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In this work, we examined the influence of F114P, F114N, and N47S mutations on the electronic structure of the T1 copper center in azurin (Az) by studying the hyperfine couplings of CβH2 protons of Cys112 using the two-dimensional (2D) pulsed EPR technique HYSCORE (hyperfine sublevel correlation). Our results show that isotropic hyperfine couplings for the two Cys112 Cβ protons in WTAz, F114NAz, and N47SAz vary within 21 ± 2 MHz, with differences of only several tenths of MHz between the two protons in a given protein despite major variations previously observed in SC112 electron density. Furthermore, significantly smaller couplings of 0.7 MHz and 7.4 MHz are observed for the F114PAz mutation—which eliminates a backbone amide H-bonding interaction with SC112—despite previous observations of a significant increase in S character in the SOMO, demonstrating a more complex relationship between Cβ–1H hyperfine and the unpaired spin density of SC112 than previously proposed. In addition, the 2D HYSCORE spectra show resolvable differences in the anisotropic couplings between the two protons in all samples, indicating inconsistencies between previously reported ENDOR and computational data for the WTAz protein. Other findings visible in 2D spectra is the distortion of the cross-ridges from Cys112 CβH2 protons with the large isotropic couplings resulted from aiso strain produced by the fluctuations of the Cβ‒H2 group orientation within θ ~ ± 2° degrees around the most populated conformation. [ABSTRACT FROM AUTHOR]

The “Histidine-brace” (His-brace) copper-binding site, composed of Cu(His)2 with a backbone amine, is found in metalloproteins with diverse functions. A primary example is lytic polysaccharide monooxygenase (LPMO), a class of enzymes that catalyze the oxidative depolymerization of polysaccharides, providing not only an energy source for native microorganisms but also a route to more effective industrial biomass conversion. Despite its importance, how the Cu His-brace site performs this unique and challenging oxidative depolymerization reaction remains to be understood. To answer this question, we have designed a biosynthetic model of LPMO by incorporating the Cu His-brace motif into azurin, an electron transfer protein. Spectroscopic studies, including ultraviolet-visible (UV–Vis) absorption and electron paramagnetic resonance, confirm copper binding at the designed His-brace site. Moreover, the designed protein is catalytically active towards both cellulose and starch, the native substrates of LPMO, generating degraded oligosaccharides with multiturnovers by C1 oxidation. It also performs oxidative cleavage of the model substrate 4-nitrophenyl-D-glucopyranoside, achieving a turnover number ~9% of that of a native LPMO assayed under identical conditions. This work presents a rationally designed artificial metalloenzyme that acts as a structural and functional mimic of LPMO, which provides a promising system for understanding the role of the Cu His-brace site in LPMO activity and potential application in polysaccharide degradation. [ABSTRACT FROM AUTHOR]

In this study, the superoxide radical O2•− formed by treating Ti(OR)4 (R = iPr, nBu) with H2O2 in the presence of KOH was detected in the EPR spectra. The g-tensor of this radical differs from the typical values reported for a superoxide on various TiO2 surfaces. On the other hand, similar g-tensor components g||(zz = 2.10 ± 0.01, g⊥ = 2.005 ± 0.003 assigned to the O2•− were previously observed for radicals in aqueous solutions in the presence of K2O, alkaline solutions of DMSO, and water/DMSO mixtures. A common factor in all these systems is the presence of alkali ions. However, there was no structural support for the possible interaction of alkali ions with a superoxide in these systems. The use of multifrequency pulsed EPR techniques in this work revealed the stabilization of the O2•− near the K+ ion and its involvement in a strong hydrogen bond with the surface. These findings are consistent with the features previously reported for superoxides on a Na pre-covered MgO surface. Interactions with a closely located 23Na and a strongly coupled 1H proton were also seen in the HYSCORE spectra but assigned to two different superoxides with various gzz values presented in the sample. [ABSTRACT FROM AUTHOR]

A selective heterogeneous catalyst with the superoxide anion O2•− stable at room temperature, generated by treating Ti(OR)4 (R = iPr, nBu) with H2O2, was previously reported and used for the oxidation of organic compounds. It was proposed that exceptional stability of the O2•− is resulted from its stabilization near Ti+4 cation on the TiO2 surface with the participation of water molecules and/or OH groups. In this article, we performed high-resolution pulsed EPR study on the O2•− in this catalyst with the aim to obtain quantitative information about its interactions with the surrounding molecules. Our data has shown that the O2•− is involved in weak hyperfine interactions with protons in the nearest environment, which do not exceed a value of 2 MHz. It corresponds to an Os...H distance of ≥ 3 Å that excludes the formation of hydrogen bonds. The analysis of possible structures of O2•− adsorption on TiO2 surface taking into account our results and lack of radical mobility at room temperature previously observed in EPR spectra led us to suggest that the O2•− is stabilized in the form of superoxo bridge coordinated with two titanium atoms. [ABSTRACT FROM AUTHOR]

The study represents the first detection of a tridentate coordination mode for triphosphate in solution. It has utilized advanced electron spin echo modulation and four-pulse- two-dimensional hyperfine sublevel correlation spectroscopic techniques to characterize the solution structure of the 1:1 triphosphate.

