Hyperfine Sublevel Correlation Spectroscopy Research Articles

Studying high-spin ferric heme proteins by pulsed EPR spectroscopy: Analysis of the ferric form of the E7Q mutant of human neuroglobin

In this work, the high-spin ferric form of the E7Q mutant of human neuroglobin (E7Q-NGB) is studied by X-band continuous-wave electron paramagnetic resonance (CW EPR) and hyperfine sublevel correlation (HYSCORE) spectroscopy. It is shown that the use of matched pulses in the HYSCORE experiment is essential to observe the nitrogen spectral contributions. The validity of approximating the high-spin Fe(III) system (S=5/2) as an effectiveS=1/2 system is tested and the consequences for the HYSCORE simulations are highlighted. Comparative HYSCORE experiments combined with deuterium exchange experiments for aquometmyoglobin and ferric E7Q-NGB clearly show that the heme iron of the latter protein is pentacoordinated, lacking the distal water. Furthermore, CW EPR experiments show that, at high pH, the E10K residue is coordinating to the heme iron in this globin. These observations are corroborated by resonance Raman experiments and could also be reproduced for other E7 mutants of human and mouse neuroglobin. Finally, the proton and nitrogen hyperfine and nuclear quadrupole parameters obtained for ferric E7Q-NGB are discussed in detail.

ENDOR and related EMR methods applied to flavoprotein radicals

Flavoproteins are involved in a wide range of biological processes, owing to the versatility of the isoalloxazine moiety of flavin, which can undergo both one- and two-electron reactions with the formation of three oxidation states. Paramagnetic semiquinone radical states are stabilised in some flavoproteins and appear as transient intermediates in the reaction of many others. The apoprotein controls the reactivity of flavin semiquinones, including redox potentials, protonation states and access to substrates. Most flavoproteins are involved in oxidation-reduction processes, but some catalyze different types of reactions involving radical intermediates. Anionic and neutral flavin radicals are found in flavoproteins and are distinguished by their line widths in X-band electron paramagnetic resonance. Electron-nuclear double resonance, electron spin echo envelope modulation and hyperfine sublevel correlation spectroscopy make it possible to observe biological electron transfer and catalysis at the level of the electronic structure of the intermediate states. They provide information about the protein environment of flavin semiquinone radicals and their interactions with nearby nuclear and electron spins. Hyperfine couplings, particularly to the 8-methyl protons on the flavin ring, are a sensitive probe of perturbations of the flavin environment. They demonstrate differences in polarity of the flavin binding site and changes that occur in flavoenzymes during binding of substrates.

Structural analysis of Cu(II) ligation to the 5′-GMP nucleotide by pulse EPR spectroscopy

Simple copper salts are known to denature poly d(GC). On the other hand, copper complexes of substituted 1,4,7,10,13-pentaazacyclohexadecane-14,16-dione are able to convert the right-handed B form of the same DNA sequence to the corresponding left-handed Z form. A research program was started in order to understand why Cu(II) as an aquated ion melts DNA and induces the conformational change to Z-DNA in the form of an azamacrocyclic complex. In this paper, we present a continuous wave and pulse electron paramagnetic resonance study of the mononucleotide model system Cu(II)-guanosine 5'-monophosphate . Pulse EPR methods like electron-nuclear double resonance and hyperfine sublevel correlation spectroscopy provide unique information about the electronic and geometric structure of this model system through an elaborate mapping of the hyperfine and nuclear quadrupole interactions between the unpaired electron of the Cu(II) ion and the magnetic nuclei of the nucleotide ligand. It was found that the Cu(II) ion is directly bound to N7 of guanosine 5'-monophosphate and indirectly bound via a water of hydration to a phosphate group. This set of experiments opens the way to more detailed structural characterization of specifically bound metal ions in a variety of nucleic acids of biological interest, in particular to understand the role of the metal-(poly)nucleotide interaction.

