Multifrequency pulsed electron paramagnetic resonance study of the S2 state of the photosystem II manganese cluster

Multifrequency electron spin-echo envelope modulation (ESEEM) spectroscopy is employed to measure the strength of the hyperfine coupling of magnetic nuclei to the paramagnetic (S = 1/2) S2 form of photosystem II (PSII). Previous X-band-frequency ESEEM studies indicated that one or more histidine nitrogens are electronically coupled to the tetranuclear manganese cluster in the S2 state of PSII. However, the spectral resolution was relatively poor at the approximately 9 GHz excitation frequency, precluding any in-depth analysis of the corresponding bonding interaction between the detected histidine and the manganese cluster. Here we report ESEEM experiments using higher X-, P-, and Ka-band microwave frequencies to target PSII membranes isolated from spinach. The X- to P-band ESEEM spectra suffer from the same poor resolution as that observed in previous experiments, while the Ka-band spectra show remarkably well-resolved features that allow for the direct determination of the nuclear quadrupolar couplings for a single I = 1(14)N nucleus. The Ka-band results demonstrate that at an applied field of 1.1 T we are much closer to the exact cancellation limit (alpha iso = 2nu(14)N) that optimizes ESEEM spectra. These results reveal hyperfine (alpha iso = 7.3 +/- 0.20 MHz and alpha dip = 0.50 +/- 0.10 MHz) and nuclear quadrupolar (e(2)qQ = 1.98 +/- 0.05 MHz and eta = 0.84 +/- 0.06) couplings for a single (14)N nucleus magnetically coupled to the manganese cluster in the S 2 state of PSII. These values are compared to the histidine imidazole nitrogen hyperfine and nuclear quadrupolar couplings found in superoxidized manganese catalase as well as (14)N couplings in relevant manganese model complexes.     

Model studies of the Cu(B) site of cytochrome c oxidase utilizing a Zn(II) complex containing an imidazole-phenol cross-linked ligand

Cytochrome c oxidase, the enzyme complex responsible for the four-electron reduction of O2 to H2O, contains an unusual histidine-tyrosine cross-link in its bimetallic heme a3-CuB active site. We have synthesised an unhindered, tripodal chelating ligand, BPAIP, containing the unusual ortho-imidazole-phenol linkage, which mimics the coordination environment of the CuB center. The ligand was used to investigate the physicochemical (pKa, oxidation potential) and coordination properties of the imidazole-phenol linkage when bound to a dication. Zn(II) coordination lowers the pKa of the phenol by 0.6 log units, and increases the potential of the phenolate/phenoxyl radical couple by approximately 50 mV. These results are consistent with inductive withdrawal of electron density from the phenolic ring. Spectroscopic data and theoretical calculations (DFT) were used to establish that the cationic complex [Zn(BPAIP)Br]+ has an axially distorted trigonal bipyramidal structure, with three coordinating nitrogen ligands (two pyridine and one imidazole) occupying the equatorial plane and the bromide and the tertiary amine nitrogen of the tripod in the axial positions. Interestingly, the Zn-Namine bonding interaction is weak or absent in [Zn(BPAIP)Br]+ and the complex gains stability in basic solutions, as indicated by 1H NMR spectroscopy. These observations are supported by theoretical calculations (DFT), which suggest that the electron-donating capacity of the equatorial imidazole ligand can be varied by modulation of the protonation and/or redox state of the cross-linked phenol. Deprotonation of the phenol makes the equatorial imidazole a stronger sigma-donor, resulting in an increased Zn-Nimd interaction and thereby leading to distortion of the axial ligand axis toward a more tetrahedral geometry.     

The interstitial atom of the nitrogenase FeMo-cofactor: ENDOR and ESEEM show it is not an exchangeable nitrogen