The two-dimensional ESEEM spectroscopy, known as HYSCORE, offers higher resolution and substantially more information compared with one-dimensional techniques for the characterization of the paramagnetic centers involving large numbers of magnetically nonequivalent nuclei. This was concluded from the application of the spectroscopic technique to the study of hyperfine couplings from nitrogens of histidine and azide ligands. The hyperfine couplings of all coordinated azide and histidine nitrogens were successfully assigned for a geometry very close to that of the parent differic state.

Electron spin-echo envelope modulation experiments were performed to characterize the nitrogens surrounding the Rieske-type (2Fe-2S) cluster in 2,4,5-trichlorophenoxyacetate monooxygenase from Burkholderia cepacia AC1100. The results showed variations in the isotropic hyperfine couplings of the 'ligated nitrogens in the two histidines coordinated to the iron of the reduced Rieske cluster.'

A set of C43(DE3) and BL21(DE3) Escherichia coli host strains that are auxotrophic for various amino acids is briefly reviewed. These strains require the addition of a defined set of one or more amino acids in the growth medium, and have been specifically designed for overproduction of membrane or water-soluble proteins selectively labelled with stable isotopes, such as 2H, 13C and 15N. The strains described here are available for use and have been deposited into public strain banks. Although they cannot fully eliminate the possibility of isotope dilution and mixing, metabolic scrambling of the different amino acid types can be minimized through a careful consideration of the bacterial metabolic pathways. The use of a suitable auxotrophic expression host strain with an appropriately isotopically labelled growth medium ensures high levels of isotope labelling efficiency as well as selectivity for providing deeper insight into protein structure–function relationships. [ABSTRACT FROM AUTHOR]

The pulsed EPR spectroscopy technique is employed to explain the interaction taking place between the various nitrogen donors and the semiquinone in the [Q.sub.B] site of bacterial reaction centers. The formation of hydrogen bonds by these semiquinone forms is also analyzed.

Continuous-wave (CW) EPR spectra of reduced [2Fe-2S][(Cys).sub.3][(His).sub.1] clusters of mammalian mitoNEET soluble domain has produced features resulting from the interaction of the electron spins of the two adjacent clusters. The studies have shown that these quantitative data may be used in the future functional and theoretical studies on the mammalian mitoNEET [2Fe-2S] cluster system.

Escherichia coli (cyt) [bo.sub.3] ubiquinol oxidase has catalyzed the two-electron oxidation of ubiquinol and the four-electron reduction of [O.sub.2] to water. The results have formed a basis for more advanced theoretical analysis of the protein-semiquinone (SQ) interaction in the [Q.sub.H] site, using the model of the neutral radical indicated by the proton hyperfine couplings.

The weakly coupled (super 15)N(sub E) and (super 15)N(sub p) nuclei around the archaeal Rieske center in the uniformly (super 15)N-labeled SDX produce readily observable signatures in the hyperfine sublevel correlation spectroscopic (HYSCORE) spectra. The HYSCORE experiment can be used to study the structure-mechanism studies of a wide range of the biological redox protein system involving these nitrogens.

The presence of two different types of proteins with distinct biological electron-transfer functions is reflected in the redox potential (Em) cluster, and its pH dependence. A comparative study of the pH-dependent RR spectral changes, of three Rieske-type [2Fe-2S] proteins with different redox properties, is presented.

Orientationally selected two-, three-, and four-pulse electron spin echo envelope modulation (ESEEM) spectra from remote 15N nuclei in a Cu2+-(15N–imidazole)4 complex have been obtained in the microwave frequency region 9.12–9.82 GHz. This range of frequencies has allowed us to reach the conditions corresponding to S and ν⊥ singularities of the lineform of basic transitions. The development of these lineform singularities in orientationally selected spectra is discussed. The knowledge of the magnetic fields corresponding to the singularities was used to find the hyperfine coupling parameters for the remote nitrogen as a=2.44 MHz and T⊥=-0.39 MHz as well as to determine the angle between the axis of the complex and the direction of the largest principal hyperfine tensor element as 40°. Additional geometrical information about the ligand orientation has been obtained from the analysis of shifts of the sum combination harmonic in proton four-pulse ESEEM spectra. [ABSTRACT FROM AUTHOR]

Canthaxanthin and 8' -apo-beta-caroten-8' -al radical cations chemically prepared on activated silica-alumina and in CH2Cl2 solution containing AlCl3 are studied by pulsed EPR and electron nuclear double resonance (ENDOR).

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 bo3 ubiquinol oxidase from Escherichia coli, we have explained the observed additional features as originating from a-strain of the isotropic hyperfine coupling. In two-dimensional spectra, the a-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, Δa, and parameter, δ = |2a + T| - 4v15N. α-strain provides a direct measure of the structural dynamics and heterogeneity of the O···H···N bond in the SQ systems. [ABSTRACT FROM AUTHOR]