Identification of hydrogen bonds to the Rieske cluster through the weakly coupled nitrogens detected by electron spin echo envelope modulation spectroscopy

The interaction of the reduced[2Fe-2S] cluster of isolated Rieske fragment from the bc1 complex of Rhodobacter sphaeroides with nitrogens (14N and 15N) from the local protein environment has been studied by X- and S-band pulsed EPR spectroscopy. The two-dimensional electron spin echo envelope modulation spectra of uniformly 15N-labeled protein show two well resolved cross-peaks with weak couplings of approximately 0.3-0.4 and 1.1 MHz in addition to couplings in the range of 6-8 MHz from two coordinating Ndelta of histidine ligands. The quadrupole coupling constants for weakly coupled nitrogens determined from S-band electron spin echo envelope modulation spectra identify them as Nepsilon of histidine ligands and peptide nitrogen (Np), respectively. Analysis of the line intensities in orientation-selected S-band spectra indicated that Np is the backbone N-atom of Leu-132 residue. The hyperfine couplings from Nepsilon and Np demonstrate the predominantly isotropic character resulting from the transfer of unpaired spin density onto the 2s orbitals of the nitrogens. Spectra also show that other peptide nitrogens in the protein environment must carry a 5-10 times smaller amount of spin density than the Np of Leu-132 residue. The appearance of the excess unpaired spin density on the Np of Leu-132 residue indicates its involvement in hydrogen bond formation with the bridging sulfur of the Rieske cluster. The configuration of the hydrogen bond therefore provides a preferred path for spin density transfer. Observation of similar splittings in the 15N spectra of other Rieske-type proteins and [2Fe-2S] ferredoxins suggests that a hydrogen bond between the bridging sulfur and peptide nitrogen is a common structural feature of [2Fe-2S] clusters.     

Hydrogen bonds involved in binding the Qi-site semiquinone in the bc1 complex, identified through deuterium exchange using pulsed EPR

Exchangeable protons in the immediate neighborhood of the semiquinone (SQ) at the Qi-site of the bc1 complex (ubihydroquinone:cytochrome c oxidoreductase (EC 1.10.2.2)) from Rhodobacter sphaeroides have been characterized using electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation spectroscopy (HYSCORE) and visualized by substitution of H2O by 2H2O. Three exchangeable protons interact with the electron spin of the SQ. They possess different isotropic and anisotropic hyperfine couplings that allow a clear distinction between them. The strength of interactions indicates that the protons are involved in hydrogen bonds with SQ. The hyperfine couplings differ from values typical for in-plane hydrogen bonds previously observed in model experiments. It is suggested that the two stronger couplings involve formation of hydrogen bonds with carbonyl oxygens, which have a significant out-of-plane character due to the combined influence of bulky substituents and the protein environment. These two hydrogen bonds are most probably to side chains suggested from crystallographic structures (His-217 and Asp-252 in R. sphaeroides). Assignment of the third hydrogen bond is more ambiguous but may involve either a bond between Asn-221 and a methoxy O-atom or a bond to water. The structural and catalytic roles of the exchangeable protons are discussed in the context of three high resolution crystallographic structures for mitochondrial bc1 complexes. Potential H-bonds, including those to water molecules, form a network connecting the quinone (ubiquinone) occupant and its ligands to the propionates of heme bH and the external aqueous phase. They provide pathways for exchange of protons within the site and with the exteriors, needed to accommodate the different hydrogen bonding requirements of different quinone species during catalysis.     

Exploration of ligands to the Qi site semiquinone in the bc1 complex using high-resolution EPR

