Structural determinants of nitroxide motion in spin-labeled proteins: tertiary contact and solvent-inaccessible sites in helix G of T4 lysozyme

A nitroxide side chain (R1) has been substituted at single sites along a helix-turn-helix motif in T4 lysozyme (residues 114-135). Together with previously published data, the new sites reported complete a continuous scan through the motif. Mutants with R1 at sites 115 and 118 were selected for crystallographic analysis to identify the structural origins of the corresponding two-component EPR spectra. At 115, R1 is shown to occupy two rotamers in the room temperature crystal structure, one of which has not been previously reported. The two components in the EPR spectrum apparently arise from differential interactions of the two rotamers with the surrounding structure, the most important of which is a hydrophobic interaction of the nitroxide ring. Interestingly, the crystal structure at 100 K reveals a single rotamer, emphasizing the possibility of rotamer selection in low-temperature crystal structures. Residue 118 is at a solvent-inaccessible site in the protein core, and the structure of 118R1, the first reported for the R1 side chain at a buried site, reveals how the side chain is accommodated in an overpacked core.     

Crystal structures of spin labeled T4 lysozyme mutants: implications for the interpretation of EPR spectra in terms of structure

High resolution (1.43-1.8 A) crystal structures and the corresponding electron paramagnetic resonance (EPR) spectra were determined for T4 lysozyme derivatives with a disulfide-linked nitroxide side chain [-CH(2)-S-S-CH(2)-(3-[2,2,5,5-tetramethyl pyrroline-1-oxyl]) identical with R1] substituted at solvent-exposed helix surface sites (Lys65, Arg80, Arg119) or a tertiary contact site (Val75). In each case, electron density is clearly resolved for the disulfide group, revealing distinct rotamers of the side chain, defined by the dihedral angles X(1) and X(2). The electron density associated with the nitroxide ring in the different mutants is inversely correlated with its mobility determined from the EPR spectrum. Residue 80R1 assumes a single g(+)()g(+)() conformation (Chi(1) = 286, X(2) = 294). Residue 119R1 has two EPR spectral components, apparently corresponding to two rotamers, one similar to that for 80R1 and the other in a tg(-)() conformation (Chi(1) = 175, X(2) = 54). The latter state is apparently stabilized by interaction of the disulfide with a Gln at i + 4, a situation also observed at 65R1. R1 residues at helix surface site 65 and tertiary contact site 75 make intra- as well as intermolecular contacts in the crystal and serve to identify the kind of molecular interactions possible for the R1 side chain. A single conformation of the entire 75R1 side chain is stabilized by a variety of interactions with the nitroxide ring, including hydrophobic contacts and two unconventional C-H.O hydrogen bonds, one in which the nitroxide acts as a donor (with tyrosine) and the other in which it acts as an acceptor (with phenylalanine). The interactions revealed in these structures provide an important link between the dynamics of the R1 side chain, reflected in the EPR spectrum, and local protein structure. A library of such interactions will provide a basis for the quantitative interpretation of EPR spectra in terms of protein structure and dynamics.     

Structural origins of nitroxide side chain dynamics on membrane protein α-helical sites

Understanding the structure and dynamics of membrane proteins in their native, hydrophobic environment is important to understanding how these proteins function. EPR spectroscopy in combination with site-directed spin labeling (SDSL) can measure dynamics and structure of membrane proteins in their native lipid environment; however, until now the dynamics measured have been qualitative due to limited knowledge of the nitroxide spin label's intramolecular motion in the hydrophobic environment. Although several studies have elucidated the structural origins of EPR line shapes of water-soluble proteins, EPR spectra of nitroxide spin-labeled proteins in detergents or lipids have characteristic differences from their water-soluble counterparts, suggesting significant differences in the underlying molecular motion of the spin label between the two environments. To elucidate these differences, membrane-exposed α-helical sites of the leucine transporter, LeuT, from Aquifex aeolicus, were investigated using X-ray crystallography, mutational analysis, nitroxide side chain derivatives, and spectral simulations in order to obtain a motional model of the nitroxide. For each crystal structure, the nitroxide ring of a disulfide-linked spin label side chain (R1) is resolved and makes contacts with hydrophobic residues on the protein surface. The spin label at site I204 on LeuT makes a nontraditional hydrogen bond with the ortho-hydrogen on its nearest neighbor F208, whereas the spin label at site F177 makes multiple van der Waals contacts with a hydrophobic pocket formed with an adjacent helix. These results coupled with the spectral effect of mutating the i ± 3, 4 residues suggest that the spin label has a greater affinity for its local protein environment in the low dielectric than on a water-soluble protein surface. The simulations of the EPR spectra presented here suggest the spin label oscillates about the terminal bond nearest the ring while maintaining weak contact with the protein surface. Combined, the results provide a starting point for determining a motional model for R1 on membrane proteins, allowing quantification of nitroxide dynamics in the aliphatic environment of detergent and lipids. In addition, initial contributions to a rotamer library of R1 on membrane proteins are provided, which will assist in reliably modeling the R1 conformational space for pulsed dipolar EPR and NMR paramagnetic relaxation enhancement distance determination.     

