Igor Gromov

Probing the role of the proximal heme ligand in cytochrome P450cam by recombinant incorporation of selenocysteine

The unique monooxygenase activity of cytochrome P450cam has been attributed to coordination of a cysteine thiolate to the heme cofactor. To investigate this interaction, we replaced cysteine with the more electron-donating selenocysteine. Good yields of the selenoenzyme were obtained by bacterial expression of an engineered gene containing the requisite UGA codon for selenocysteine and a simplified yet functional selenocysteine insertion sequence (SECIS). The sulfur-to-selenium substitution subtly modulates the structural, electronic, and catalytic properties of the enzyme. Catalytic activity decreases only 2-fold, whereas substrate oxidation becomes partially uncoupled from electron transfer, implying a more complex role for the axial ligand than generally assumed.

The continuous wave electron paramagnetic resonance experiment revisited.

When the modulation frequency used in continuous wave electron paramagnetic resonance (cw EPR) spectroscopy exceeds the linewidth, modulation sidebands appear in the spectrum. It is shown theoretically and experimentally that these sidebands are actually multiple photon transitions, sigma(+)+kxpi, where one microwave (mw) sigma(+) photon is absorbed from the mw radiation field and an arbitrary number k of radio frequency (rf) pi photons are absorbed from or emitted to the modulation rf field. Furthermore, it is demonstrated that both the derivative shape of the lines in standard cw EPR spectra and the distortions due to overmodulation are caused by the unresolved sideband pattern of these lines. The single-photon transition does not even give a contribution to the first-harmonic cw EPR signal. Multiple photon transitions are described semiclassically in a toggling frame and their existence is proven using second quantization. With the toggling frame approach and perturbation theory an effective Hamiltonian for an arbitrary sideband transition is derived. Based on the effective Hamiltonians an expression for the steady-state density operator in the singly rotating frame is derived, completely describing all sidebands in all modulation frequency harmonics of the cw EPR signal. The relative intensities of the sidebands are found to depend in a very sensitive way on the actual rf amplitude and the saturation of single sidebands is shown to depend strongly on the effective field amplitude of the multiple photon transitions. By comparison with the analogous solutions for frequency-modulation EPR it is shown that the field-modulation and the frequency-modulation technique are not equivalent. The experimental data fully verify the theoretical predictions with respect to intensities and lineshapes.

A multi-frequency pulse EPR and ENDOR approach to study strongly coupled nuclei in frozen solutions of high-spin ferric heme proteins.

In spite of the tremendous progress in the field of pulse electron paramagnetic resonance (EPR) in recent years, these techniques have been scarcely used to investigate high-spin (HS) ferric heme proteins. Several technical and spin-system-specific reasons can be identified for this. Additional problems arise when no single crystals of the heme protein are available. In this work, we use the example of a frozen solution of aquometmyoglobin (metMb) to show how a multi-frequency pulse EPR approach can overcome these problems. In particular, the performance of the following pulse EPR techniques are tested: Davies electron nuclear double resonance (ENDOR), hyperfine correlated ENDOR (HYEND), electron-electron double resonance (ELDOR)-detected NMR, and several variants of hyperfine sublevel correlation (HYSCORE) spectroscopy including matched and SMART HYSCORE. The pulse EPR experiments are performed at X-, Q- and W-band microwave frequencies. The advantages and drawbacks of the different methods are discussed in relation to the nuclear interaction that they intend to reveal. The analysis of the spectra is supported by several simulation procedures, which are discussed. This work focuses on the analysis of the hyperfine and nuclear-quadrupole tensors of the strongly coupled nuclei of the first coordination sphere, namely, the directly coordinating heme and histidine nitrogens and the 17O nucleus of the distal water ligand. For the latter, 17O-isotope labeling was used. The accuracy of our results and the spectral resolution are compared in detail to an earlier single-crystal continuous-wave ENDOR study on metMb, and it will be shown how additional information can be obtained from the multi-frequency approach. The current work is therefore prone to become a template for future EPR/ENDOR investigations of HS ferric heme proteins for which no single crystals are available.

