Ryszard J. Gurbiel

Metalloenzyme Active-Site Structure and Function through Multifrequency CW and Pulsed ENDOR

Electron paramagnetic resonance (EPR) techniques have long been major tools in efforts to determine the structure and function of metalloen-zyme active sites (Beinert et al., 1962; Hoff, 1989). Much of the information EPR provides about the composition, structure, and bonding of a paramagnetic metal center is obtained by analysis of hyperfine coupling constants (Abragam and Bleaney, 1970; Atherton, 1973) that arise from interactions between the spin of the unpaired electron(s) and the spins of nuclei associated with the metal center, endogenous ligands, or bound substrate. At the most basic level, the observation of hyperfine coupling and its assignment to one or more nuclei (e.g., 1H, 14N) provide information about the chemical composition of the center. Detailed analysis of these couplings can provide information about its geometry or about substrate binding, as well as deep insights into chemical bonding. In principle these coupling constants can be calculated from splittings in the EPR spectrum. However, as illustrated in Fig. 1, for most metalloproteins these splittings cannot be resolved, and thus the chemical information they carry is lost.

Polycrystalline ENDOR patterns from centers with axial EPR spectra. General formulas and simple analytic expressions for deriving geometric information from dipolar couplings

Abstract We present a formulation for rapid simulations of polycrystalline ENDOR patterns for systems whose EPR spectra exhibit axial-resolved magnetic interactions (g tensor and “central”-nucleus hyperfine couplings). The ENDOR response of interest is from a nucleus with nonaxial hyperfine couplings and unresolved splittings. The equations simplify when the hyperfine tensor of the nucleus being observed in ENDOR has axial symmetry in its principal axis frame, as would occur for an I = 1 2 nucleus (1H or 19F) interacting by a through-space, electron-nuclear dipolar interaction. In such cases the field dependence of the ENDOR frequencies can be described by simple analytic functions, well suited to least-squares determination of metrical parameters that define the nuclear position. Sample calculations are given for a proton interacting with a nitroxide radical.

The effects of in vivo and in vitro non-enzymatic glycosylation and glycoxidation on physico-chemical properties of haemoglobin in control and diabetic patients

The erythrocyte deformability, which is related to erythrocyte internal viscosity, was suggested to depend upon the physico-chemical properties of haemoglobin. In the present study we employed ESR spectroscopy on order to explore further the extent to which the in vivo or in vitro glycation and/or glycoxidation might affect haemoglobin structure on conformation. We revealed that under both in vivo and in vitro conditions the attachment of glucose induced a mobilization of thiol groups in the selected domains of haemoglobin molecules ( the increased h+1/h0 parameter of maleimide spin label, MSL; 0.277 +/- 0.021 in diabetics vs 0.338 +/- 0.017 in controls, n = 12, P < 0.0001). The relative rotational correlation time (tau c) of two spin labels, TEMPONE and TEMPAMINE, respectively, in erythrocyte insides (5.22 +/- 0.42 in diabetics, n = 21 vs 4.79 +/- 0.38, n = 16 in controls, P < 0.005) and in the solutions of in vitro glycated haemoglobin, were increased. Neither oxidation nor crosslinking of thiol groups was evidenced in glycated and/or oxidized haemoglobin. In addition, erythrocyte deformability was found to be reduced in type 2 diabetic patients (6.71 +/- 1.08, n = 28 vs 7.31 +/- 0.96, n = 21, P < 0.015). In conclusion, these observations suggest that: the attachment of glucose to haemoglobin might have decreased the mobility of the Lys-adjacent Cys residues, thus leading to the increased h+1/h0 parameter of MSL. Such structural changes in haemoglobin owing to non-enzymatic glycosylation may contribute to the increased viscosity of haemoglobin solutions (r = 0.497, P < 0.0035) and the enhanced internal viscosity of diabetic erythrocytes (r = 0.503, P < 0.003). We argue that such changes in haemoglobin, and consequently in red blood cells, might contribute to the handicapped oxygen release under tissue hypoxia in the diabetic state.

