Probing the Menasemiquinone Binding Mode to Nitrate Reductase†A by Selective 2 H and 15 N Labeling, HYSCORE Spectroscopy, and DFT Modeling.

In vivo specific isotope labeling at the residue or substituent level is used to probe menasemiquinone (MSK) binding to the quinol oxidation site of respiratory nitrate reductase A (NarGHI) from E. coli. 15 N selective labeling of His15 Nδ or Lys15 Nζ in combination with hyperfine sublevel correlation (HYSCORE) spectroscopy unambiguously identified His15 Nδ as the direct hydrogen-bond donor to the radical. In contrast, an essentially anisotropic coupling to Lys15 Nζ consistent with a through-space magnetic interaction was resolved. This suggests that MSK does not form a hydrogen bond with the side chain of the nearby Lys86 residue. In addition, selective 2 H labeling of the menaquinone methyl ring substituent allows unambiguous characterization of the 2 H-and hence of the 1 H-methyl isotropic hyperfine coupling by 2 H HYSCORE. DFT calculations show that a simple molecular model consisting of an imidazole Nδ atom in a hydrogen-bond interaction with a MSK radical anion satisfactorily accounts for the available spectroscopic data. These results support our previously proposed one-sided binding model for MSK to NarGHI through a single short hydrogen bond to the Nδ of His66, one of the distal heme axial ligands. This work establishes the basis for future investigations aimed at determining the functional relevance of this peculiar binding mode.

Nucleotides and substrates trigger the dynamics of the Toc34 GTPase homodimer involved in chloroplast preprotein translocation.

GTPases are molecular switches that control numerous crucial cellular processes. Unlike bona fide GTPases, which are regulated by intramolecular structural transitions, the less well studied GAD-GTPases are activated by nucleotide-dependent dimerization. A member of this family is the translocase of the outer envelope membrane of chloroplast Toc34 involved in regulation of preprotein import. The GTPase cycle of Toc34 is considered a major circuit of translocation regulation. Contrary to expectations, previous studies yielded only marginal structural changes of dimeric Toc34 in response to different nucleotide loads. Referencing PELDOR and FRET single-molecule and bulk experiments, we describe a nucleotide-dependent transition of the dimer flexibility from a tight GDP- to a flexible GTP-loaded state. Substrate binding induces an opening of the GDP-loaded dimer. Thus, the structural dynamics of bona fide GTPases induced by GTP hydrolysis is replaced by substrate-dependent dimer flexibility, which likely represents a general regulatory mode for dimerizing GTPases.

Probing the ATP hydrolysis cycle of the ABC multidrug transporter LmrA by pulsed EPR spectroscopy.

Members of the ATP binding cassette (ABC) transporter superfamily translocate various types of molecules across the membrane at the expense of ATP. This requires cycling through a number of catalytic states. Here, we report conformational changes throughout the catalytic cycle of LmrA, a homodimeric multidrug ABC transporter from L. lactis. Using site-directed spin labeling and pulsed electron-electron double resonance (PELDOR/DEER) spectroscopy, we have probed the reorientation of the nucleotide binding domains and transmembrane helix 6 which is of particular relevance to drug binding and part of the dimerization interface. Our data show that LmrA samples a very large conformational space in its apo state, which is significantly reduced upon nucleotide binding. ATP binding but not hydrolysis is required to trigger this conformational change, which results in a relatively fixed orientation of both the nucleotide binding domains and transmembrane helices 6. This orientation is maintained throughout the ATP hydrolysis cycle until the protein cycles back to its apo state. Our data present strong evidence that switching between two dynamically and structurally distinct states is required for substrate translocation.

Determination of the proton environment of high stability Menasemiquinone intermediate in Escherichia coli nitrate reductase A by pulsed EPR.