Structural origin of weakly ordered nitroxide motion in spin‐labeled proteins

A disulfide-linked nitroxide side chain (R1) used in site-directed spin labeling of proteins often exhibits an EPR spectrum characteristic of a weakly ordered z-axis anisotropic motion at topographically diverse surface sites, including those on helices, loops and edge strands of β-sheets. To elucidate the origin of this motion, the first crystal structures of R1 that display simple z-axis anisotropic motion at solvent-exposed helical sites (131 and 151) and a loop site (82) in T4 lysozyme have been determined. Structures of 131R1 and 151R1 determined at cryogenic or ambient temperature reveal an intraresidue C_α—H···S_δ interaction that immobilizes the disulfide group, consistent with a model in which the internal motions of R1 are dominated by rotations about the two terminal bonds (Columbus, Kálai, Jeko, Hideg, and Hubbell, Biochemistry 2001;40:3828–3846). Remarkably, the 131R1 side chain populates two rotamers equally, but the EPR spectrum reflects a single dominant dynamic population, showing that the two rotamers have similar internal motion determined by the common disulfide-backbone interaction. The anisotropic motion for loop residue 82R1 is also accounted for by a common disulfide-backbone interaction, showing that the interaction does not require a specific secondary structure. If the above observations prove to be general, then significant variations in order and rate for R1 at noninteracting solvent-exposed helical and loop sites can be assigned to backbone motion because the internal motion is essentially constant.     

Rotameric preferences of a protein spin label at edge‐strand β‐sheet sites

Protein spin labeling to yield the nitroxide‐based R1 side chain is a powerful method to measure protein dynamics and structure by electron spin resonance. However, R1 measurements are complicated by the flexibility of the side chain. While analysis approaches for solvent‐exposed α‐helical environment have been developed to partially account for flexibility, similar work in β‐sheets is lacking. The goal of this study is to provide the first essential steps for understanding the conformational preferences of R1 within edge β‐strands using X‐ray crystallography and double electron electron resonance (DEER) distance measurements. Crystal structures yielded seven rotamers for a non‐hydrogen‐bonded site and three rotamers for a hydrogen‐bonded site. The observed rotamers indicate contextual differences in R1 conformational preferences compared to other solvent‐exposed environments. For the DEER measurements, each strand site was paired with the same α‐helical site elsewhere on the protein. The most probable distance observed by DEER is rationalized based on the rotamers observed in the crystal structure. Additionally, the appropriateness of common molecular modeling methods that account for R1 conformational preferences are assessed for the β‐sheet environment. These results show that interpretation of R1 behavior in β‐sheets is difficult and indicate further development is needed for these computational methods to correctly relate DEER distances to protein structure at edge β‐strand sites.     

Molecular motion of spin labeled side chains in the C-terminal domain of RGL2 protein: a SDSL-EPR and MD study

Five singly spin labeled side chains at surface sites in the C-terminal domain of RGL2 protein have been analyzed to investigate the general relationship between nitroxide side chain mobility and protein structure. At these sites, the structural perturbation produced by replacement of a native residue with a nitroxide side chain appears to be very slight at the level of the backbone fold. The primary determinants of the nitroxide side chain mobility are backbone dynamics and tertiary interactions. On the exposed surfaces of alpha-helices, the side chain mobility is not restricted by tertiary interactions but appears to be determined by backbone dynamics, while in loop sites, the side chain mobility is even higher. For a better understanding of the changes in the EPR spectral line shape, molecular dynamics simulations were performed and found in agreement with EPR spectral data.     