Pulsed EPR spectroscopy was used to explore the structural neighborhood of the semiquinone (SQ) stabilized at the Qi site of the bc1 complex of Rhodobacter sphaeroides (EC 1.10.2.2) and to demonstrate that the nitrogen atom of a histidine imidazole group donates an H-bond to the SQ. Crystallographic structures show two different configurations for the binding of ubiquinone at the Qi site of mitochondrial bc1 complexes in which histidine (His-201 in bovine sequence) is either a direct H-bond donor or separated by a bridging water. The paramagnetic properties of the SQ formed at the site provide an independent method for studying the liganding of this intermediate species. The antimycin-sensitive SQ formed at the Qi site by either equilibrium redox titration, reduction of the oxidized complex by ascorbate, or addition of decylubihydroquinone to the oxidized complex in the presence of myxothiazol all showed similar properties. The electron spin echo envelope modulation spectra in the 14N region were dominated by lines with frequencies at 1.7 and 3.1 MHz. Hyperfine sublevel correlation spectroscopy spectra showed that these were contributed by a single nitrogen. Further analysis showed that the 14N nucleus was characterized by an isotropic hyperfine coupling of approximately 0.8 MHz and a quadrupole coupling constant of approximately 0.35 MHz. The nitrogen was identified as the N-epsilon or N-delta imidazole nitrogen of a histidine (it is likely to be His-217, or His-201 in bovine sequence). A distance of 2.5-3.1 A for the O-N distance between the carbonyl of SQ and the nitrogen was estimated. The mechanistic significance is discussed in the context of a dynamic role for the movement of His-217 in proton transfer to the site.     

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. ¹⁴N and ¹⁵N ESEEM and HYSCORE measurements carried out at X-band (∼9.7 GHz) on the wild-type enzyme or the enzyme uniformly labeled with ¹⁵N nuclei reveal an interaction between the semiquinone and a single nitrogen nucleus. The isotropic hyperfine coupling constant A_iso(¹⁴N) ∼0.8 MHz shows that it occurs via an H-bond to one of the quinone carbonyl group. Using ¹⁴N ESEEM and HYSCORE spectroscopies at a lower frequency (S-band, ∼3.4 GHz), the ¹⁴N nuclear quadrupolar parameters of the interacting nitrogen nucleus (κ = 0.49, η = 0.50) were determined and correspond to those of a histidine N_δ, assigned to the heme b_D ligand His-66 residue. Moreover S-band ¹⁵N ESEEM spectra enabled us to directly measure the anisotropic part of the nitrogen hyperfine interaction (T(¹⁵N) = 0.16 MHz). A distance of ∼2.2 Åbetween 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.     

Catalytic roles for two water bridged residues (Asp-98 and His-255) in the active site of copper-containing nitrite reductase

Two active site residues, Asp-98 and His-255, of copper-containing nitrite reductase (NIR) from Alcaligenes faecalis have been mutated to probe the catalytic mechanism. Three mutations at these two sites (D98N, H255D, and H255N) result in large reductions in activity relative to native NIR, suggesting that both residues are involved intimately in the reaction mechanism. Crystal structures of these mutants have been determined using data collected to better than 1. 9-A resolution. In the native structure, His-255 Nepsilon2 forms a hydrogen bond through a bridging water molecule to the side chain of Asp-98, which also forms a hydrogen bond to a water or nitrite oxygen ligated to the active site copper. In the D98N mutant, reorientation of the Asn-98 side chain results in the loss of the hydrogen bond to the copper ligand water, consistent with a negatively charged Asp-98 directing the binding and protonation of nitrite in the native enzyme. An additional solvent molecule is situated between residues 255 and the bridging water in the H255N and H255D mutants and likely inhibits nitrite binding. The interaction of His-255 with the bridging water appears to be necessary for catalysis and may donate a proton to reaction intermediates in addition to Asp-98.     

Insights into the nature of the hydrogen bonding of *Tyr272 in apo-galactose oxidase

The synthesis and structure of an o-methylthio-phenol-imidazole, 2-(2'-(4'-tert-butyl-6'-methylsulfanyl)-hydroxyphenyl))-4,5-diphenyl-imidazole ((MeS)LH), is reported; X-ray crystallographic studies have shown that (MeS)LH involves an O-H...N(+) hydrogen bond between the phenol and an imidazole nitrogen. (MeS)LH undergoes a reversible, one-electron, oxidation to form the radical cation [(MeS)LH](*)(+) the EPR spectrum of which is remarkably similar to that of (*)Tyr(272) in Cu-free, oxidized, apo-GO. Density Functional Theory calculations, have shown that the proton-transferred (R-O(*)...H-N(+)) form of [(MeS)LH](*)(+) has a spin density distribution--with a substantial delocalization of the unpaired electron spin density onto the ortho sulfur atom--and EPR properties that are in good agreement with those of (*)Tyr(272) in Cu-free, oxidized, apo-GO whereas the non-proton-transferred (R-O(*)(+)-H...N) form does not. The results reported herein are a further demonstration of the influence of hydrogen bonding on the nature and properties of phenoxyl radicals and strongly suggest that the phenoxyl oxygen of (*)Tyr(272) in Cu-free, oxidized, apo-GO is involved in a O(*)...H-O/N hydrogen bond.     