A recent high-resolution X-ray crystallographic study (1.16 A) of the Azotobacter vinelandii nitrogenase MoFe protein revealed a previously undetected electron density associated with the active site FeMo-cofactor. The density is located inside the cluster at the center of the "trigonal prism" of six irons and is assigned to a species "X". The identity of species X was not resolved, although the electron density is consistent with a single N, O, or C atom. One proposal is that X is an N atom that derives from and exchanges with N from N2 during catalysis. In the present study, we have examined this possibility by employing 14N and 15N isotopes of N2 along with ENDOR and ESEEM spectroscopies. The WT MoFe protein and alpha-359Arg-->Lys and alpha-381Phe-->Leu variants were allowed to turn over in the presence of 14N2 or 15N2, and then were examined as resting enzymes by ENDOR and ESEEM at X- and Q-bands to look for all 14N and 15N signals coupled to the electron spin of the FeMo-cofactor and to determine if any exchanged during turnover. We have found five peaks in Q-band pulsed ENDOR spectra that appear to arise not only from previously reported N1/N2, which give rise to the ESEEM, but also from one or two additional coupled nitrogens. None of the ENDOR and ESEEM signals vanish or are altered by catalytic turnover with 15N2, and no new 15N signal is detected, leading to the conclusion that if species X is a nitrogen atom, it does not exchange during dinitrogen reduction.     

Investigation of the calcium-binding site of the oxygen evolving complex of photosystem II using 87Sr ESEEM spectroscopy

The proximity of the calcium/strontium binding site of the oxygen evolving complex (OEC) of photosystem II (PSII) to the paramagnetic Mn cluster is explored with (87)Sr three-pulse electron spin-echo envelope modulation (ESEEM) spectroscopy. CW-EPR spectra of Sr(2+)-substituted Ca(2+)-depleted PSII membranes show the modified g = 2 multiline EPR signal as previously reported. We performed three-pulse ESEEM on this modified multiline signal of the Mn cluster using natural abundance Sr and (87)Sr, respectively. Three-pulse ESEEM of the natural abundance Sr sample exhibits no detectable modulation by the 7% abundance (87)Sr. On the other hand, that of the (87)Sr enriched (93%) sample clearly reveals modulation arising from the I = (9)/(2) (87)Sr nucleus weakly magnetically coupled to the Mn cluster. Using a simple point dipole approximation for the electron spin, analysis of the (87)Sr ESEEM modulation depth via an analytic expression suggests a Mn-Ca (Sr) distance of 4.5 A. Simulation of three-pulse ESEEM with a numerical matrix diagonalization procedure gave good agreement with this analytical result. A more appropriate tetranuclear magnetic/structural model for the Mn cluster converts the 4.5 A point dipole distance to a 3.8-5.0 A range of distances. DFT calculations of (43)Ca and (87)Sr quadrupolar interactions on Ca (and Sr substituted) binding sites in various proteins suggest that the lack of the nuclear quadrupole induced splitting in the ESEEM spectrum of (87)Sr enriched PSII samples is related to a very high degree of symmetry of the ligands surrounding the Sr(2+) ion in the substituted Ca site. Numerical simulations show that moderate (87)Sr quadrupolar couplings decrease the envelope modulation relative to the zero quadrupole case, and therefore we consider that the 3.8-5.0 A range obtained without quadrupolar coupling included in the simulation represents an upper limit to the actual manganese-calcium distance. This (87)Sr pulsed EPR spectroscopy provides independent direct evidence that the calcium/strontium binding site is close to the Mn cluster in the OEC of PSII.     

Nitramine anion fragmentation: A mass spectrometric and Ab initio study

The fragment ion formation characteristics of the radical anions generated from hexahydro-1,3,5-trinitrotriazine (RDX) and its three nitroso metabolites were studied using GC/MS with negative chemical ionization (NCI) to understand the fragmentation mechanisms responsible for the formation of the most abundant ions observed in their NCI mass spectra. Ab initio and density functional theory calculations were used to calculate relative free energies for different fragment ion structures suggested by the m/z values of the most abundant ions observed in the NCI mass spectra. The NCI mass spectra of the four nitramines are dominated by ions formed by the cleavage of nitrogen-nitrogen and carbon-nitrogen bonds in the atrazine ring. The most abundant anions in the NCI mass spectra of these nitramines have the general formulas C(2)H(4)N(3)O (m/z 86) and C(2)H(4)N(3)O(2) (m/z 102). The analyses of isotope-labeled standards indicate that these two ions are formed by neutral losses that include two exocylic nitrogens and one atrazine ring nitrogen. Our calculations and observations of the nitramine mass spectra suggest that the m/z 86 and m/z 102 ions are formed from either the (M--NO)(-) or (M--NO(2))(-) fragment anions by a single fragmentation reaction producing neutral losses of CH(2)N(2)O or CH(2)N(2)O(2) rather than a set of sequential reactions involving neutral losses of HNO(2) or HNO and HCN.     