High-Resolution Structure of a Protein Spin-Label in a Solvent-Exposed β-Sheet and Comparison with DEER Spectroscopy

X-ray crystallography has been a useful tool in the development of site-directed spin labeling by resolving rotamers of the nitroxide spin-label side chain in a variety of α-helical environments. In this work, the crystal structure of a doubly spin-labeled N8C/K28C mutant of the B1 immunoglobulin-binding domain of protein G (GB1) was solved. The double mutant formed a domain-swapped dimer under crystallization conditions. Two rotameric states of the spin-label were resolved at the solvent-exposed α-helical site, at residue 28; these are in good agreement with rotamers previously reported for helical structures. The second site, at residue 8 on an interior β-strand, shows the presence of three distinct solvent-exposed side-chain rotamers. One of these rotamers is rarely observed within crystal structures of R1 sites and suggests that the H(α) and S(δ) hydrogen bond that is common to α-helical sites is absent at this interior β-strand residue. Variable temperature continuous wave (CW) experiments of the β-strand site showed two distinct components that were correlated to the rotameric states observed in crystallography. Interestingly, the CW data at room temperature could be fit without the use of an order parameter, which is consistent with the lack of the H(α) and S(δ) interaction. Additionally, double electron electron resonance (DEER) spectroscopy was performed on the GB1 double mutant in its monomeric form and yielded a most probable interspin distance of 25 ± 1 ?. In order to evaluate the accuracy of the measured DEER distance, the rotamers observed in the crystal structure of the domain-swapped GB1 dimer were modeled into a high-resolution structure of the wild type monomeric GB1. The distances generated in the resulting GB1 structural models match the most probable DEER distance within ～2 ?. The results are interesting as they indicate by direct experimental measurement that the rotameric states of R1 found in this crystal provide a very close match to the most probable distance measured by DEER.     

Determination of interspin distances between spin labels attached to insulin: comparison of electron paramagnetic resonance data with the X-ray structure

A method was developed to determine the interspin distances of two or more nitroxide spin labels attached to specific sites in proteins. This method was applied to different conformations of spin-labeled insulins. The electron paramagnetic resonance (EPR) line broadening due to dipolar interaction is determined by fitting simulated EPR powder spectra to experimental data, measured at temperatures below 200 K to freeze the protein motion. The experimental spectra are composed of species with different relative nitroxide orientations and interspin distances because of the flexibility of the spin label side chain and the variety of conformational substates of proteins in frozen solution. Values for the average interspin distance and for the distance distribution width can be determined from the characteristics of the dipolar broadened line shape. The resulting interspin distances determined for crystallized insulins in the R6 and T6 structure agree nicely with structural data obtained by x-ray crystallography and by modeling of the spin-labeled samples. The EPR experiments reveal slight differences between crystal and frozen solution structures of the B-chain amino termini in the R6 and T6 states of hexameric insulins. The study of interspin distances between attached spin labels can be applied to obtain structural information on proteins under conditions where other methods like two-dimensional nuclear magnetic resonance spectroscopy or x-ray crystallography are not applicable.     

Osmolyte perturbation reveals conformational equilibria in spin‐labeled proteins

Recent evidence suggests that proteins at equilibrium can exist in a manifold of conformational substates, and that these substates play important roles in protein function. Therefore, there is great interest in identifying regions in proteins that are in conformational exchange. Electron paramagnetic resonance spectra of spin‐labeled proteins containing the nitroxide side chain (R1) often consist of two (or more) components that may arise from slow exchange between conformational substates (lifetimes > 100 ns). However, crystal structures of proteins containing R1 have shown that multicomponent spectra can also arise from equilibria between rotamers of the side chain itself. In this report, it is shown that these scenarios can be distinguished by the response of the system to solvent perturbation with stabilizing osmolytes such as sucrose. Thus, site‐directed spin labeling (SDSL) emerges as a new tool to explore slow conformational exchange in proteins of arbitrary size, including membrane proteins in a native‐like environment. Moreover, equilibrium between substates with even modest differences in conformation is revealed, and the simplicity of the method makes it suitable for facile screening of multiple proteins. Together with previously developed strategies for monitoring picosecond to millisecond backbone dynamics, the results presented here expand the timescale over which SDSL can be used to explore protein flexibility.     