Characterization of the exchangeable protons in the immediate vicinity of the semiquinone radical at the QH site of the cytochrome bo3 from Escherichia coli

The cytochrome bo3 ubiquinol oxidase from Escherichia coli resides in the bacterial cytoplasmic membrane and catalyzes the two-electron oxidation of ubiquinol-8 and four-electron reduction of O2 to water. The one-electron reduced semiquinone forms transiently during the reaction, and the enzyme has been demonstrated to stabilize the semiquinone. Two-dimensional electron spin echo envelope modulation has been applied to explore the exchangeable protons involved in hydrogen bonding to the semiquinone by substitution of 1H2O by 2H2O. Three exchangeable protons possessing different isotropic and anisotropic hyperfine couplings were identified. The strength of the hyperfine interaction with one proton suggests a significant covalent O-H binding of carbonyl oxygen O1 that is a characteristic of a neutral radical, an assignment that is also supported by the unusually large hyperfine coupling to the methyl protons. The second proton with a large anisotropic coupling also forms a strong hydrogen bond with a carbonyl oxygen. This second hydrogen bond, which has a significant out-of-plane character, is from an NH2 or NH nitrogen, probably from an arginine (Arg-71) known to be in the quinone binding site. Assignment of the third exchangeable proton with smaller anisotropic coupling is more ambiguous, but it is clearly not involved in a direct hydrogen bond with either of the carbonyl oxygens. The results support a model that the semiquinone is bound to the protein in a very asymmetric manner by two strong hydrogen bonds from Asp-75 and Arg-71 to the O1 carbonyl, while the O4 carbonyl is not hydrogen-bonded to the protein.     

EPR, ENDOR, and TRIPLE resonance spectroscopy on the neutral flavin radical in Escherichia coli DNA photolyase

Ultraviolet radiation promotes the formation of a cyclobutane ring between adjacent pyrimidine residues on the same DNA strand to form a pyrimidine dimer. Such dimers may be restored to their monomeric forms through the action of a light-absorbing enzyme named DNA photolyase. The redox-active cofactor involved in the light-induced electron transfer reactions of DNA repair and enzyme photoactivation is a noncovalently bound FAD. In this paper, the FAD cofactor of Escherichia coli DNA photolyase was characterized as the neutral flavin semiquinone by EPR spectroscopy at 9.68 and 94.5 GHz. From the high-frequency/high-field EPR spectrum, the principal values of the axially symmetric g-matrix of FADH(*) were extracted. Both EPR spectra show an emerging hyperfine splitting of 0.85 mT that could be assigned to the isotropic hyperfine coupling constant (hfc) of the proton at N(5). To obtain more information about the electron spin density distribution ENDOR and TRIPLE resonance spectroscopies were applied. All major proton hfc's could be measured and unambiguously assigned to molecular positions at the isoalloxazin moiety of FAD. The isotropic hfc's of the protons at C(8alpha) and C(6) are among the smallest values reported for protein-bound neutral flavin semiquinones so far, suggesting a highly restricted delocalization of the unpaired electron spin on the isoalloxazin moiety. Two further hfc's have been detected and assigned to the inequivalent protons at C(1'). Some conclusions about the geometrical arrangement of the ribityl side chain with respect to the isoalloxazin ring could be drawn: Assuming tetrahedral angles at C(1') the dihedral angle between the C(1')-C(2') bond and the 2p(z)() orbital at N(10) has been estimated to be 170.4 degrees +/- 1 degrees.     