Identification of the nitrogen donor hydrogen bonded with the semiquinone at the Q(H) site of the cytochrome bo3 from Escherichia coli

The selective (15)N isotope labeling was used for the identification of the nitrogen involved in a hydrogen bond formation with the semiquinone in the high-affinity Q(H) site in the cytochrome bo(3) ubiquinol oxidase. This nitrogen produces dominating contribution to X-Band (14)N ESEEM spectra. The 2D ESEEM (HYSCORE) experiments with the Q(H) site SQ in the series of selectively (15)N labeled bo(3) oxidase proteins have directly identified the N(epsilon) of R71 as an H-bond donor. In addition, selective (15)N labeling has allowed us for the first time to determine weak hyperfine couplings with the side-chain nitrogens from all residues around the SQ. Those are reflecting a distribution of the unpaired spin density over the protein in the SQ state of the quinone processing site.     

Characterization of the phenoxyl radical in model complexes for the Cu(B) site of cytochrome c oxidase: steady-state and transient absorption measurements, UV resonance raman spectroscopy, EPR spectroscopy, and DFT calculations for M-BIAIP

Physicochemical properties of the covalently cross-linked tyrosine-histidine-Cu(B) (Tyr-His-Cu(B)) unit, which is a minimal model complex [M(II)-BIAIPBr]Br (M = Cu(II), Zn(II)) for the Cu(B) site of cytochrome c oxidase, were investigated with steady-state and transient absorption measurements, UV resonance Raman (UVRR) spectroscopy, X-band continuous-wave electron paramagnetic resonance (EPR) spectroscopy, and DFT calculations. The pH dependency of the absorption spectra reveals that the pK(a) of the phenolic hydroxyl is ca. 10 for the Cu(II) model complex (Cu(II)-BIAIP) in the ground state, which is similar to that of p-cresol (tyrosine), contrary to expectations. The bond between Cu(II) and nitrogen of cross-linked imidazole cleaves at pH 4.9. We have successfully obtained UVRR spectra of the phenoxyl radical form of BIAIPs and have assigned bands based on the previously reported isotope shifts of Im-Ph (2-(1-imidazoyl)-4-methylphenol) (Aki, M.; Ogura, T.; Naruta, Y.; Le, T. H.; Sato, T.; Kitagawa, T. J. Phys. Chem. A 2002, 106, 3436-3444) in combination with DFT calculations. The upshifts of the phenoxyl vibrational frequencies for 8a (C-C stretching), 7a' (C-O stretching), and 19a, and the Raman-intensity enhancements of 19b, 8b, and 14 modes indicate that UVRR spectra are highly sensitive to imidazole-phenol covalent linkage. Both transient absorption measurements and EPR spectra suggest that the Tyr-His-Cu(B) unit has only a minor effect on the electronic structure of the phenoxyl radical form, although our experimental results appear to indicate that the cross-linked Tyr radical exhibits no EPR. The role of the Tyr-His-Cu(B) unit in the enzyme is discussed in terms of the obtained spectroscopic parameters of the model complex.     

Optical spectra of protected diamine 10-bond-bridged intervalence radical cations related to N,N,N'N'-tetraalkylbenzidine

The optical absorption spectra of the delocalized intervalence radical cations of seven o,o'-linked benzidine derivatives that have the nitrogens protected as 9-(9-aza-bicyclo[3.3.1]nonan-3-one) derivatives are discussed and compared with that of the p-phenylene radical cation. The linking units are CH2, CH2CH2, NMe, S, SO2, and C=O, and we also studied H,H (the unlinked benzidine). The lowest-energy absorption band is assigned as the transition from the antibonding combination of symmetrical N and aromatic orbitals to the antibonding combination of the antisymmetric N and aromatic orbitals using TD-DFT calculations, and a good correlation between the observed transition energies and those calculated using the simple Koopmans theorem-based "neutral in-cation geometry" calculations on the UB3LYP/6-31G* structures is found. The use of the two-state model that equates the electronic interaction through the bridge between the amino groups with half of the lowest transition energy is seriously incorrect for these and other delocalized intervalence compounds. The problem of extracting the electronic interactions that actually are involved from calculated transition energies is discussed.     

