Andrei V. Astashkin

Pulsed dipolar spectroscopy distance measurements in biomacromolecules labeled with Gd(III) markers

This work demonstrates the feasibility of using Gd(III) tags for long-range Double Electron Electron Resonance (DEER) distance measurements in biomacromolecules. Double-stranded 14- base pair Gd(III)-DNA conjugates were synthesized and investigated at K(a) band. For the longest Gd(III) tag the average distance and average deviation between Gd(III) ions determined from the DEER time domains was about 59±12Å. This result demonstrates that DEER measurements with Gd(III) tags can be routinely carried out for distances of at least 60Å, and analysis indicates that distance measurements up to 100Å are possible. Compared with commonly used nitroxide labels, Gd(III)-based labels will be most beneficial for the detection of distance variations in large biomacromolecules, with an emphasis on large scale changes in shape or distance. Tracking the folding/unfolding and domain interactions of proteins and the conformational changes in DNA are examples of such applications.

Pulsed EPR Determination of the Distance between Heme Iron and FMN Centers in a Human Inducible Nitric Oxide Synthase

Mammalian nitric oxide synthase (NOS) is a homodimeric flavo-hemoprotein that catalyzes the oxidation of L-arginine to nitric oxide (NO). Regulation of NO biosynthesis by NOS is primarily through control of interdomain electron transfer (IET) processes in NOS catalysis. The IET from the flavin mononucleotide (FMN) to heme domains is essential in the delivery of electrons required for O(2) activation in the heme domain and the subsequent NO synthesis by NOS. The NOS output state for NO production is an IET-competent complex of the FMN-binding domain and heme domain, and thereby it facilitates the IET from the FMN to the catalytic heme site. The structure of the functional output state has not yet been determined. In the absence of crystal structure data for NOS holoenzyme, it is important to experimentally determine the Fe...FMN distance to provide a key calibration for computational docking studies and for the IET kinetics studies. Here we used the relaxation-induced dipolar modulation enhancement (RIDME) technique to measure the electron spin echo envelope modulation caused by the dipole interactions between paramagnetic FMN and heme iron centers in the [Fe(III)][FMNH(*)] (FMNH(*): FMN semiquinone) form of a human inducible NOS (iNOS) bidomain oxygenase/FMN construct. The FMNH(*)...Fe distance has been directly determined from the RIDME spectrum. This distance (18.8 +/- 0.1 A) is in excellent agreement with the IET rate constant measured by laser flash photolysis [Feng, C. J.; Dupont, A.; Nahm, N.; Spratt, D.; Hazzard, J. T.; Weinberg, J.; Guillemette, J.; Tollin, G.; Ghosh, D. K. J. Biol. Inorg. Chem. 2009, 14, 133-142].

High-field pulsed EPR and ENDOR of Gd3+ complexes in glassy solutions

In this work D-band pulsed electron paramagnetic resonance was used to record the field-sweep spectra of several Gd3+ complexes in glassy water-methanol solutions. These spectra were analyzed by a specially developed stochastic superposition model that predicted the essential features of the distribution of the crystal field interaction (CFI) parameters in glassy systems. As a result of this analysis, the CFI distributions for the studied complexes were evaluated. The D-band Mims1H electron-nuclear double resonance spectra were free from CFI-related distortions, which allowed us to accurately determine the hyperfine interaction (HFI) parameters for water ligand protons and to unequivocally establish that the HFI distribution is solely related to the distribution of the Gd−H distances.

The distances from tyrosine D to redox-active components on the donor side of Photosystem II determined by pulsed electron-electron double resonance

Abstract A pulsed electron-electron double resonance (ELDOR) method was applied to measyre the dipole interactions between paramagnetic species on the donor side of Photosystem II. The distance between the Mn cluster and the redox-active tyrosine residue YD was determined to be 27 ± 0.2 A in the S2 state of the oxygen-evolving Photosystem II. In Ca2+-depleted preparations the same distance was obtained from the measurement of the modified multiline signal. The distance between YD and the paramagnetic species giving rise to the split S3 signal was found to be 30 ± 0.2 A .

Comparative modulation analysis of electron spin echo signals from oxidized chlorophyll a in vitro and P 700 centres in chloroplasts

Abstract A comparative analysis of electron spin echo modulation effects from 14 N nuclei of pyrrole rings for Chla + and P 700 + has been performed, yielding parameters of quadrupole and isotropic hyperfine couplings with nitrogen nuclei in these paramagnetic centres.

Spectrometer dead time: effect on electron spin echo modulation spectra in disordered systems

Abstract The effect of spectrometer dead time on line shapes in ESE modulation spectra for disordered systems with S = 1 2 , I = 1 2 has been studied by means of numerical calculations.