Characterization of mutants that change the hydrogen bonding of the semiquinone radical at the QH site of the cytochrome bo3 from Escherichia coli

The cytochrome bo3 ubiquinol oxidase catalyzes the two-electron oxidation of ubiquinol in the cytoplasmic membrane of Escherichia coli, and reduces O2 to water. This enzyme has a high affinity quinone binding site (QH), and the quinone bound to this site acts as a cofactor, necessary for rapid electron transfer from substrate ubiquinol, which binds at a separate site (QL), to heme b. Previous pulsed EPR studies have shown that a semiquinone at the QH site formed during the catalytic cycle is a neutral species, with two strong hydrogen bonds to Asp-75 and either Arg-71 or Gln-101. In the current work, pulsed EPR studies have been extended to two mutants at the QH site. The D75E mutation has little influence on the catalytic activity, and the pattern of hydrogen bonding is similar to the wild type. In contrast, the D75H mutant is virtually inactive. Pulsed EPR revealed significant structural changes in this mutant. The hydrogen bond to Arg-71 or Gln-101 that is present in both the wild type and D75E mutant oxidases is missing in the D75H mutant. Instead, the D75H has a single, strong hydrogen bond to a histidine, likely His-75. The D75H mutant stabilizes an anionic form of the semiquinone as a result of the altered hydrogen bond network. Either the redistribution of charge density in the semiquinone species, or the altered hydrogen bonding network is responsible for the loss of catalytic function.     

Asymmetric binding of the high-affinity Q(H)(*)(-) ubisemiquinone in quinol oxidase (bo3) from Escherichia coli studied by multifrequency electron paramagnetic resonance spectroscopy

Ubiquinone-2 (UQ-2) selectively labeled with (13)C (I =(1)/(2)) at either the position 1- or the 4-carbonyl carbon is incorporated into the ubiquinol oxidase bo(3) from Escherichia coli in which the native quinone (UQ-8) has been previously removed. The resulting stabilized anion radical in the high-affinity quinone-binding site (Q(H)(*)(-)) is investigated using multifrequency (9, 34, and 94 GHz) electron paramagnetic resonance (EPR) spectroscopy. The corresponding spectra reveal dramatic differences in (13)C hyperfine couplings indicating a strongly asymmetric spin density distribution over the quinone headgroup. By comparison with previous results on labeled ubisemiquinones in proteins as well as in organic solvents, it is concluded that Q(H)(*)(-) is most probably bound to the protein via a one-sided hydrogen bond or a strongly asymmetric hydrogen-bonding network. This observation is discussed with regard to the function of Q(H) in the enzyme and contrasted with the information available on other protein-bound semiquinone radicals.     

The binding of imidazole in an azurin-like blue-copper site

 Frozen solutions of the azurin mutant His117Gly in the presence of excess of methyl-substituted imidazoles have been investigated by electron spin-echo envelope modulation (ESEEM) spectroscopy at 9 GHz. The addition of imidazole is known to reconstitute a blue-copper site and variation of the non-protein bound ligand [N-methyl-, 2-methyl-, 4(5)-methylimidazole] has allowed the study of the copper-imidazole binding as a model for histidine binding in such sites. Quadrupole and hyperfine tensors of the remote nitrogen of the imidazoles have been determined. The quadrupole tensors indicate that the methyl-substituted imidazoles in the mutant adopt the same orientation relative to copper as the histidine-117 in the wild-type protein. Analysis of the hyperfine tensors in terms of spin densities reveals that the spin density on the remote nitrogen of the substituted imidazole has σ and a variable π character, depending on the position of the methyl group. For azurin the corresponding spin density is of virtually pure σ character. In conclusion, blue-copper sites show subtle variations as regards the histidine/imidazole centred part of the wavefunction of the unpaired electron. Received: 27 October 1998 / Accepted: 9 February 1999     

Density functional calculated spin densities and hyperfine couplings for hydrogen bonded 1,4-naphthosemiquinone and phyllosemiquinone anion radicals: a model for the A1 free radical formed in photosystem I

The B3LYP hybrid density functional method is used to calculate spin densities and hyperfine couplings for the 1,4-naphthosemiquinone anion radical and a model of the phyllosemiquinone anion radical. The effect of hydrogen bonding on the spin density distribution is shown to lead to a redistribution of pi spin density from the semiquinone carbonyl oxygens to the carbonyl carbon atoms. The effect of in plane and out of plane hydrogen bonding is examined. Out of plane hydrogen bonding is shown to give rise to a significant delocalisation of spin density on to the hydrogen bond donor heavy atom. Excellent agreement is observed between calculated and experimental hyperfine couplings. Comparison of calculated hyperfine couplings with experimental determinations for the A1 phyllosemiquinone anion radical present in Photosystem I (PS I) of higher plant photosynthesis indicates that the in vivo radical may have a hydrogen bond to the O4 atom only as opposed to hydrogen bonds to each oxygen atom in alcohol solvents. The hydrogen bonding situation appears to be the reverse of that observed for QA in the bacterial type II reaction centres where the strong hydrogen bond occurs to the quinone O1 oxygen atom. For different types of reaction centre the presence or absence of the non-heme Fe(II) atom may well determine which type of hydrogen bonding situation prevails at the primary quinone site which in turn may influence the direction of subsequent electron transfer.     