The phenoxy/phenol/copper cation: a minimalistic model of bonding relations in active centers of mononuclear copper enzymes

The "bare" complex [Cu(PhOH)(PhO)](+) with a phenol (PhOH) and a phenoxy (PhO) ligand bound to copper is studied both experimentally and computationally. The binding energies and structure of this complex are probed by mass spectrometry, infrared multi-photon dissociation, and DFT calculations. Further, the monoligated complexes [Cu(PhO)](+) and [Cu(PhOH)](+) are investigated for comparison. DFT calculations on the [Cu(PhOH)(PhO)](+) complex predict that a phenolate anion interacts with copper(II) preferentially through the oxygen atom, and the bonding is associated with electron transfer to the metal center resulting in location of the unpaired electron at the aromatic moiety. Neutral phenol, on the other hand, interacts with copper preferentially through the aromatic ring. The same arrangements are also found in the monoligated complexes [Cu(PhO)](+) and [Cu(PhOH)](+). The calculations further indicate that the bond strength between the copper atom and the oxygen atom of the phenoxy radical is weakened by the presence of neutral phenol from 2.6 eV in bare [Cu(PhO)](+) to 2.1 eV in [Cu(PhOH)(PhO)](+).     

Spectroscopic study of 2-, 4- and 5-substituents on pKa values of imidazole heterocycles prone to intramolecular proton-electrons transfer

New 2-(1H-imidazol-2-yl)phenols (L1Et–L8tBuPt) bearing a phenolic proton in the vicinity of the imidazole base were prepared and characterized. Experimental studies of the dependence of their protonation/deprotonation equilibrium on substituent identities and intramolecular hydrogen bonding tendencies were carried out using electronic absorption spectroscopy at varying pH values. In order to make comparison, 2-(anthracen-10-yl)-4,5-diphenyl-1H-imidazole (L9Anthr) bearing no phenolic proton and 4,5-diphenyl-2-(4,5-diphenyl-1H-imidazol-2-yl)-1H-imidazole (L10BisIm) bearing two symmetrical imidazole base fragments were also prepared and experimentally investigated. DFT calculations were carried out to study frontier orbitals of the investigated molecules. While electron-releasing substituents produced increase in protonation–deprotonation pK_as for the hydroxyl group, values for the imidazole base were mainly affected by polarization of the imidazole ring aromaticity across the 2-imidazole carbon and the 4,5-imidazole carbons axis of the imidazole ring. It was concluded that electron-releasing substituents on the phenol ring and/or electron-withdrawing substituents on 4,5-imidazole carbons negatively affects donor strengths/coordination chemistries of 2-(1H-imidazol-2-yl)phenols, and vice versa. Change of substituents on the phenol ring significantly altered the donor strength of the imidazole base. The understanding of pK_a variation on account of electronic effects of substituents in this work should aid the understanding of biochemical properties and substituent environments of imidazole-containing biomacromolecules.     

Modeling the active site of cytochrome oxidase: synthesis and characterization of a cross-linked histidine-phenol

A cross-linked histidine-phenol compound was synthesized as a chemical analogue of the active site of cytochrome c oxidase. The structure of the cross-linked compound (compound 1) was verified by IR, (1)H and (13)C NMR, mass spectrometry, and single-crystal X-ray analysis. Spectrophotometric titrations indicated that the pK(a) of the phenolic proton on compound 1 (8.34) was lower than the pK(a) of tyrosine (10.1) or of p-cresol (10.2). This decrease in pK(a) is consistent with the hypothesis that a cross-linked histidine-tyrosine may facilitate proton delivery to the binuclear site in cytochrome c oxidase. Time-resolved optical absorption spectra of compound 1 at room temperature, generated by excitation at 266 nm in the presence and absence of dioxygen, indicated a species with absorption maxima at approximately 330 and approximately 500 nm, which we assign to the phenoxyl radical of compound 1. The electron paramagnetic resonance (EPR) spectra of compound 1, obtained after UV photolysis, confirmed the generation of a paramagnetic species at low temperature. Because the cross-linked compound lacks beta-methylene protons, the EPR line shape was dramatically altered when compared to that of the tyrosyl radical. However, simulation of the EPR line shape and measurement of the isotropic g value was consistent with a small coupling to the imidazole nitrogen and with little spin density perturbation in the phenoxyl ring. The ground-state Fourier transform infrared (FT-IR) spectrum of compound 1 showed that addition of the imidazole ring perturbs the frequency of the tyrosine ring stretching vibrations. The difference FT-IR spectrum, associated with the oxidation of the cross-linked compound, detected significant perturbations of the phenoxyl radical vibrational bands. We postulate that phenol oxidation produces a small delocalization of spin density onto the imidazole nitrogen of compound 1, which may explain its unique optical spectral properties.     