Escherichia coli nitrate reductase A (NarGHI) is a membrane-bound enzyme that couples quinol oxidation at a periplasmically oriented Q-site (Q(D)) to proton release into the periplasm during anaerobic respiration. To elucidate the molecular mechanism underlying such a coupling, endogenous menasemiquinone-8 intermediates stabilized at the Q(D) site (MSQ(D)) of NarGHI have been studied by high-resolution pulsed EPR methods in combination with (1)H2O/2H2O exchange experiments. One of the two non-exchangeable proton hyperfine couplings resolved in hyperfine sublevel correlation (HYSCORE) spectra of the radical displays characteristics typical from quinone methyl protons. However, its unusually small isotropic value reflects a singularly low spin density on the quinone carbon α carrying the methyl group, which is ascribed to a strong asymmetry of the MSQ(D) binding mode and consistent with single-sided hydrogen bonding to the quinone oxygen O1. Furthermore, a single exchangeable proton hyperfine coupling is resolved, both by comparing the HYSCORE spectra of the radical in 1H2O and 2H2O samples and by selective detection of the exchanged deuterons using Q-band 2H Mims electron nuclear double resonance (ENDOR) spectroscopy. Spectral analysis reveals its peculiar characteristics, i.e. a large anisotropic hyperfine coupling together with an almost zero isotropic contribution. It is assigned to a proton involved in a short ∼1.6 Å in-plane hydrogen bond between the quinone O1 oxygen and the Nδ of the His-66 residue, an axial ligand of the distal heme b(D). Structural and mechanistic implications of these results for the electron-coupled proton translocation mechanism at the Q(D) site are discussed, in light of the unusually high thermodynamic stability of MSQ(D).

Optimization of transversal relaxation of nitroxides for pulsed electron-electron double resonance spectroscopy in phospholipid membranes.

Pulsed electron-electron double resonance (PELDOR) spectroscopy is increasingly applied to spin-labeled membrane proteins. However, after reconstitution into liposomes, spin labels often exhibit a much faster transversal relaxation (T(m)) than in detergent micelles, thus limiting application of the method in lipid bilayers. In this study, the main reasons for enhanced transversal relaxation in phospholipid membranes were investigated systematically by use of spin-labeled derivatives of stearic acid and phosphatidylcholine as well as spin-labeled derivatives of the channel-forming peptide gramicidin A under the conditions typically employed for PELDOR distance measurements. Our results clearly show that dephasing due to instantaneous diffusion that depends on dipolar interaction among electron spins is an important contributor to the fast echo decay in cases of high local concentrations of spin labels in membranes. The main difference between spin labels in detergent micelles and membranes is their local concentration. Consequently, avoiding spin clustering and suppressing instantaneous diffusion is the key step for maximizing PELDOR sensitivity in lipid membranes. Even though proton spin diffusion is an important relaxation mechanism, only in samples of low local concentrations does deuteration of acyl chains and buffer significantly prolong T(m). In these cases, values of up to 7 µs have been achieved. Furthermore, our study revealed that membrane composition and labeling position in the membrane can also affect T(m), either by promoting the segregation of spin-labeled species or by altering their exposure to matrix protons. Effects of other experimental parameters including temperature (<50 K), presence of oxygen, and cryoprotectant type are negligible under our experimental conditions.

HYSCORE evidence that endogenous mena- and ubisemiquinone bind at the same Q site (Q(D)) of Escherichia coli nitrate reductase A.

Through the use of an Escherichia coli strain deficient in menaquinone biosynthesis, purified nitrate reductase A (NarGHI)-enriched inner membrane vesicles were titrated and monitored by electron paramagnetic resonance (EPR) spectroscopy, revealing the formation of protein-bound ubisemiquinone (USQ) species. Two-dimensional ESEEM (HYSCORE) experiments on these radicals revealed the same magnetic interaction with an (14)N nucleus as found for menasemiquinone stabilized at the Q(D) site of E. coli NarGHI and assigned to His66 N(delta), a distal heme axial ligand. Moreover, this signature was lost in the NarGHI(H66Y) mutant, which is known to be unable to react with quinols. These findings demonstrate that NarGHI-bound USQ can be formed and detected by EPR. They also provide the first direct experimental evidence for similar binding of natural menasemiquinones and ubisemiquinones within the same protein Q site of NarGHI.

Direct evidence for nitrogen ligation to the high stability semiquinone intermediate in Escherichia coli nitrate reductase A.