The cupric site in nitrous oxide reductase contains a mixed-valence [Cu(II),Cu(I)] binuclear center: a multifrequency electron paramagnetic resonance investigation

Multifrequency electron paramagnetic resonance (EPR) spectra of the Cu(II) site in nitrous oxide reductase (N2OR) from Pseudomonas stutzeri confirm the assignment of the low field g value at 2.18 consistent with the seven line pattern observed at 9.31 GHz, 10 K. S-band spectra at 20 K are better resolved than the X-band spectra recorded at 10 K. The features observed at 2.4, 3.4, 9.31 and 35 GHz are explained by a mixed-valence [Cu(1.5)..Cu(1.5)] S = 1/2 species with the unpaired electron delocalized between two equivalent Cu nuclei. The resemblance of the N2OR S-band spectra to the spectra for the EPR-detectable Cu of cytochrome c oxidase suggests that the S-band spectrum for cytochrome c oxidase measured below 30 K may also contain hyperfine splittings from two approximately equivalent Cu nuclei.     

Stabilization of the imidazole ring of His-195 at the active site of chloramphenicol acetyltransferase

The imidazole of His-195 plays an essential role in the proposed general base mechanism of chloramphenicol acetyltransferase (CAT). The structure of the binary complex of CATIII and chloramphenicol suggests that two unusual interactions might determine the conformation of the side chain of His-195: (i) an intraresidue hydrogen bond between its main chain carbonyl and the protonated N delta 1 of the imidazole ring and (ii) face-to-face van der Waals contact between the His-195 imidazole group and the aromatic side chain of Tyr-25. Tyr-25 also makes a hydrogen bond, via its phenolic hydroxyl, to the carbonyl oxygen of the substrate chloramphenicol. Replacement of Tyr-25 of CATIII by phenylalanine results in a modest increase in the Km for chloramphenicol (from 11.6 to 14.6 microM) and a 2-fold fall in kcat (599 to 258 s-1), indicative of a free energy contribution to transition state binding of 0.6 kcal mol-1 for the hydrogen bond between Tyr-25 and chloramphenicol. In contrast, substitution of Tyr-25 by alanine yields an enzyme that is dramatically impaired in its ability to bind chloramphenicol (Km = 173 microM). As kcat for Ala-25 CAT is also reduced (130 s-1), the loss of the aryl group results in a 69-fold decrease in kcat/Km, corresponding to a free energy contribution to binding and catalysis of 2.5 kcal mol-1. In addition to the loss of the hydrogen bond between Tyr-25 and chloramphenicol, the loss of substrate affinity in Ala-25 CAT may be a direct consequence of reduced hydrophobicity of the chloramphenicol-binding site and/or the loss of critical constraints on the precise conformation of the catalytic imidazole. However, as with wild type CAT, inactivation of Ala-25 CAT by the affinity reagent 3-(bromoacetyl) chloramphenicol is accompanied by modification solely at N epsilon 2 of His-195. Hence, the results demonstrate that tautomeric stabilization of the imidazole ring persists in the absence of van der Waals interactions with the side chain of Tyr-25, probably as a consequence of hydrogen bonding between the protonated N delta 1 and the carbonyl oxygen of His-195.     

