The R2 protein of class I ribonucleotide reductase generates and stores a tyrosyl radical essential for ribonucleotide reduction and, thus, DNA synthesis. X-ray structures of the protein have enabled detailed mechanistic suggestions, but no structural information has been available for the active radical-containing state of the protein. Here we report on methods to generate the functional tyrosyl radical in single crystals of R2 from Escherichia coli (Y122 • ). We further report on subsequent high-field EPR experiments on the radical-containing crystals. A full rotational pattern of the spectra was collected and the orientation of the g-tensor axes were determined, which directly reflect the orientation of the radical in the crystal frame. The EPR data are discussed in comparison with a 1.42-Å x-ray structure of the met (oxidized) form of the protein, also presented in this paper. Comparison of the orientation of the radical Y122 • obtained from high-field EPR with that of the reduced tyrosine Y122-OH reveals a significant rotation of the tyrosyl side chain, away from the diiron center, in the active radical state. Implications for the radical transfer connecting the diiron site in R2 with the substrate-binding site in R1 are discussed. In addition, the present study demonstrates that structural and functional information about active radical states can be obtained by combined x-ray and high-field EPR crystallography. R ibonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides and are thus essential for DNA synthesis. Class I RNRs consist of two homodimeric proteins: R1, which contains the active site and binding site for allosteric regulators, and R2, which generates and harbors the tyrosyl radical Y122• (Escherichia coli numbering) needed for catalysis. The catalytic reaction in R1 is believed to be initiated by a reversible radical transfer from Tyr 122 in R2 to an active-site cysteine in R1.The tyrosyl radical on the R2 subunit is generated by means of the reductive cleavage of molecular oxygen at a diiron center. Crystal structures of E. coli R2 are available for both the reduced diferrous (Fe 2). The structural data have, together with kinetic data and theoretical calculations, served as the basis for the formulation of proposals for the mechanism of radical generation and radical migration in the overall RNR reaction (3-9). However, no structure has been available for the radical-containing form, hence, the orientation and location of the active radical Y122• have not been known. The diiron center is in the active enzyme in the diferric form and couples antiferromagnetically to an S ϭ 0 ground state (3-5, 10). Detailed high-field EPR experiments have been performed on Y122• in frozen R2 solutions (11-15). The obtained g-tensor values were found to be indicators for the polarity of the radical environment. In particular, it was found that the tyrosyl radical is hydrogen-bonded in RNR of mouse and herpes simplex virus (12, 14), whereas in E. coli it is not (11-15).In...

Tryptophan radicals, which are generated in the reconstitution reaction of mutants Y122F and Y177W of subunit R2 apoprotein of E. coli and mouse ribonucleotide reductase (RNR), respectively, with Fe(2+) and oxygen, are investigated by high-field EPR at 94 GHz and compared with the tyrosine radicals occurring in the respective wild-type proteins. For the first time, accurate g-values are obtained for protein-associated neutral tryptophan free radicals, which show only a small anisotropy. The apparent hyperfine patterns observed in frozen solutions are very similar for tryptophan and tyrosine radicals in mouse subunit R2 at conventional X-band EPR. The radicals can, however, be discriminated by their different g-tensors using high-field EPR. Tryptophan radicals were postulated as reaction intermediates in the proposed radical transfer pathway of RNR. Furthermore, the data obtained here for the electronic structure of protein-associated tryptophan neutral free radicals are important for identification and understanding of the functional important tryptophan radicals which occur in other enzymes, e.g., DNA photolyase and cytochrome c peroxidase, where they are magnetically coupled to other radicals or to a metal center.

The photocycle of bacterial photosynthetic reaction centers (RCs) involves electron transfer between two quinone molecules, QA and QB. The semiquinone biradical QA -•QB -• forms an intermediate state in this process. We trapped the biradical at low temperature (77 K) and investigated its EPR spectra at three microwave frequencies, 9.6, 35, and 94 GHz, at temperatures between 1.5 and 100 K. The spectra were described with a spin Hamiltonian that contained, in addition to the Zeeman terms, dipolar and exchange interactions, and were fitted using the simulated annealing method (Kirkpatrick et al. Science 1983, 220, 671). From the parameters derived from the fit, information about the spatial and electronic structure was obtained. The relative position and orientation of the two quinones, determined from the EPR spectra, compared well with those obtained from X-ray diffraction of RCs in the QAQB -• state (Stowell et al. Science 1997, 276, 812). The values of the dipolar coupling and of the exchange interaction obtained from the fits were E d/h = (10.3 ± 0.1) MHz and J o/h = (−60 ± 20) MHz, respectively. The value of J o was used to estimate a maximum electron-transfer rate, k ET, (QA -•QB -• → QAQB =) of ∼109 s-1. This agrees within an order of magnitude with the value derived from kinetics experiments (Graige et al. Biochemistry 1999, 38, 11465).