Effects of Interdomain Tether Length and Flexibility on the Kinetics of Intramolecular Electron Transfer in Human Sulfite Oxidase

Sulfite oxidase (SO) is a vitally important molybdenum enzyme that catalyzes the oxidation of toxic sulfite to sulfate. The proposed catalytic mechanism of vertebrate SO involves two intramolecular one-electron transfer (IET) steps from the molybdenum cofactor to the iron of the integral b-type heme and two intermolecular one-electron steps to exogenous cytochrome c. In the crystal structure of chicken SO [Kisker, C., et al. (1997) Cell 91, 973-983], which is highly homologous to human SO (HSO), the heme iron and molybdenum centers are separated by 32 A and the domains containing these centers are linked by a flexible polypeptide tether. Conformational changes that bring these two centers into greater proximity have been proposed [Feng, C., et al. (2003) Biochemistry 42, 5816-5821] to explain the relatively rapid IET kinetics, which are much faster than those theoretically predicted from the crystal structure. To explore the proposed role(s) of the tether in facilitating this conformational change, we altered both its length and flexibility in HSO by site-specific mutagenesis, and the reactivities of the resulting variants have been studied using laser flash photolysis and steady-state kinetics assays. Increasing the flexibility of the tether by mutating several conserved proline residues to alanines did not produce a discernible systematic trend in the kinetic parameters, although mutation of one residue (P105) to alanine produced a 3-fold decrease in the IET rate constant. Deletions of nonconserved amino acids in the 14-residue tether, thereby shortening its length, resulted in more drastically reduced IET rate constants. Thus, the deletion of five amino acid residues decreased IET by 70-fold, so that it was rate-limiting in the overall reaction. The steady-state kinetic parameters were also significantly affected by these mutations, with the P111A mutation causing a 5-fold increase in the sulfite K(m) value, perhaps reflecting a decrease in the ability to bind sulfite. The electron paramagnetic resonance spectra of these proline to alanine and deletion mutants are identical to those of wild-type HSO, indicating no significant change in the Mo active site geometry.

Electron spin echo envelope modulation theory for high electron spin systems in weak crystal field

Electron spin echo envelope modulation (ESEEM) experiments with aqueous complexes of Gd3+ and Mn2+ have shown that a common and unusual feature of the primary ESEEM spectra of such high spin/weak crystal field systems is an extremely low intensity of the sum combination line. Numerical simulations of the ESEEM spectra based on the existing theory [Coffino and Peisach, J. Chem. Phys. 97, 3072 (1992); Larsen and Singel, J. Chem. Phys. 98, 6704 (1993)] could not reproduce these ESEEM spectra. In this work the theoretical description of the ESEEM was revised and corrected, and new expressions were derived for the ESEEM from high electron spin (S>1/2) systems in a weak crystal field, interacting with a nuclear spin I=1/2. The corrections primarily affected the shape and intensity of the sum combination line, whose position was found to be sensitive to the product of the crystal field and anisotropic hyperfine interaction constants. These theoretical improvements resulted in a successful simulation of the prima...

Molecular basis for enzymatic sulfite oxidation: How three conserved active site residues shape enzyme activity

Sulfite dehydrogenases (SDHs) catalyze the oxidation and detoxification of sulfite to sulfate, a reaction critical to all forms of life. Sulfite-oxidizing enzymes contain three conserved active site amino acids (Arg-55, His-57, and Tyr-236) that are crucial for catalytic competency. Here we have studied the kinetic and structural effects of two novel and one previously reported substitution (R55M, H57A, Y236F) in these residues on SDH catalysis. Both Arg-55 and His-57 were found to have key roles in substrate binding. An R55M substitution increased Km(sulfite)(app) by 2–3 orders of magnitude, whereas His-57 was required for maintaining a high substrate affinity at low pH when the imidazole ring is fully protonated. This effect may be mediated by interactions of His-57 with Arg-55 that stabilize the position of the Arg-55 side chain or, alternatively, may reflect changes in the protonation state of sulfite. Unlike what is seen for SDHWT and SDHY236F, the catalytic turnover rates of SDHR55M and SDHH57A are relatively insensitive to pH (∼60 and 200 s–1, respectively). On the structural level, striking kinetic effects appeared to correlate with disorder (in SDHH57A and SDHY236F) or absence of Arg-55 (SDHR55M), suggesting that Arg-55 and the hydrogen bonding interactions it engages in are crucial for substrate binding and catalysis. The structure of SDHR55M has sulfate bound at the active site, a fact that coincides with a significant increase in the inhibitory effect of sulfate in SDHR55M. Thus, Arg-55 also appears to be involved in enabling discrimination between the substrate and product in SDH.

Electron spin echo envelope modulation spectroscopy (ESEEM) of the radical cations of 14N and 15N bacteriochlorophyll a

Abstract An electron spin echo envelope modulation frequency analysis is performed of the 14 N and 15 N bacteriochlorophyll a + cation in solid solution. Hyperfine couplings of 2.0 and 3.1 MHz are derived which correspond to the perpendicular component of the axial hyperfine coupling tensor. Seven quadrupole frequencies were measured, six of which group in two sets of three zero-field quadrupole splittings.