Pseudoequivalent nitrogen atoms in aqueous imidazole distinguished by chemical shifts in photoelectron spectroscopy

The photoelectron spectra of aqueous imidazole are presented, and the N 1s and C 1s binding energies are assigned aided by density functional theory calculations. The chemical equivalency of the two nitrogens of the cationic form is directly identified by the occurrence of a single N 1s photoelectron peak, which results from the delocalization of the positive charge over the molecule as a consequence of the Cv symmetry of the system. In contrast to NMR measurements, the photoemission process is faster than the rapid proton exchange in the aqueous environment, making the pseudoequivalent nitrogens of the neutral state clearly distinguishable with a N 1s binding energy shift of 1.7 eV.     

N-tert-Butoxy-1-aminopyrenyl Radicals. Isolation,Electronic Structure,and Magnetic Characterization

N-tert-Butoxy-2,7-di-tert-butyl-1-pyrenylaminyl (4), N-tert-butoxy-2-tert-butyl-1-pyrenylaminyl (5), and N-tert-butoxy-7-tert-butyl-1-pyrenylaminyl (6) free radicals were generated by the reaction of the lithium amides of the corresponding 1-aminopyrenes with tert-butyl peroxybenzoate in THF at -78 degrees C. Although 6 could not be isolated due to the gradual decomposition in solution, 4 and 5 were quite persistent and could be isolated as monomeric radical crystals. The X-ray crystallographic analyses for the isolated free radicals were successfully carried out, indicating that the N and O atoms are almost coplanar with the pyrene ring. The ESR spectra of 4 and 5 were very complex due to the presence of many magnetically unequivalent protons. Therefore, the proton hyperfine coupling (hfc) constants were determined by (1)H ENDOR/TRIPLE resonance spectroscopy. To assign the hfc constants for the pyrene ring protons, a partially deuterated radical, 4-d(4), was prepared and the ENDOR and ESR spectra were measured. To discuss the spin density distribution for 4 and 5 ab initio molecular orbital calculations were performed by the DFT UBecke 3LYP method, using the STO 6-31G basis set. Magnetic susceptibility measurements were carried out for 4 and 5 with a SQUID magnetometer. For 4 a weak antiferromagnetic interaction was observed, and for 5 a very strong antiferromagnetic interaction was observed. The antiferromagnetic interactions were explained by their crystal structures.     

Reaction of phenols with the 2,2-diphenyl-1-picrylhydrazyl radical. Kinetics and DFT calculations applied to determine ArO-H bond dissociation enthalpies and reaction mechanism

The formal H-atom abstraction by the 2,2-diphenyl-1-picrylhydrazyl (dpph(*)) radical from 27 phenols and two unsaturated hydrocarbons has been investigated by a combination of kinetic measurements in apolar solvents and density functional theory (DFT). The computed minimum energy structure of dpph(*) shows that the access to its divalent N is strongly hindered by an ortho H atom on each of the phenyl rings and by the o-NO(2) groups of the picryl ring. Remarkably small Arrhenius pre-exponential factors for the phenols [range (1.3-19) x 10(5) M(-1) s(-1)] are attributed to steric effects. Indeed, the entropy barrier accounts for up to ca. 70% of the free-energy barrier to reaction. Nevertheless, rate differences for different phenols are largely due to differences in the activation energy, E(a,1) (range 2 to 10 kcal/mol). In phenols, electronic effects of the substituents and intramolecular H-bonds have a large influence on the activation energies and on the ArO-H BDEs. There is a linear Evans-Polanyi relationship between E(a,1) and the ArO-H BDEs: E(a,1)/kcal x mol(-1) = 0.918 BDE(ArO-H)/kcal x mol(-1) - 70.273. The proportionality constant, 0.918, is large and implies a "late" or "product-like" transition state (TS), a conclusion that is congruent with the small deuterium kinetic isotope effects (range 1.3-3.3). This Evans-Polanyi relationship, though questionable on theoretical grounds, has profitably been used to estimate several ArO-H BDEs. Experimental ArO-H BDEs are generally in good agreement with the DFT calculations. Significant deviations between experimental and DFT calculated ArO-H BDEs were found, however, when an intramolecular H-bond to the O(*) center was present in the phenoxyl radical, e.g., in ortho semiquinone radicals. In these cases, the coupled cluster with single and double excitations correlated wave function technique with complete basis set extrapolation gave excellent results. The TSs for the reactions of dpph(*) with phenol, 3- and 4-methoxyphenol, and 1,4-cyclohexadiene were also computed. Surprisingly, these TS structures for the phenols show that the reactions cannot be described as occurring exclusively by either a HAT or a PCET mechanism, while with 1,4-cyclohexadiene the PCET character in the reaction coordinate is much better defined and shows a strong pi-pi stacking interaction between the incipient cyclohexadienyl radical and a phenyl ring of the dpph(*) radical.     