The membrane-bound heterotrimeric nitrate reductase A (NarGHI) catalyzes the oxidation of quinols in the cytoplasmic membrane of Escherichia coli and reduces nitrate to nitrite in the cytoplasm. The enzyme strongly stabilizes a menasemiquinone intermediate at a quinol oxidation site (Q(D)) located in the vicinity of the distal heme b(D). Here molecular details of the interaction between the semiquinone radical and the protein environment have been provided using advanced multifrequency pulsed EPR methods. (14)N and (15)N ESEEM and HYSCORE measurements carried out at X-band ( approximately 9.7 GHz) on the wild-type enzyme or the enzyme uniformly labeled with (15)N nuclei reveal an interaction between the semiquinone and a single nitrogen nucleus. The isotropic hyperfine coupling constant A(iso)((14)N) approximately 0.8 MHz shows that it occurs via an H-bond to one of the quinone carbonyl group. Using (14)N ESEEM and HYSCORE spectroscopies at a lower frequency (S-band, approximately 3.4 GHz), the (14)N nuclear quadrupolar parameters of the interacting nitrogen nucleus (kappa = 0.49, eta = 0.50) were determined and correspond to those of a histidine N(delta), assigned to the heme b(D) ligand His-66 residue. Moreover S-band (15)N ESEEM spectra enabled us to directly measure the anisotropic part of the nitrogen hyperfine interaction (T((15)N) = 0.16 MHz). A distance of approximately 2.2 Abetween the carbonyl oxygen and the nitrogen could then be calculated. Mechanistic implications of these results are discussed in the context of the peculiar properties of the menasemiquinone intermediate stabilized at the Q(D) site of NarGHI.

Multifrequency pulsed electron paramagnetic resonance on metalloproteins.

Metalloproteins often contain metal centers that are paramagnetic in some functional state of the protein; hence electron paramagnetic resonance (EPR) spectroscopy can be a powerful tool for studying protein structure and function. Dipolar spectroscopy allows the determination of the dipole-dipole interactions between metal centers in protein complexes, revealing the structural arrangement of different paramagnetic centers at distances of up to 8 nm. Hyperfine spectroscopy can be used to measure the interaction between an unpaired electron spin and nuclear spins within a distance of 0.8 nm; it therefore permits the characterization of the local structure of the paramagnetic center's ligand sphere with very high precision. In this Account, we review our laboratory's recent applications of both dipolar and hyperfine pulsed EPR methods to metalloproteins. We used pulsed dipolar relaxation methods to investigate the complex of cytochrome c and cytochrome c oxidase, a noncovalent protein-protein complex involved in mitochondrial electron-transfer reactions. Hyperfine sublevel correlation spectroscopy (HYSCORE) was used to study the ligand sphere of iron-sulfur clusters in complex I of the mitochondrial respiratory chain and substrate binding to the molybdenum enzyme polysulfide reductase. These examples demonstrate the potential of the two techniques; however, they also highlight the difficulties of data interpretation when several paramagnetic species with overlapping spectra are present in the protein. In such cases, further approaches and data are very useful to enhance the information content. Relaxation filtered hyperfine spectroscopy (REFINE) can be used to separate the individual components of overlapping paramagnetic species on the basis of differences in their longitudinal relaxation rates; it is applicable to any kind of pulsed hyperfine or dipolar spectroscopy. Here, we show that the spectra of the iron-sulfur clusters in complex I can be separated by this method, allowing us to obtain hyperfine (and dipolar) information from the individual species. Furthermore, performing pulsed EPR experiments at different magnetic fields is another important tool to disentangle the spectral components in such complex systems. Despite the fact that high magnetic fields do not usually lead to better spectral separation for metal centers, they provide additional information about the relative orientation of different paramagnetic centers. Our high-field EPR studies on cytochrome c oxidase reveal essential information regarding the structural arrangement of the binuclear Cu(A) center with respect to both the manganese ion within the enzyme and the cytochrome in the protein-protein complex with cytochrome c.

Dynamic nuclear polarization with a rigid biradical.