Protein-cofactor interactions in bacterial reaction centers from Rhodobacter sphaeroides R-26: II. Geometry of the hydrogen bonds to the primary quinone formula by 1H and 2H ENDOR spectroscopy

The geometry of the hydrogen bonds to the two carbonyl oxygens of the semiquinone Q(A)(. -) in the reaction center (RC) from the photosynthetic purple bacterium Rhodobacter sphaeroides R-26 were determined by fitting a spin Hamiltonian to the data derived from (1)H and (2)H ENDOR spectroscopies at 35 GHz and 80 K. The experiments were performed on RCs in which the native Fe(2+) (high spin) was replaced by diamagnetic Zn(2+) to prevent spectral line broadening of the Q(A)(. -) due to magnetic coupling with the iron. The principal components of the hyperfine coupling and nuclear quadrupolar coupling tensors of the hydrogen-bonded protons (deuterons) and their principal directions with respect to the quinone axes were obtained by spectral simulations of ENDOR spectra at different magnetic fields on frozen solutions of deuterated Q(A)(. -) in H(2)O buffer and protonated Q(A)(. -) in D(2)O buffer. Hydrogen-bond lengths were obtained from the nuclear quadrupolar couplings. The two hydrogen bonds were found to be nonequivalent, having different directions and different bond lengths. The H-bond lengths r(OH) are 1.73 +/- 0.03 Angstrom and 1.60 +/- 0.04 Angstrom, from the carbonyl oxygens O(1) and O(4) to the NH group of Ala M260 and the imidazole nitrogen N(delta) of His M219, respectively. The asymmetric hydrogen bonds of Q(A)(. -) affect the spin density distribution in the quinone radical and its electronic structure. It is proposed that the H-bonds play an important role in defining the physical properties of the primary quinone, which affect the electron transfer processes in the RC.     

Ultrahigh resolution drug design. II. Atomic resolution structures of human aldose reductase holoenzyme complexed with Fidarestat and Minalrestat: implications for the binding of cyclic imide inhibitors

The X-ray structures of human aldose reductase holoenzyme in complex with the inhibitors Fidarestat (SNK-860) and Minalrestat (WAY-509) were determined at atomic resolutions of 0.92 A and 1.1 A, respectively. The hydantoin and succinimide moieties of the inhibitors interacted with the conserved anion-binding site located between the nicotinamide ring of the coenzyme and active site residues Tyr48, His110, and Trp111. Minalrestat's hydrophobic isoquinoline ring was bound in an adjacent pocket lined by residues Trp20, Phe122, and Trp219, with the bromo-fluorobenzyl group inside the "specificity" pocket. The interactions between Minalrestat's bromo-fluorobenzyl group and the enzyme include the stacking against the side-chain of Trp111 as well as hydrogen bonding distances with residues Leu300 and Thr113. The carbamoyl group in Fidarestat formed a hydrogen bond with the main-chain nitrogen atom of Leu300. The atomic resolution refinement allowed the positioning of hydrogen atoms and accurate determination of bond lengths of the inhibitors, coenzyme NADP+ and active-site residue His110. The 1'-position nitrogen atom in the hydantoin and succinimide moieties of Fidarestat and Minalrestat, respectively, form a hydrogen bond with the Nepsilon2 atom of His 110. For Fidarestat, the electron density indicated two possible positions for the H-atom in this bond. Furthermore, both native and anomalous difference maps indicated the replacement of a water molecule linked to His110 by a Cl-ion. These observations suggest a mechanism in which Fidarestat is bound protonated and becomes negatively charged by donating the proton to His110, which may have important implications on drug design.     

Interactions of intermediate semiquinone with surrounding protein residues at the Q(H) site of wild-type and D75H mutant cytochrome bo3 from Escherichia coli

Selective (15)N isotope labeling of the cytochrome bo(3) ubiquinol oxidase from Escherichia coli with auxotrophs was used to characterize the hyperfine couplings with the side-chain nitrogens from residues R71, H98, and Q101 and peptide nitrogens from residues R71 and H98 around the semiquinone (SQ) at the high-affinity Q(H) site. The two-dimensional ESEEM (HYSCORE) data have directly identified N(ε) of R71 as an H-bond donor carrying the largest amount of unpaired spin density. In addition, weaker hyperfine couplings with the side-chain nitrogens from all residues around the SQ were determined. These hyperfine couplings reflect a distribution of the unpaired spin density over the protein in the SQ state of the Q(H) site and the strength of interaction with different residues. The approach was extended to the virtually inactive D75H mutant, where the intermediate SQ is also stabilized. We found that N(ε) of a histidine residue, presumably H75, carries most of the unpaired spin density instead of N(ε) of R71, as in wild-type bo(3). However, the detailed characterization of the weakly coupled (15)N atoms from selective labeling of R71 and Q101 in D75H was precluded by overlap of the (15)N lines with the much stronger ~1.6 MHz line from the quadrupole triplet of the strongly coupled (14)N(ε) atom of H75. Therefore, a reverse labeling approach, in which the enzyme was uniformly labeled except for selected amino acid types, was applied to probe the contribution of R71 and Q101 to the (15)N signals. Such labeling has shown only weak coupling with all nitrogens of R71 and Q101. We utilize density functional theory-based calculations to model the available information about (1)H, (15)N, and (13)C hyperfine couplings for the Q(H) site and to describe the protein-substrate interactions in both enzymes. In particular, we identify the factors responsible for the asymmetric distribution of the unpaired spin density and ponder the significance of this asymmetry to the quinone's electron transfer function.     