Axial Coordination of Heme in Ferric CcmE Chaperone Characterized by EPR Spectroscopy

In Escherichia coli cytochrome c maturation requires a set of eight proteins including the heme chaperone CcmE, which binds heme transiently, yet covalently. Several variants of CcmE were purified and analyzed by continuous-wave electron paramagnetic resonance, electron nuclear double resonance, and hyperfine sublevel correlation spectroscopy to investigate the heme axial coordination. Results reveal the presence of a number of coordination environments, two high-spin heme centers with different rhombicities, and at least one low-spin heme center. The low-spin species was shown to be an artifact induced by the presence of available histidines in the vicinity of the iron. Both of the high-spin forms are five-coordinated, and comparison of the spectra of the wild-type CcmE with those of the mutant CcmE Y134H proves that the higher-rhombicity form is coordinated by Tyr 134. The low-rhombicity (axial) form does not have a histidine residue or a water molecule as an axial ligand. However, we identified exchangeable protons coupled to the iron ion. We propose that the axial form can be coordinated by a carboxyl group of an acidic residue in the flexible domain of the protein. The two species would represent two different conformations of the flexible α-helix domain surrounding the heme. This conformational flexibility confers CcmE special dynamic properties that are certainly important for its function.

Dynamical effects and ergodicity in the dipolar glass phase:evidence from time-domain EPR and phase memory time studies ofAsO44− inRb1−x(NH4)xH2PO4 (x = 0,0.5,1)

Three-pulse electron spin echo envelope modulation (ESEEM), hyperfinesublevel correlation spectroscopy (HYSCORE) investigations andtwo-pulse electron spin echo (ESE) measurements of phase memory timeTM, were carried out, in the 20–200 K temperature range, on anAsO44− paramagneticprobe stabilized in RbH2PO4 (RDP), NH4H2PO4 (ADP),and dipolar glass Rb0.5(NH4)0.5H2PO4 (RADP). The results obtained on ADP revealed hyperfine interaction of the probe ion with the14N of the ammonium ion, the coupling constant satisfying the condition of ‘cancellation’ at afield of 480 mT. The ammonium ion was found to be in two different sites in ADP,which became indistinguishable on the formation of dipolar glass RADP. Theseresults were confirmed by HYSCORE spectral measurements. The fast Fouriertransform (FFT) spectra of three-pulse ESEEM decays have clearly revealed theinteraction with protons in the bond both in ADP and RDP; and in RADP with an averaged couplingconstant. The phase memory times in RADP exhibited strong temperaturedependence and were found to be dependent on the nuclear spin quantum numbermI of75As. The temperaturedependence of TM exhibited a well-defined maximum around 90 K, coinciding with the temperature of onset of ‘freezing’in Rb0.5(NH4)0.5H2PO4. This is symptomatic of dynamic fluctuations in the dipolar glass phase, with onset around150 K, going through a maximum around 90 K and slowing down on further cooling. Theseresults suggest that in RADP, a dynamical mechanism with progressive slowing downbelow 90 K is operative in the glass formation. This implies that the RADP system, withx = 0.5, exists in an ergodic relaxor (R)-state in the 20–200 K temperature range wherein everyfluctuating monodomain can be viewed as statistically representative of the whole sample.

Hydrogen Bonding Affects the [NiFe] Active Site ofDesulfovibriovulgarisMiyazaki F Hydrogenase: A Hyperfine Sublevel Correlation Spectroscopy and Density Functional Theory Study

Pulse electron paramagnetic resonance and hyperfine sublevel correlation spectroscopy have been used to investigate nitrogen coordination of the active site of [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F in its oxidized "ready" state. The obtained (14)N hyperfine (A = [+1.32, +1.32, +2.07] MHz) and nuclear quadrupole (e(2)qQ/h = -1.9 MHz, eta = 0.37) coupling constants were assigned to the N(epsilon) of a highly conserved histidine (His88) by studying a hydrogenase preparation in which the histidines were (15)N labeled. The histidine is hydrogen-bonded via its N(epsilon)-H to the nickel-coordinating sulfur of a cysteine (Cys549) that carries an appreciable amount of spin density. Through the hydrogen bond a small fraction of the spin density ( approximately 1%) is delocalized onto the histidine ring giving rise to an isotropic (14)N hyperfine coupling constant of about 1.6 MHz. These conclusions are supported by density functional calculations. The measured (14)N quadrupole coupling constants are related to the polarization of the N(epsilon)-H bond, and the respective hydrogen bond can be classified as being weak.

15N HYSCORE Characterization of the Fully Deprotonated, Reduced Form of the Archaeal Rieske [2Fe−2S] Center

The hyperfine couplings for strongly and weakly coupled 15N nuclei around a reduced Rieske [2Fe-2S] center of uniformly 15N-labeled, hyperthermostable archaeal Rieske protein at pH 13.3 were determined by hyperfine sublevel correlation (HYSCORE) spectroscopy and compared with those at physiological pH. Significant changes in the hyperfine couplings of the terminal histidine Ndelta ligands and Nepsilon nuclei were observed between them, which can be explained by not only the redistribution of the unpaired electron spin density over the ligands but also the difference in the mixed-valence state of the fully deprotonated, reduced cluster. These quantitative data can be used in theoretical analysis for the selection of an appropriate model of the mixed-valence state of the reduced Rieske center at very alkaline pH.