Effect of Ca2+/Sr2+ substitution on the electronic structure of the oxygen-evolving complex of photosystem II: a combined multifrequency EPR, 55Mn-ENDOR, and DFT study of the S2 state

The electronic structures of the native Mn(4)O(x)Ca cluster and the biosynthetically substituted Mn(4)O(x)Sr cluster of the oxygen evolving complex (OEC) of photosystem II (PSII) core complexes isolated from Thermosynechococcus elongatus, poised in the S(2) state, were studied by X- and Q-band CW-EPR and by pulsed Q-band (55)Mn-ENDOR spectroscopy. Both wild type and tyrosine D less mutants grown photoautotrophically in either CaCl(2) or SrCl(2) containing media were measured. The obtained CW-EPR spectra of the S(2) state displayed the characteristic, clearly noticeable differences in the hyperfine pattern of the multiline EPR signal [Boussac et al. J. Biol. Chem.2004, 279, 22809-22819]. In sharp contrast, the manganese ((55)Mn) ENDOR spectra of the Ca and Sr forms of the OEC were remarkably similar. Multifrequency simulations of the X- and Q-band CW-EPR and (55)Mn-pulsed ENDOR spectra using the Spin Hamiltonian formalism were performed to investigate this surprising result. It is shown that (i) all four manganese ions contribute to the (55)Mn-ENDOR spectra; (ii) only small changes are seen in the fitted isotropic hyperfine values for the Ca(2+) and Sr(2+) containing OEC, suggesting that there is no change in the overall spin distribution (electronic coupling scheme) upon Ca(2+)/Sr(2+) substitution; (iii) the changes in the CW-EPR hyperfine pattern can be explained by a small decrease in the anisotropy of at least two hyperfine tensors. It is proposed that modifications at the Ca(2+) site may modulate the fine structure tensor of the Mn(III) ion. DFT calculations support the above conclusions. Our data analysis also provides strong support for the notion that in the S(2) state the coordination of the Mn(III) ion is square-pyramidal (5-coordinate) or octahedral (6-coordinate) with tetragonal elongation. In addition, it is shown that only one of the currently published OEC models, the Siegbahn structure [Siegbahn, P. E. M. Acc. Chem. Res.2009, 42, 1871-1880, Pantazis, D. A. et al. Phys. Chem. Chem. Phys.2009, 11, 6788-6798], is consistent with all data presented here. These results provide important information for the structure of the OEC and the water-splitting mechanism. In particular, the 5-coordinate Mn(III) is a potential site for substrate 'water' (H(2)O, OH(-)) binding. Its location within the cuboidal structural unit, as opposed to the external 'dangler' position, may have important consequences for the mechanism of O-O bond formation.     

Reversibly reducible cis-dichloroplatinum(II) and cis-dichloropalladium(II) complexes of bis(1-methylimidazol-2-yl)glyoxal

Complexes cis-MCl2(big), big=bis(1-methylimidazol-2-yl)glyoxal, M=Pt, Pd, were prepared and characterized through electrochemistry, spectroscopy, and for M=Pt, by X-ray structure analysis. The seven-membered chelate ring formed through N,N' coordination of the ligand big shows a boat conformation in agreement with density functional theory (DFT) calculation results. No significant intermolecular interactions were observed for the platinum compound. Both the PdII and PtII complexes undergo reversible one-electron reduction in CH2Cl2/ 0.1 M Bu4NPF6; the reduced palladium compound disintegrates above -30 degrees C. Electron paramagnetic resonance (EPR), UV-vis, and IR spectroelectrochemistry studies were employed to study the monoanions. The anion radical complex [cis-PtCl2(big)]*- exhibits a well-resolved EPR spectrum with small but well-detectable g anisotropy and an isotropic 195Pt hyperfine coupling of 12.2 G. DFT calculations confirm the spin concentration in the alpha-semidione part of the radical complex with small delocalization to the bis(imidazolyl)metal section. The results show that EPR and electroactive moieties can be linked to the cis-dichloroplatinum(II) group via imidazole coordination.     