The charge separated state P 700•+ A 1•-(P 700 ) primary electron donor, A 1 ) phylloquinone electron acceptor) in photosystem I of oxygenic photosynthesis has been investigated by EPR spectroscopy in frozen solution and single crystals. The transient EPR spectra of P 700•+ A 1 •recorded in frozen solution of fully deuterated samples at X-, Q-, and W-band frequencies are shown to contain sufficient information to yield the orientation of the g-tensors of both A 1•and P 700•+ with respect to the axis connecting both spins. So far incomplete information on the orientation of A 1•relative to the membrane plane has been complemented by data from time-resolved EPR on single crystals measured at Q-band. The phylloquinone headgroup orientation evaluated from the EPR data in the charge-separated state P 700•+ A 1 •is compared with the presently available X-ray structural model. The g-tensor of P 700 •+ has also been determined from cw-EPR experiments at W-band on single crystals, independent of the orientational data of the P 700 •+ g-tensor from the time-resolved EPR experiments.The direction of the principal axes of g(P 700•+ ) differ from the molecular axes system of the chlorophylls comprising P 700 as found previously in the case of P 865•+ in bacterial reaction centers. The implications of the complete structural model from the A 1 •and P 700•+ molecular magnetic interaction tensors in the active charge separated state P 700•+ A 1 •in PS I are discussed.

Articles you may be interested inA kilowatt pulsed 94 GHz electron paramagnetic resonance spectrometer with high concentration sensitivity, high instantaneous bandwidth, and low dead time Rev. Sci. Instrum. A quasioptical transient electron spin resonance spectrometer operating at 120 and 240 GHz Rev. Sci. Instrum. 76, 074101 (2005);High-field/high-frequency electron spin resonance ͑ESR͒ offers improved sensitivity and resolution compared to ESR at conventional fields and frequencies. However, most high-field/high-frequency ESR spectrometers suffer from limited mm-wave power, thereby requiring long mm-wave pulses. This precludes their use when relaxation times are short, e.g., in fluid samples. Low mm-wave power is also a major factor limiting the achievable spectral coverage and thereby the multiplex advantage of Fourier transform ESR ͑FTESR͒ experiments. High-power pulses are needed to perform two-dimensional ͑2D͒ FTESR experiments, which can unravel the dynamics of a spin system in great detail, making it an excellent tool for studying spin and molecular dynamics. We report on the design and implementation of a high-power, high-bandwidth, pulsed ESR spectrometer operating at 95 GHz. One of the principal design goals was the ability to investigate dynamic processes in aqueous samples at physiological temperatures with the intent to study biological systems. In initial experiments on aqueous samples at room temperature, we achieved 200 MHz spectral coverage at a sensitivity of 1.1ϫ10 10 ͱs spins and a dead time of less than 50 ns.2D-electron-electron double resonance experiments on aqueous samples are discussed to demonstrate the practical application of such a spectrometer.

Using frequency-modulation atomic force microscopy ͑FM-AFM͒ at sub-nanometer vibration amplitudes, we find in the system n-dodecanol/graphite that solvation layers may extend for several nanometers into the bulk liquid. These layers maintain crystalline order which can be imaged using FM-AFM. The energy dissipation of the vibrating tip can peak sharply upon penetration of molecular layers. The tip shape appears critical for this effect.A notable property of liquid near the liquid-solid interface is the presence of solvation layers, i.e., ordering of liquid molecules due to boundary conditions imposed by the solid. Such layering has been observed via oscillatory solvation forces when confining the liquid between the substrate and a solid probe using the surface force apparatus 1 and atomic force microscopy ͑AFM͒. 2,3 While solvation forces are routinely detected via the modulation of the force, the lateral structure of the solvation shells-beyond the first adsorbed monolayer-has rarely been directly observed. One exception are long-chain alkanes adsorbed from solution, for which a second layer has been imaged using scanning tunneling microscopy ͑STM͒. 4 Unfortunately, STM does not lend itself to imaging of "higher" layers as the tunneling current decreases exponentially with the thickness of the confined ͑nonconductive͒ liquid. The lamellar structure of a second solvation layer of hexadecane was imaged recently using a tuning fork AFM. 5 There remain open questions about the structure of higher solvation layers, namely, whether they exhibit lateral order. These questions will become increasingly important for high-resolution AFM imaging in liquid, in particular, for hydration layers in biological systems. 6,7 The development of instrumentation capable of smallamplitude, frequency-modulated AFM ͑FM-AFM͒ ͑Ref. 8͒ in liquid environments 9 has dramatically increased the resolution and sensitivity achievable in AFM studies in liquid. We apply FM-AFM to a linear alcohol ͑dodecanol͒ on an atomically flat graphite substrate slightly above the bulk freezing temperature. We find not only spectroscopic evidence of multiple solvation layers, i.e., force oscillations but also obtain real-space topography images of the alcohol molecules in higher layers, demonstrating that the solvation layers in this system have a crystalline structure. Further we sometimes observe, depending on the condition of the tip, sharp peaks in the mechanical dissipation just as a solvation layer is squeezed out of the tip-sample gap.To achieve the sensitivity necessary for molecular resolution AFM in liquid, a commercial AFM ͑Molecular Imaging Picoscope͒ was modified. Changes to the optical beam deflection sensor include replacement of the standard laser source with a home-built, rf-modulated diode laser 9 and modification of the focusing optics to achieve a smaller numerical aperture. Using standard silicon cantilevers ͑type NCLR, Nanosensors, Neuchatel, Switzerland and type ACLA, AppNano, Santa Clara, CA͒ with a spring constant of k c Ϸ 40 N / m, a ...