Electronic Structure of Binuclear Mixed Valence Copper Azacryptates Derived from Integrated Advanced EPR and DFT Calculations

Binuclear, mixed valence copper complexes with a [Cu(+1)(.5), Cu(+1)(.5)] redox state and S = (1)/(2) can be stabilized with rigid azacryptand ligands. In this system the unpaired electron is delocalized equally over the two copper ions, and it is one of the very few synthetic models for the electron mediating Cu(A) site of nitrous oxide reductase and cytochrome c oxidase. The spatial and electronic structures of the copper complex in frozen solution were obtained from the magnetic interactions, namely the g-tensor and the (63,65)Cu, (14)N, (2)H, and (1)H hyperfine couplings, in combination with density functional theory (DFT) calculations. The magnetic interactions were determined from continuous wave (CW) electron paramagnetic resonance (EPR), pulsed electron nuclear double resonance (ENDOR), two-dimensional TRIPLE, and hyperfine sublevel correlation spectroscopy (HYSCORE) carried out at W-band or/and X-band frequencies. The DFT calculated g and Cu hyperfine values were in good agreement with the experimental values showing that the structure in solution is indeed close to that of the optimized structure. Then, the DFT calculated hyperfine parameters were used as guidelines and starting points in the simulations of the various experimental ENDOR spectra. A satisfactory agreement with the experimental results was obtained for the (14)N hyperfine and quadrupole interactions. For (1)H the DFT calculations gave good predictions for the hyperfine tensor orientations and signs, and they were also successful in reproducing trends in the magnitude of the various proton hyperfine couplings. These, in turn, were very useful for ENDOR signals assignments and served as constraints on the simulation parameters.

Location of Vanadium(IV) in VAPO-5 as Studied by Hyperfine Sublevel Correlation Spectroscopy

Abstract Vanadium(IV) framework substitution in aluminophosphate molecular sieves has been explored by electron spin resonance (ESR) and hyperfine sublevel correlation (HYSCORE) spectroscopy. In both as-synthesized and hydrated materials V(IV) exists as VO2+ species that coordinates to two framework oxygen atoms and presumably three water molecules in a distorted octahedral geometry.

Mn2+-adenosine nucleotide complexes in the presence of the nitrogenase iron-protein: detection of conformational rearrangements directly at the nucleotide binding site by EPR and 2D-ESEEM (two-dimensional electron spin-echo envelope modulation spectroscopy).

Both ATP and a bivalent nucleotide-bound metal activator, normally Mg2+, are required for nitrogenase activity. EPR and ESEEM (electron spin-echo envelope modulation) measurements have been carried out on adenosine nucleotides in which the Mg2+ ion that is usually bound is replaced by Mn2+ in the presence of Kp2 (nitrogenase Fe-protein from Klebsiella pneumoniae). The Mn2+ zero-field splitting parameters have been determined from the EPR-spectrum to be |D|=0.0125 cm(-1) with a rhombicity lambda=E/D=0.31 by direct diagonalization of the complete spin Hamiltonian. ESEEM spectra of the Fe-protein with MnADP and MnATP both show an ESEEM line pair with one signal component at about 3.6 MHz and a relatively broad resonance at 8 MHz originating from a superhyperfine coupling to a 31P nuclear spin from one or more directly co-ordinated phospho group(s) of the nucleotide. A pronounced resonance overlapping the low-frequency component of the (31)P-signal at about 3.5 MHz is attributed to an interaction of Mn2+ with univalent 23Na nuclei. ESEEM lines at frequencies <3.5 MHz have been ascribed to interactions with 14N nuclei. Differences in the 14N features that depend on the type of nucleotide are consistent with substantial conformational rearrangements at the nucleotide-binding site upon hydrolysis. In addition, four-pulse HYSCORE (hyperfine sublevel correlation spectroscopy) experiments not only confirm the three-pulse ESEEM results, but also achieve significantly better spectral deconvolution, especially of the 31P-couplings, and demonstrate that the nucleotide is at least a unidentate ligand of Mn2+. Moreover it was also possible to identify peaks from an 14N interaction more clearly; these most probably arise from outer-sphere interactions with nitrogen atom(s) of non-co-ordinated residues which are affected by conformational rearrangements upon nucleotide hydrolysis. In addition, different redox states of the [4Fe-4S] cluster of the Fe-protein show disparate conformations of the metal-nucleotide co-ordination environment, demonstrating that also the cluster site communicates with the nucleotide binding site.