Motion of spin-labeled side chains in T4 lysozyme: effect of side chain structure

Previous studies have shown that the mobility of nitroxide side chains in a protein, inferred from the electron paramagnetic resonance (EPR) spectra, can be used to classify particular sites as helix surface sites, tertiary contact sites, buried sites, or loop sites. In addition, the sequence dependence of mobility can identify regular secondary structure. However, in the most widely used side chain, an apparent interaction of the nitroxide ring with the protein at some helix surface sites gives rise to EPR spectra degenerate with those at tertiary contact sites. In the present study, we use selected sites in T4 lysozyme to evaluate novel nitroxide side chains designed to resolve this degeneracy. The results indicate that the reagent 3-(methanesulfonylthiomethyl)-2,2, 5,5-tetramethylpyrrolidin-1-yloxy reacts with cysteine to give a nitroxide side chain that has a high contrast in mobility between helix surface and tertiary contact sites, effectively resolving the degeneracy. The reagent 3-(iodomercuriomethyl)-2,2,5,5-tetramethyl-2, 5-dihydro-1H-pyrrol-1-yloxy reacts with cysteine to provide a mercury-linked nitroxide that also shows reduced interaction with the protein at most helix surface sites. Thus, these new side chains may be the preferred choices for structure determination using site-directed spin labeling.     

Molecular origin of electron paramagnetic resonance line shapes on β-barrel membrane proteins: the local solvation environment modulates spin-label configuration

In this work, electron paramagnetic resonance (EPR) spectroscopy and X-ray crystallography were used to examine the origins of EPR line shapes from spin-labels at the protein-lipid interface on the β-barrel membrane protein BtuB. Two atomic-resolution structures were obtained for the methanethiosulfonate spin-label derivatized to cysteines on the membrane-facing surface of BtuB. At one of these sites, position 156, the label side chain resides in a pocket formed by neighboring residues; however, it extends from the protein surface and yields a single-component EPR spectrum in the crystal that results primarily from fast rotation about the fourth and fifth bonds linking the spin-label to the protein backbone. In lipid bilayers, site 156 yields a multicomponent spectrum resulting from different rotameric states of the labeled side chain. Moreover, changes in the lipid environment, such as variations in bilayer thickness, modulate the EPR spectrum by modulating label rotamer populations. At a second site, position 371, the labeled side chain interacts with a pocket on the protein surface, leading to a highly immobilized single-component EPR spectrum that is not sensitive to hydrocarbon thickness. This spectrum is similar to that seen at other sites that are deep in the hydrocarbon, such as position 170. This work indicates that the rotameric states of spin-labels on exposed hydrocarbon sites are sensitive to the environment at the protein-hydrocarbon interface, and that this environment may modulate weak interactions between the labeled side chain and the protein surface. In the case of BtuB, lipid acyl chain packing is not symmetric around the β-barrel, and EPR spectra from labeled hydrocarbon-facing sites in BtuB may reflect this asymmetry. In addition to facilitating the interpretation of EPR spectra of membrane proteins, these results have important implications for the use of long-range distance restraints in protein structure refinement that are obtained from spin-labels.     

Motion of spin label side chains in cellular retinol-binding protein: correlation with structure and nearest-neighbor interactions in an antiparallel beta-sheet

A goal in the development of site-directed spin labeling in proteins is to correlate the motion of a nitroxide side chain with local structure, interactions, and dynamics. Significant progress toward this goal has been made using alpha-helical proteins of known structure, and the present study is the first step in a similar exploration of a beta-sheet protein, cellular retinol-binding protein (CRBP). Nitroxide side chains were introduced along both interior and edge strands. At sites in interior strands, the side-chain motion is strongly influenced by interactions with side chains of neighboring strands, giving rise to a rich variety of dynamic modes (weakly ordered, strongly ordered, immobilized) and complex electron paramagnetic resonance spectra that are modulated by strand twist. The interactions giving rise to the dynamic modes are explored using mutagenesis, and the results demonstrate the particular importance of the non-hydrogen-bonded neighbor residue in giving rise to highly ordered states. Along edge strands of the beta-sheet, the motion of the side chain is simple and weakly ordered, resembling that at solvent-exposed surfaces of an alpha-helix. A simple working model is proposed that can account for the wide variety of dynamic modes encountered. Collectively, the results suggest that the nitroxide side chain is an effective probe of side-chain interactions, and that site-directed spin labeling should be a powerful means of monitoring conformational changes that involve changes in beta-sheet topology.     