Electron paramagnetic resonance (EPR) spectroscopy at 94 GHz is used to study the dark-stable tyrosine radical Y D• in single crystals of photosystem II core complexes (cc) isolated from the thermophilic cyanobacterium Synechococcus elongatus. These complexes contain at least 17 subunits, including the water-oxidizing complex (WOC), and 32 chlorophyll a molecules͞PS II; they are active in light-induced electron transfer and water oxidation. I n oxygenic photosynthesis, two photosystems (PS I and PS II) function in sequence to convert light into energy-rich chemical compounds (1, 2). PS I uses energy from the absorption of a photon to reduce NADP ϩ to NADPH, which is required for CO 2 reduction. The electrons for this process are donated by PS II. On light excitation of PS II, an electron is transferred from the primary donor P 680 , a chlorophyll species, via an intermediate pheophytin Pheo a to the plastoquinone acceptors Q A and Q B . Two sequential univalent redox steps and concomitant protonation events lead to plastohydroquinol Q B H 2 , which leaves PS II and provides electrons to PS I via the cytochrome b 6 f complex (for review, see ref.2). The photooxidized cation radical P 680•ϩ has the highest oxidation potential of all cofactors known in nature (Նϩ1.1 V), which is sufficient for water oxidation. P 680•ϩ extracts an electron from a redox active tyrosine Y Z . The intermediate tyrosine radical Y Z• , in turn, oxidizes the water-oxidizing complex (WOC), a tetranuclear manganese cluster. The WOC passes through a cycle of four one-electron oxidation steps in which water is oxidized and protons and O 2 are released (for reviews, see refs. 3-7). The exact water-splitting mechanism is still unknown.The cofactors involved in the electron transfer chain of PS II are bound to two protein subunits, D 1 and D 2 . From amino acid sequence homology (8-10), two-dimensional electron crystallography (11), and computer modeling (12), D 1 and D 2 are assumed to be arranged analogously to the L and M subunits in the reaction center of purple bacteria. This analogy has been supported recently by x-ray crystallographic studies of the PS II single crystals (13). Whereas Y Z in D 1 connects P 680•ϩ to the WOC in the electron transfer chain, the homologous Y D in D 2 does not seem to take part in the charge separation process. However, Y D is also coupled to the WOC and, under illumination, forms a dark-stable radical Y D• (Fig. 1). The functional role of Y D is not understood in detail; it may be necessary for assembly of the PS II complex (14, 15). Recent results also suggest that it may play a role in preventing photoinhibition during activation of the PS II complex (16).In the light-induced single electron transfer process and in the water-splitting cycle, various paramagnetic species are formed (17) that have been studied by conventional X-band (9 GHz) electron paramagnetic resonance (EPR) techniques during the last decade. Most of these species can be observed only in the freeze-trapped state in frozen PS II solutions. ...

High-field ESR offers many advantages in exploring fundamental questions of structure and dynamics in chemical, biological and physical samples. We provide a review of recent work performed at ACERT demonstrating the utility and flexibility of our methods for extracting both qualitative and quantitative information from a variety of systems. In particular, we emphasize the utility of multi-frequency ESR techniques for unraveling the details of the complex dynamical modes of proteins in solution and in heterogeneous systems such as lipid bilayers. We also include indications of directions for future work where appropriate.