Pulsed ENDOR Study of Cu(I)−NO Adsorption Complexes in Cu−L Zeolite

The local environments of Cu(I)-NO adsorption complexes formed in zeolites Cu-L and Cu-ZSM-5 were studied by electron spin resonance (ESR), pulsed electron nuclear double resonance (ENDOR), and hyperfine sublevel correlation spectroscopy (HYSCORE). Cu(I)-NO complexes have attracted special interest because they are important intermediates in the catalytic decomposition of nitric oxide over copper exchanged zeolites. Recently, detailed structures of the complexes in Cu-ZSM-5 zeolites, O2-Al-O2-Cu(I)-NO, have been proposed on the basis of quantum chemical calculations (Pietrzyk, et al. J. Phys. Chem. B 2003, 107, 6105. Dedecek, et al. Phys. Chem. Chem. Phys. 2002, 4, 5406). 27Al pulsed ENDOR and HYSCORE experiments allowed the hyperfine coupling parameters of an aluminum nuclei found in the vicinity of the Cu(I)-NO complex formed in zeolite Cu-L to be estimated. The data indicate that the aluminum atom is located in the third coordination sphere of the adsorbed NO molecule in agreement with the suggested geometry of the adsorption sites. Broad distributions of aluminum nuclear quadrupole and hyperfine coupling parameters and short electron spin relaxation times of the Cu(I)-NO species prevented the determination of the 27Al hyperfine couplings for zeolite Cu-ZSM-5.

An orientation-selected ENDOR and HYSCORE study of the Ni-C active state of Desulfovibrio vulgaris Miyazaki F hydrogenase

Electron nuclear double resonance (ENDOR) and hyperfine sublevel correlation spectroscopy (HYSCORE) are applied to study the active site of catalytic [NiFe]-hydrogenase from Desulfovibrio vulgaris Miyazaki F in the reduced Ni-C state. These techniques offer a powerful tool for detecting nearby magnetic nuclei, including a metal-bound substrate hydrogen, and for mapping the spin density distribution of the unpaired electron at the active site. The observed hyperfine couplings are assigned via comparison with structural data from X-ray crystallography and knowledge of the complete g-tensor in the Ni-C state (Foerster et al. (2003) J Am Chem Soc 125:83-93). This is found to be in good agreement with density functional theory calculations. The two most strongly coupled protons (a(iso)=13.7, 11.8 MHz) are assigned to the beta-CH(2) protons of the nickel-coordinating cysteine 549, and a third proton (a(iso)=8.9 MHz) is assigned to a beta-CH(2) proton of cysteine 546. Using D(2)O exchange experiments, the presence of a hydride in the bridging position between the nickel and iron-recently been detected for a regulatory hydrogenase (Brecht et al. (2003) J Am Chem Soc 125:13075-13083)-is experimentally confirmed for the first time for catalytic hydrogenases. The hydride exhibits a small isotropic hyperfine coupling constant (a(iso)=-3.5 MHz) since it is bound to Ni in a direction perpendicular to the z-axis of the Ni (3d(z)(2)) orbital. Nitrogen signals that belong to the nitrogen N(epsilon) of His-88 have been identified. This residue forms a hydrogen bond with the spin-carrying Ni-coordinated sulfur of Cys-549. Comparison with other hydrogenases reveals that the active site is essentially the same in all proteins, including a regulatory hydrogenase.

17O ESEEM Evidence for Exchange of the Axial Oxo Ligand in the Molybdenum Center of the High pH Form of Sulfite Oxidase

A 17O ESEEM investigation of the high pH form of chicken sulfite oxidase using hyperfine sublevel correlation (HYSCORE) spectroscopy at 29.25 GHz has revealed a new type of exchangeable 17O ligand that is characterized by a significantly smaller hyperfine interaction ( approximately 5 MHz) than that previously detected by CW EPR. This new type of exchangeable oxygen ligand is assigned to the axial oxo group of the Mo(V) center.