Structure of the KcsA potassium channel from Streptomyces lividans: a site-directed spin labeling study of the second transmembrane segment.

KcsA is a prokaryotic potassium channel. The present study employs cysteine scanning mutagenesis and site-directed spin labeling to investigate the structure of the second transmembrane segment (residues 82-120) in functional tetrameric channels reconstituted in lipid bilayers. Spin-spin interactions are observed between nitroxide side chains at symmetry-related sites close to the 4-fold axis of symmetry. To aid in quantitative analysis of these interactions, a new diamagnetic analogue of the nitroxide side chain is used to prepare magnetically dilute samples with constant structure. Using constraints imposed by the spin-spin interactions, a packing model for this segment is deduced that is in excellent agreement with the recently reported crystal structure [Doyle, D., et al. (1998) Science 280, 69-77]. The relatively immobilized state of the nitroxide side chains suggests that the channel is rigid on the electron paramagnetic resonance time scale. Moreover, the poor sulfhydryl reactivity of the cysteine at many locations indicates that the channel is not subject to the low-frequency fluctuations that permit reaction of buried cysteines. At sites expected to be located in the pore, the accessibility of the side chains to collision with O(2) or nickel(II) ethylenediaminediacetate is low. This inaccessibility, together with the generally low mobility of the side chains throughout the sequence, makes it difficult to detect the presence of the pore based on these measurements. However, the presence of a solvated pore can be directly demonstrated using a polarity parameter deduced from the EPR spectra recorded at low temperature. These measurements also reveal the presence of a polarity gradient in the phospholipid bilayer.     

Structure and function of the visual arrestin oligomer

A distinguishing feature of rod arrestin is its ability to form oligomers at physiological concentrations. Using visible light scattering, we show that rod arrestin forms tetramers in a cooperative manner in solution. To investigate the structure of the tetramer, a nitroxide side chain (R1) was introduced at 18 different positions. The effects of R1 on oligomer formation, EPR spectra, and inter-spin distance measurements all show that the structures of the solution and crystal tetramers are different. Inter-subunit distance measurements revealed that only arrestin monomer binds to light-activated phosphorhodopsin, whereas both monomer and tetramer bind microtubules, which may serve as a default arrestin partner in dark-adapted photoreceptors. Thus, the tetramer likely serves as a 'storage' form of arrestin, increasing the arrestin-binding capacity of microtubules while readily dissociating to supply active monomer when it is needed to quench rhodopsin signaling.     

Probing the structure of membrane proteins with electron spin echo envelope modulation spectroscopy

A new approach has been developed to probe the structural properties of membrane peptides and proteins using the pulsed electron paramagnetic resonance technique of electron spin echo envelope modulation (ESEEM) spectroscopy and the α-helical M2δ subunit of the acetylcholine receptor incorporated into phospholipid bicelles. To demonstrate the practicality of this method, a cysteine-mutated nitroxide spin label (SL) is positioned 1, 2, 3, and 4 residues away from a fully deuterated Val side chain (denoted i + 1 to i + 4). The characteristic periodicity of the α-helical structure gives rise to a unique pattern in the ESEEM spectra. In the i + 1 and i + 2 samples, the 2H nuclei are too far away to be detected. However, with the 3.6 residue per turn pattern of an α-helix, the i + 3 and i + 4 samples reveal a strong signal from the 2H nuclei of the Val side chain. Modeling studies verify these data suggesting that the closest 2H-labeled Val to SL distance would in fact be expected in the i + 3 and i + 4 samples. This technique is very advantageous, because it provides pertinent qualitative structural information on an inherently difficult system like membrane proteins in a short period of time (minutes) with small amounts of protein (μg).     

Estimation of inter-residue distances in spin labeled proteins at physiological temperatures: experimental strategies and practical limitations.

Magnetic dipolar interactions between pairs of solvent-exposed nitroxide side chains separated by approximately one to four turns along an alpha-helix in T4 lysozyme are investigated. The interactions are analyzed both in frozen solution (rigid lattice conditions) and at room temperature as a function of solvent viscosity. At room temperature, a novel side chain with hindered internal motion is used, along with a more commonly employed nitroxide side chain. The results suggest that methods developed for rigid lattice conditions can be used to analyze dipolar interactions between nitroxides even in the presence of motion of the individual spins, provided the rotational correlation time of the interspin vector is sufficiently long. The distribution of distances observed for the various spin pairs is consistent with rotameric equilibria in the nitroxide side chain, as observed in crystal structures. The existence of such distance distributions places important constraints on the interpretation of internitroxide distances in terms of protein structure and structural changes.     