Contributions of the 8-methyl group to the vibrational normal modes of flavin mononucleotide and its 5-methyl semiquinone radical

Resonance Raman spectroscopy is a powerful tool to investigate flavins and flavoproteins, and a good understanding of the flavin vibrational normal modes is essential for the interpretation of the Raman spectra. Isotopic labeling is the most effective tool for the assignment of vibrational normal modes, but such studies have been limited to labeling of rings II and III of the flavin isoalloxazine ring. In this paper, we report the resonance and pre-resonance Raman spectra of flavin mononucleotide (FMN) and its N5-methyl neutral radical semiquinone (5-CH 3FMN(*)), of which the 8-methyl group of ring I has been deuterated. The experiments indicate that the Raman bands in the low-frequency region are the most sensitive to 8-methyl deuteration. Density functional theory (DFT) calculations have been performed on lumiflavin to predict the isotope shifts, which are used to assign the calculated normal modes to the Raman bands of FMN. A first assignment of the low-frequency Raman bands on the basis of isotope shifts is proposed. Partial deuteration of the 8-methyl group reveals that the changes in the Raman spectra do not always occur gradually. These observations are reproduced by the DFT calculations, which provide detailed insight into the underlying modifications of the normal modes that are responsible for the changes in the Raman spectra. Two types of isotopic shift patterns are observed: either the frequency of the normal mode but not its composition changes or the composition of the normal mode changes, which then appears at a new frequency. The DFT calculations also reveal that the effect of H/D-exchange in the 8-methyl group on the composition of the vibrational normal modes is affected by the position of the exchanged hydrogen, i.e., whether it is in or out of the isoalloxazine plane.     

1H, 13C and 15N NMR assignments of phenazopyridine derivatives

Phenazopyridine hydrochloride (1), a drug in clinical use for many decades, and some derivatives were studied by one- and two-dimensional (1)H, (13)C and (15)N NMR methodology. The assignments, combined with DFT calculations, reveal that the preferred protonation site of the drug is the pyridine ring nitrogen atom. The chemoselective acetylation of phenazopyridine (2) and its influence on the polarization of the azo nitrogen atoms were evidenced by the (15)N NMR spectra. Molecular calculations of the phenazopyridines 2-4 show that the pyridine and phenyl groups are oriented in an antiperiplanar conformation with intramolecular hydrogen bonding between the N-b atom and the C-2 amino group preserving the E-azo stereochemistry.     

A tetracoordinated rhodium aminyl radical complex

A [16 + 1] valence electron configured rhodium aminyl radical complex could be synthesized and characterized in detail by pulse EPR spectroscopy and DFT calculations. The unpaired electron is delocalized over the metal center and two adjacent nitrogens. H-abstraction reactions from thiols and triethylsilane show that the spin density is predominantly localized on both nitrogens.     

14N Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy of the Cation Radical P840+, the Primary Electron Donor of the Chlorobium limicola Reaction Center

The electronic structure of the oxidized primary chlorophyll electron donor, P840^+., of the green sulfur bacterium Chlorobium limicola has been investigated using electron spin echo envelope modulation (ESEEM) spectroscopy. This ESEEM investigation of the electron spin density distribution in the radical cation P840^+. in membranes isolated from C. limicola confirms that the electron spin is shared eqully between the two bacteriochlorophyll a molecules. Observation of the small hyperfine couplings to the ring nitrogens by ESEEM gives results that are in agreement with those obtained from ENDOR measurements (S. E. J. Rigby, R. Thapar, M. C. W. Evans and P. Heathcote, FEBS Lett. 350,24–28, 1994) of the large hyperfine couplings to the methyl group protons. These results in combination with the Raman spectroscopy of P840 (U. Feiler, D. Albouy, B. Robert and T. A. Mattioli, Biochemistry 34,11099–11105, 1995) all indicate that the reaction center of green sulfur photosynthetic bacteria is functionally a protein homodimer providing a symmetrical protein environment for the primary electron donor.