Structural Investigation of Vanadyl-Acetylacetonate-Containing Precursors of TiOx−VOxMixed Oxides on SBA-15

TiOx−VOx mixed-oxide supported SBA-15 catalysts were prepared by the molecular designed dispersion method (MDD) using acetylacetonate complexes. Two different synthesis route depositions are presented to prepare these mixed-oxide catalysts in a very controlled way. Continuous wave and pulse EPR spectroscopy is employed to determine the location of the VO(acac)2 complexes in the mixed-oxide precursors (before calcination) in comparison to other supports. It is shown that the different synthesis pathways lead to differences in the local structure of the VO2+ complexes and to a distinctive degree of spreading of the metals on the support surface. This is confirmed by additional characterizations of the final catalyst in terms of chemical composition and porosity. Depending on the way of preparation of the mixed oxides, the final catalysts present significant differences in pore structure, showing partially blocked or open mesopores. In the case of the mixed-oxide samples a higher dispersion of the metals is observed compared to the disposition of VO(acac)2 on the pure SBA-15 support. Both hyperfine sublevel correlation (HYSCORE) and IR spectroscopy show that the VO(acac)2 complex, in contrast to the TiO(acac)2 complex, has a great tendency to increase its coordination sphere by coordinating water molecules, but that this tendency is highly reduced when VO(acac)2 is present in a Ti environment.

Two-Dimensional Pulsed EPR Studies of Vanadium-Exchanged ZSM-5

The pulsed electron paramagnetic resonance (EPR) technique of hyperfine sublevel correlation spectroscopy (HYSCORE) was used to obtain structural information about vanadium(VO2+)-exchanged ZSM-5. HYSCORE spectra were obtained for vanadium-exchanged ZSM-5 before and after dehydration and after the adsorption of ammonia. For the hydrated samples, proton hyperfine coupling constants were measured and assigned to equatorial water ligands with orientations perpendicular and parallel to the equatorial plane. Nitrogen hyperfine coupling constants for adsorbed ammonia were also determined from the HYSCORE spectra. The results were compared with previous density functional theory (DFT) calculations of hyperfine coupling constants for vanadyl model complexes.

FT-EPR spectroscopy in the borate system

80-mol% B2O3 20-mol% Li2O glass has been subjected to γ-irradiation at room temperature and subsequently studied by CW EPR (continuous wave electron paramagnetic resonance), ECHO-fs (field sweep), 4p-ESEEM (electron spin echo envelop modulation spectroscopy), 2D-HYSCORE (hyperfine sublevel correlation) spectroscopy and pulse ENDOR (electron nuclear double resonance) spectroscopies. The variation of the EPR signal with the conditions of measurements suggests that several centers contribute to the signal. The Aiso-couplings of the next neighbor have been determined by these methods. One may suggest on this ground the existence of the tetraborate, diborate and boron network non-bridging oxygen (BN-nbo) structures, though this preliminary effort needs supplementary investigational work to be convinced about it.

Determination of functional center local environment in copper-modified Pb[Zr0.54Ti0.46]O3 ceramics

Ferroelectric copper(II)-modified polycrystalline Pb[Zr0.54Ti0.46]O3 ceramics with a dopant concentration of 0.25 mol % were investigated by means of electron paramagnetic resonance and hyperfine sublevel correlation spectroscopy. Special emphasis was put on the resolution of the Pb207 hyperfine couplings, which were resolved in the first and second coordination sphere. The results prove that copper is incorporated at the octahedrally coordinated [Zr,Ti] site, acting as an acceptor and disfavor a directly coordinated oxygen vacancy to the copper center for charge compensation.

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.

133Cesium HYSCORE Investigation of the Di-tert-butyl Nitroxide−Cs+ Adsorption Complex in CsNaY Zeolite

133Cs hyperfine sublevel correlation (HYSCORE) spectroscopy has been employed to characterize the structure of adsorption complexes of di-tert-butyl nitroxide (DTBN) with cesium cations in zeolite CsNaY. The experimental 133Cs HYSCORE data proved the direct coordination of the adsorbed DTBN molecules to the Cs+ ions and revealed nonambiguously the existence of two different types of adsorption complexes. Evaluation of the orientation selective 133Cs HYSCORE spectra provided the 133Cs hyperfine coupling tensors and thus information about the geometrical structure of those complexes. For one type of adsorption complexes, a complex geometry was obtained where the Cs+ ion is located within the molecular mirror plane of the DTBN radical with an oxygen−cesium cation bond length of 0.25 nm. For this, the isotropic Cs hyperfine coupling was negative. The second Cs+−DTBN complex is characterized by a bent structure. With an isotropic 133Cs hyperfine coupling of 9 MHz, the unpaired electron spin density in this Cs+...

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.