methoxy group relative to the ring plane. This is an attribute unique to ubiquinone among natural quinones and could account for its widespread function with many different redox complexes. In this work, we use the photosynthetic reaction center as a model system for understanding the role of methoxy conformations in determining the redox potential of the ubiquinone/semiquinone couple. Despite the abundance of X-ray crystal structures for the reaction center, quinone site resolution has thus far been too low to provide a reliable measure of the methoxy dihedral angles of the primary and secondary quinones, Q(A) and Q(B). We performed 2D ESEEM (HYSCORE) on isolated reaction centers with ubiquinones (13)C-labeled at the headgroup methyl and methoxy substituents, and have measured the (13)C isotropic and anisotropic components of the hyperfine tensors. Hyperfine couplings were compared to those derived by DFT calculations as a function of methoxy torsional angle allowing estimation of the methoxy dihedral angles for the semiquinones in the Q(A) and Q(B) sites. Based on this analysis, the orientation of the 2-methoxy groups are distinct in the two sites, with Q(B) more out of plane by 20-30°. This corresponds to an ≈50 meV larger electron affinity for the Q(B) quinone, indicating a substantial contribution to the experimental difference in redox potentials (60-75 mV) of the two quinones. The methods developed here can be readily extended to ubiquinone-binding sites in other protein complexes

Determination of the Complete Spin Density Distribution in ¹³C-Labeled Protein-Bound Radical Intermediates Using Advanced 2D Electron Paramagnetic Resonance Spectroscopy and Density Functional Theory

Determining the complete electron spin density distribution for protein-bound radicals, even with advanced pulsed electron paramagnetic resonance (EPR) methods, is a formidable task. Here we present a strategy to overcome this problem combining multifrequency HYSCORE and ENDOR measurements on site-specifically ¹³C-labeled samples with DFT calculations on model systems. As a demonstration of this approach, pulsed EPR experiments are performed on the primary Q_A and secondary Q_B ubisemiquinones of the photosynthetic reaction center from <i>Rhodobacter sphaeroides</i> ¹³C-labeled at the ring and tail positions. Despite the large number of nuclei interacting with the unpaired electron in these samples, two-dimensional X- and Q-band HYSCORE and orientation selective Q-band ENDOR resolve and allow for a characterization of the eight expected ¹³C resonances from significantly different hyperfine tensors for both semiquinones. From these results we construct, for the first time, the most complete experimentally determined maps of the <i>s</i>- and <i>p</i>_π-orbital spin density distributions for any protein organic cofactor radical to date. This work lays a foundation for understanding the relationship between the electronic structure of semiquinones and their functional properties, and introduces new techniques for mapping out the spin density distribution that are readily applicable to other systems