Single-crystal EPR and ENDOR study of nitrogenase from clostridium pasteurianum

Abstract The molybdenum-iron (MoFe) protein of nitrogenase from Clostridium pasteurianum forms monoclinic crystals in space group P21. The unit cell comprises two molecules, each of which possesses a molecular twofold symmetry axis and binds two chemically identical molybdenum-iron cofactors (M centers). Thus, there are four magnetically distinct M centers within the unit cell related by two different symmetry operations: a molecular twofold axis of symmetry in the bc ∗ plane relates the two EPR centers within a molecule; a crystallographic twofold screw axis of symmetry along the b axis of the unit cell relates the two different molecules in a unit cell. Single-crystal EPR studies at X-band (9.5 GHz) and Q-band (35 GHz) microwave frequencies have been employed to determine g tensors in the crystallographic frame for the four magnetically distinct M centers. Determination of these tensors has been achieved by a novel procedure that relies heavily on the symmetry relations between sites; it rests primarily on measurements that only involve rotation of the magnetic field parallel to a single plane of the crystal and uses g values from frozen-solution samples instead of additional rotations. Single-crystal 1H ENDOR spectra of the M centers are presented.

The ups and downs of Feher-style ENDOR

A new transient variation of the “Feher-style” electron-nuclear double resonance (ENDOR) method is examined. In this technique, the passage-mode electron paramagnetic resonance (EPR) signal is monitored following the application of a pulsed radio frequency (RF). Continuous-wave and transient proton ENDOR experiments have been conducted on the nonheme iron center from the protein nitrile hydratase. These experiments show that the transient ENDOR signal response exhibits a complex response with multiple phases in the time evolution of the ENDOR signal. Both increases and decreases in the passage-mode EPR signals are observed at different times following the RF pulse that induces an ENDOR transition. A simple model based on a packet-shifting ENDOR mechanism for a nonadiabatic passage EPR signal is proposed. This model describes many of the features seen in the transient ENDOR experiments and provides new insight into the traditional Feher-style ENDOR measurements. This new model shows that a packet-shifting mechanism can account for many of the “negative ENDOR” effects commonly seen in Feher-style ENDOR, which suggests that more exotic ENDOR mechanisms may not be required to explain these observations.

Nailfold capillaroscopy assessment of microcirculation abnormalities and endothelial dysfunction in children with primary or secondary Raynaud syndrome

Raynaud syndrome (RS) manifests as episodes of transient spasms of peripheral blood vessels, most often in response to cold. The reason of that symptom (primary RS (pRS)) usually cannot be found but may be accompanied by some autoimmune diseases (secondary RS (sRS)). In this study, we assessed microcapillary status and serum concentrations of chosen cytokines, adhesive molecules, and nitric oxide (NO) in patients with pRS and sRS in comparison with healthy children. Eighty-six patients with RS were enrolled into the study, including 52 with pRS and 34 with sRS. The control group consisted of 29 healthy children. A decrease in myorelaxative and anticoagulant abilities was observed, with simultaneous prevalence of vasopressor substances and procoagulative activity. Therefore, several important factors such as endothelin-1 (ET-1), E-selectin (E-sel), interleukin-18 (IL-18), and nitrogen oxide (NO) were also analyzed. Two types of capillaroscopy status were determined: normal and microangiopathic. There was a significant relationship between presence of microangiopathy and higher serum ET-1 (p = 0.018) and E-sel (p = 0.021) levels. Similarly, we have found a correlation between presence of ANA and higher ET-1 (p = 0.005), but not E-sel (p = 0.241). In patients with pRS, we found significant relationship between ANA and higher ET-1 (p = 0.008). No such relations were observed in sRS patients. Our data indicates that external factor-induced vasoconstrictive effects dominated in pRS, whereas in sRS in the course of connective tissue diseases, it was accompanied by coexistent vasodilation due to endothelial dysfunction. The latter phenomenon is at least partially dependent on insufficient NO release.

Cytochrome c Oxidase: A Brief Introduction and Some New Results from High Field Endor Studies of The CuA and CuB Sites

A Mitchellian view of mitochondrial respiration is presented in Scheme I.1,2 This illustrates electron transfer from an electron rich material, NH, through quinone, Q, through Complex HI, into cytochrome c, and through Complex IV(cytochrome c oxidase) to O2, with accompanying proton translocations across the lipid bilayer.

B14: FPGA-based fast ADC interface and preprocessing unit

Abstract The paper describes a design of the FPGA-based unique device for the Electron Paramagnetic Resonance spectrometer. The design includes a digital interface for fast, 800Msps analog-to-digital converter. Important part of the design is a digital signal preprocessing unit, allowing fast accumulation of multiple data for separating weak signals from the noise. Additionally, the unit provides certain means to control the remaining part of the measurement device. The paper shortly introduces the experiment environment, focusing on interesting FPGA design issues. Results of experiments, proving the measurement quality of the whole device are presented as well.