Tautomeric state and pKa of the phosphorylated active site histidine in the N-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system.

The phosphorylated form of the N-terminal domain of enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli has been investigated by one-bond and long-range 1H-15N correlation spectroscopy. The active site His 189 is phosphorylated at the Nepsilon2 position and has a pKa of 7.3, which is one pH unit higher than that of unphosphorylated His 189. Because the neutral form of unphosphorylated His 189 is in the Ndelta1-H tautomer, and its Nepsilon2 atom is solvent inaccessible and accepts a hydrogen bond from the hydroxyl group of Thr 168, both protonation and phosphorylation of His 189 must be accompanied by a change in the side-chain conformation of His 189, specifically from a chi(2) angle in the g+ conformer in the unphosphorylated state to the g- conformer in the phosphorylated state.     

Electron spin-echo spectroscopic studies of Escherichia coli fumarate reductase

Electron spin-echo envelope modulation (ESEEM) spectroscopy was applied to the study of reduced Centre 1 of Escherichia coli fumarate reductase (succinate:(acceptor) oxidoreductase, EC 1.3.99.1). The ESEEM spectrum derived from stimulated (3-pulse) echo envelopes obtained at 8.8 GHz contained lines at 0.9, 2.1, 3.0 and 4.2 MHz in the g = 1.94 region. When studied at 11.4 GHz, these low-frequency components scale with magnetic field in a manner indicating interaction between the unpaired electron spin of the Fe-S cluster and a weakly coupled 14N nucleus. Spectral simulations of these ESEEM data yield nuclear quadrupole interaction parameters indicative of peptide nitrogen. For oxidized protein, the magnetic-field dependence of the linear electric-field effect (LEFE) for Centre 3 was measured, and the results confirm the presence of a [3Fe-4S] cluster in the protein.     

Tautomeric states of the active-site histidines of phosphorylated and unphosphorylated IIIGlc, a signal-transducing protein from Escherichia coli, using two-dimensional heteronuclear NMR techniques.

IIIGlc is an 18.1-kDa signal-transducing phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system from Escherichia coli. The 1H, 15N, and 13C histidine ring NMR signals of both the phosphorylated and unphosphorylated forms of IIIGlc have been assigned using two-dimensional 1H-15N and 1H-13C heteronuclear multiple-quantum coherence (HMQC) experiments and a two-dimensional 13C-13C-1H correlation spectroscopy via JCC coupling experiment. The data were acquired on uniformly 15N-labeled and uniformly 15N/13C-labeled protein samples. The experiments rely on one-bond and two-bond J couplings that allowed for assignment of the signals without the need for the analysis of through-space (nuclear Overhauser effect spectroscopy) correlations. The 15N and 13C chemical shifts were used to determine that His-75 exists predominantly in the N epsilon 2-H tautomeric state in both the phosphorylated and unphosphorylated forms of IIIGlc, and that His-90 exists primarily in the N delta 1-H state in the unphosphorylated protein. Upon phosphorylation of the N epsilon 2 nitrogen of His-90, the N delta 1 nitrogen remains protonated, resulting in the formation of a charged phospho-His-90 moiety. The 1H, 15N, and 13C signals of the phosphorylated and unphosphorylated proteins showed only minor shifts in the pH range from 6.0 to 9.0. These data indicate that the pK alpha values for both His-75 and His-90 in IIIGlc and His-75 in phospho-IIIGlc are less than 5.0, and that the pK alpha value for phospho-His-90 is greater than 10. The results are presented in relation to previously obtained structural data on IIIGlc, and implications for proposed mechanisms of phosphoryl transfer are discussed.