Analysis of side-chain rotamers in transmembrane proteins

We measured the frequency of side-chain rotamers in 14 alpha-helical and 16 beta-barrel membrane protein structures and found that the membrane environment considerably perturbs the rotamer frequencies compared to soluble proteins. Although there are limited experimental data, we found statistically significant changes in rotamer preferences depending on the residue environment. Rotamer distributions were influenced by whether the residues were lipid or protein facing, and whether the residues were found near the N- or C-terminus. Hydrogen-bonding interactions with the helical backbone perturbs the rotamer populations of Ser and His. Trp and Tyr favor side-chain conformations that allow their side chains to extend their polar atoms out of the membrane core, thereby aligning the side-chain polarity gradient with the polarity gradient of the membrane. Our results demonstrate how the membrane environment influences protein structures, providing information that will be useful in the structure prediction and design of transmembrane proteins.     

Side-chain modeling with an optimized scoring function

Modeling side-chain conformations on a fixed protein backbone has a wide application in structure prediction and molecular design. Each effort in this field requires decisions about a rotamer set, scoring function, and search strategy. We have developed a new and simple scoring function, which operates on side-chain rotamers and consists of the following energy terms: contact surface, volume overlap, backbone dependency, electrostatic interactions, and desolvation energy. The weights of these energy terms were optimized to achieve the minimal average root mean square (rms) deviation between the lowest energy rotamer and real side-chain conformation on a training set of high-resolution protein structures. In the course of optimization, for every residue, its side chain was replaced by varying rotamers, whereas conformations for all other residues were kept as they appeared in the crystal structure. We obtained prediction accuracy of 90.4% for chi(1), 78.3% for chi(1 + 2), and 1.18 A overall rms deviation. Furthermore, the derived scoring function combined with a Monte Carlo search algorithm was used to place all side chains onto a protein backbone simultaneously. The average prediction accuracy was 87.9% for chi(1), 73.2% for chi(1 + 2), and 1.34 A rms deviation for 30 protein structures. Our approach was compared with available side-chain construction methods and showed improvement over the best among them: 4.4% for chi(1), 4.7% for chi(1 + 2), and 0.21 A for rms deviation. We hypothesize that the scoring function instead of the search strategy is the main obstacle in side-chain modeling. Additionally, we show that a more detailed rotamer library is expected to increase chi(1 + 2) prediction accuracy but may have little effect on chi(1) prediction accuracy.     

Orientation of the tyrosyl radical in Salmonella typhimurium class Ib ribonucleotide reductase determined by high field EPR of R2F single crystals

The R2 protein of class I ribonucleotide reductase (RNR) generates and stores a tyrosyl radical, located next to a diferric iron center, which is essential for ribonucleotide reduction and thus DNA synthesis. X-ray structures of class Ia and Ib proteins from various organisms served as bases for detailed mechanistic suggestions. The active site tyrosine in R2F of class Ib RNR of Salmonella typhimurium is located at larger distance to the diiron site, and shows a different side chain orientation, as compared with the tyrosine in R2 of class Ia RNR from Escherichia coli.No structural information has been available for the active tyrosyl radical in R2F. Here we report on high field EPR experiments of single crystals of R2F from S. typhimurium, containing the radical Tyr-105*. Full rotational pattern of the spectra were recorded, and the orientation of the g-tensor axes were determined, which directly reflect the orientation of the radical Tyr-105* in the crystal frame. Comparison with the orientation of the reduced tyrosine Tyr-105-OH from the x-ray structure reveals a rotation of the tyrosyl side chain, which reduces the distance between the tyrosyl radical and the nearest iron ligands toward similar values as observed earlier for Tyr-122* in E. coli R2. Presence of the substrate binding subunit R1E did not change the EPR spectra of Tyr-105*, indicating that binding of R2E alone induces no structural change of the diiron site. The present study demonstrates that structural and functional information about active radical states can be obtained by combining x-ray and high-field-EPR crystallography.