Corrin nitrogens and remote dimethylbenzimidazole nitrogen interactions in Cob(II)alamin studied with HYSCORE at X- and Q-band

Abstract A continuous wave and pulse electron paramagnetic resonance study of the base-on form of cob(II)alamin diluted in hydroxocob(III)alamin powder is presented. HYSCORE spectroscopy at X- and Q-band was used to study the weakly coupled corrin nitrogens and the remote nitrogen of the axial dimethylbenzimidazole ligand. Simulations of the spectra measured at different field positions enabled the following parameters to be determined; for the corrin nitrogens the hyperfine principal values are A 1 =−4.5 MHz, A 2 =−3.4 MHz and A 3 =(−2.2 to −2.5) MHz with Euler angles [ α , β , γ ]=[ n 90,25,0]° and the nuclear quadrupole parameters are e 2 qQ/h=1.75 MHz and η =0.95 with Euler angles [ α , β , γ ]=[90+ n 90,90,12.5]°, where n =0,1,2,3. The remote nitrogen of the dimethylbenzimidazole ligand has A 1 =1.75 MHz , A 2 =1.75 MHz and A 3 =2.10 MHz with Euler angles [ α , β , γ ]=[−45,−10,0]° and e 2 qQ/h=−3.3 MHz and η =0.1 with Euler angles [ α , β , γ ]=[45,90,67]°.

Multiple-photon transitions in EPR spectroscopy

An overview of the various multiple-photon processes in electron paramagnetic resonance (EPR) spectroscopy is given. First, we describe different types of multiple-photon transitions that can be observed with monochromatic and bichromatic microwave fields in spin systems with unequally spaced energy levels and in two-level systems. Then we discuss multiple-photon processes that are based on a bichromatic radiation field consisting of a transverse microwave field and a longitu- dinal radio frequency field. Two semiclassical methods, namely the tilted frame and the toggling frame approach, as well as the quantized radiation field formalism (sec- ond quantization), and Floquet theory are used for the theoretical description of the effects. We discuss how these processes manifest in the conventional field modulated continuous wave EPR experiment and demonstrate how multiple-photon transition can be observed in pulse EPR. Finally, new features of multiple-photon transitions induced by bichromatic radiation fields are introduced. We particularly stress on the phenomenon of π-photon-induced transparency and describe the characteristics of these transitions and their potential applications.

Probehead operating at 35GHz for continuous wave and pulse electron paramagnetic resonance applications

The design, construction, numerical modeling, and performance of a probehead for electron paramagnetic resonance (EPR) spectroscopy at 34–36GHz is described. A classical cylindrical cavity operating in the TE011 mode with adjustable frequency and coupling has been found to be well suited for continuous wave and pulse EPR studies of frozen solutions of transition metal complexes at low temperature. The highest attention is given to the probehead performance in the pulse mode. The implemented design has been analyzed in detail using numerical modeling. The distribution of the electromagnetic fields, eigenfrequencies, quality factors, coupling coefficients, and conversion factors are calculated and compared with experimental data. The results demonstrate the effectiveness of the design and can serve as a guide for probehead optimization.

Applications of π-photon-induced transparency in two-frequency pulse electron paramagnetic resonance experiments

An approach to pulse electron paramagnetic resonance (EPR) experiments which are based on two different resonance fields is introduced. Instead of using two microwave (mw) sources or a magnetic field jump, bichromatic pulses consisting of a transverse microwave field with frequency omega(mw) and a longitudinal radio frequency field with frequency omega(rf) are employed. Such bichromatic pulses excite a number of multiple photon transitions at frequencies omega(mw)+komega(rf) (k in Z). The pi-photon-induced transparency phenomenon is used to select the required transitions. This approach is used in the stimulated soft electron spin echo envelope modulation and the four-pulse double electron-electron resonance experiments. The results obtained using the bichromatic pulse approach are in agreement with those obtained with the standard pulse EPR techniques. It is shown that applying bichromatic pulses is straightforward and advantageous in several respects.

Radio frequencies in EPR: Conventional and advanced use

This mini-review focuses on various aspects of the application of radio frequency (rf) irradiation in electron paramagnetic resonance (EPR). The development of the electron-nuclear double resonance (ENDOR) technique is briefly described, and we highlight the use of circularly polarized rf fields and pulse ENDOR methodology in one- and two-dimensional experiments. The capability of pulse ENDOR at Q-band is illustrated with interesting experimental examples. Electron spin echo envelope modulation effects induced by an rf field in liquid samples demonstrate another role which rf fields can play. Technical achievements in the design of ENDOR resonators are illustrated by the example of a bridged loop-gap resonator. Finally, the influence of longitudinal rf fields on the dynamics of EPR transitions is explained using a dressed spin resonance treatment.