A new polarizing agent with superior performance in dynamic nuclear polarization experiments is introduced, and utilizes two TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) moieties connected through a rigid spiro tether (see structure). The observed NMR signal intensities were enhanced by a factor of 1.4 compared to those of TOTAPOL, a previously described TEMPO-based biradical with a flexible tether.

Protein-protein interactions studied by EPR relaxation measurements: cytochrome c and cytochrome c oxidase.

The complex formed between cytochrome c oxidase from Paracoccus denitrificans and its electron-transfer partner cytochrome c has been studied by multi-frequency pulse electron paramagnetic resonance spectroscopy. The dipolar relaxation of a fast-relaxing paramagnetic center induced on a more slowly relaxing center can be used to measure their distance in the range of 1-4 nm. This method has been used here for the first time to study transient protein-protein complex formation, employing soluble fragments for both interacting species. We observed significantly enhanced transversal relaxation of the CuA center in cytochrome c oxidase due to the fast-relaxing iron of cytochrome c upon complex formation. The possibility to measure cytochrome c oxidase in the presence and absence of cytochrome c permitted us to separate the dipolar relaxation from other relaxation contributions. This allowed a quantitative simulation and interpretation of the relaxation data. The specific temperature dependence of the dipolar relaxation together with the high orientational selectivity achieved at high magnetic field values may provide detailed information on distance and relative orientation of the two proteins with respect to each other in the complex. Our experimental results cannot be explained by any single well-defined structure of the complex of cytochrome c oxidase with cytochrome c, but rather suggest that a broad distribution in distances and relative orientations between the two proteins exist within this complex.

Tyrosine 167: the origin of the radical species observed in the reaction of cytochrome c oxidase with hydrogen peroxide in Paracoccus denitrificans.

Determination of the three-dimensional structure of cytochrome c oxidase, the terminal enzyme of the respiratory chain, from Paracoccus denitrificans offers the possibility of site-directed mutagenesis studies to investigate the relationship between the structure and the catalytic function of the enzyme. The mechanism of electron-coupled proton transfer is still, however, poorly understood. The P(M) intermediate of the catalytic cycle is an oxoferryl state the generation of which requires one additional electron, which cannot be provided by the two metal centers. It is suggested that the missing electron is donated to this binuclear site by a tyrosine residue that forms a radical species, which can then be detected in both the P(M) and F(*) intermediates of the catalytic cycle. One possibility to produce P(M) and F(*) intermediates artificially in cytochrome c oxidase is the addition of hydrogen peroxide to the fully oxidized enzyme. Using electron paramagnetic resonance (EPR) spectroscopy, we assign a radical species detected in this reaction to a tyrosine residue. To address the question, which tyrosine residue is the origin of the radical species, several tyrosine variants of subunit I are investigated. These variants are characterized by their turnover rates, as well as using EPR and optical spectroscopy. From these experiments, it is concluded that the origin of the radical species appearing in P(M) and F(*) intermediates produced with hydrogen peroxide is tyrosine 167. The significance of this finding for the catalytic function of the enzyme is discussed.

Multifrequency cw-EPR investigation of the catalytic molybdenum cofactor of polysulfide reductase from Wolinella succinogenes.

Electron paramagnetic resonance (EPR) spectra of the molybdenum centre in polysulfide reductase (Psr) from Wolinella succinogenes with unusually high G-tensor values have been observed for the first time. Three different Mo(V) states have been generated (by the addition of the substrate polysulfide and different redox agents) and analysed by their G- and hyperfine tensors using multifrequency (S-, X- and Q-band) cw-EPR spectroscopy. The unusually high G-tensor values are attributed to a large number of sulfur ligands. Four sulfur ligands are assumed to arise from two pterin cofactors; one additional sulfur ligand was identified from mutagenesis studies to be a cysteine residue of the protein backbone. One further sulfur ligand is proposed for two of the Mo(V) states, based on the experimentally observed shift of the g(av) value. This sixth sulfur ligand is postulated to belong to the polysulfide substrate consumed within the catalytic reaction cycle of the enzyme. The influence of the co-protein sulfur transferase on the Mo(V) G-tensor supports this assignment.