ENDOR Spectra Research Articles

Density Functional Calculations of 55Mn, 14N and 13C Electron Paramagnetic Resonance Parameters Support an Energetically Feasible Model System for the S2 State of the Oxygen‐Evolving Complex of Photosystem II

Metal and ligand hyperfine couplings of a previously suggested, energetically feasible Mn(4)Ca model cluster (SG2009(-1)) for the S(2) state of the oxygen-evolving complex (OEC) of photosystem II (PSII) have been studied by broken-symmetry density functional methods and compared with other suggested structural and spectroscopic models. This was carried out explicitly for different spin-coupling patterns of the S=1/2 ground state of the Mn(III)(Mn(IV))(3) cluster. By applying spin-projection techniques and a scaling of the manganese hyperfine couplings, computation of the hyperfine and nuclear quadrupole coupling parameters allows a direct evaluation of the proposed models in comparison with data obtained from the simulation of EPR, ENDOR, and ESEEM spectra. The computation of (55)Mn hyperfine couplings (HFCs) for SG2009(-1) gives excellent agreement with experiment. However, at the current level of spin projection, the (55)Mn HFCs do not appear sufficiently accurate to distinguish between different structural models. Yet, of all the models studied, SG2009(-1) is the only one with the Mn(III) site at the Mn(C) center, which is coordinated by histidine (D1-His332). The computed histidine (14)N HFC anisotropy for SG2009(-1) gives much better agreement with ESEEM data than the other models, in which Mn(C) is an Mn(IV) site, thus supporting the validity of the model. The (13)C HFCs of various carboxylates have been compared with (13)C ENDOR data for PSII preparations with (13)C-labelled alanine.

Organometallic mechanism of action and inhibition of the 4Fe-4S isoprenoid biosynthesis protein GcpE (IspG)

We report the results of a series of chemical, EPR, ENDOR, and HYSCORE spectroscopic investigations of the mechanism of action (and inhibition) of GcpE, E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate (HMBPP) synthase, also known as IspG, an Fe(4)S(4) cluster-containing protein. We find that the epoxide of HMBPP when reduced by GcpE generates the same transient EPR species as observed on addition of the substrate, 2-C-methyl-D-erythritol-2, 4-cyclo-diphosphate. ENDOR and HYSCORE spectra of these transient species (using (2)H, (13)C and (17)O labeled samples) indicate formation of an Fe-C-H containing organometallic intermediate, most likely a ferraoxetane. This is then rapidly reduced to a ferracyclopropane in which the HMBPP product forms an eta(2)-alkenyl pi- (or pi/sigma) complex with the 4th Fe in the Fe(4)S(4) cluster, and a similar "metallacycle" also forms between isopentenyl diphosphate (IPP) and GcpE. Based on this metallacycle concept, we show that an alkyne (propargyl) diphosphate is a good (K(i) approximately 300 nM) GcpE inhibitor, and supported again by EPR and ENDOR results (a (13)C hyperfine coupling of approximately 7 MHz), as well as literature precedent, we propose that the alkyne forms another pi/sigma metallacycle, an eta(2)-alkynyl, or ferracyclopropene. Overall, the results are of broad general interest because they provide new mechanistic insights into GcpE catalysis and inhibition, with organometallic bond formation playing, in both cases, a key role.

Characterization of the Semiquinone Radical Stabilized by the Cytochrome aa3-600 Menaquinol Oxidase of Bacillus subtilis

Cytochrome aa(3)-600 is one of the principle respiratory oxidases from Bacillus subtilis and is a member of the heme-copper superfamily of oxygen reductases. This enzyme catalyzes the two-electron oxidation of menaquinol and the four-electron reduction of O(2) to 2H(2)O. Cytochrome aa(3)-600 is of interest because it is a very close homologue of the cytochrome bo(3) ubiquinol oxidase from Escherichia coli, except that it uses menaquinol instead of ubiquinol as a substrate. One question of interest is how the proteins differ in response to the differences in structure and electrochemical properties between ubiquinol and menaquinol. Cytochrome bo(3) has a high affinity binding site for ubiquinol that stabilizes a ubi-semiquinone. This has permitted the use of pulsed EPR techniques to investigate the protein interaction with the ubiquinone. The current work initiates studies to characterize the equivalent site in cytochrome aa(3)-600. Cytochrome aa(3)-600 has been cloned and expressed in a His-tagged form in B. subtilis. After isolation of the enzyme in dodecylmaltoside, it is shown that the pure enzyme contains 1 eq of menaquinone-7 and that the enzyme stabilizes a mena-semiquinone. Pulsed EPR studies have shown that there are both similarities as well as significant differences in the interactions of the mena-semiquinone with cytochrome aa(3)-600 in comparison with the ubi-semiquinone in cytochrome bo(3). Our data indicate weaker hydrogen bonds of the menaquinone in cytochrome aa(3)-600 in comparison with ubiquinone in cytochrome bo(3). In addition, the electronic structure of the semiquinone cyt aa(3)-600 is more shifted toward the anionic form from the neutral state in cyt bo(3).

Probing the ternary complexes of indoleamine and tryptophan 2,3-dioxygenases by cryoreduction EPR and ENDOR spectroscopy.

We have applied cryoreduction/EPR/ENDOR techniques to characterize the active-site structure of the ferrous-oxy complexes of human (hIDO) and Shewanella oneidensis (sIDO) indoleamine 2,3-dioxygenases, Xanthomonas campestris (XcTDO) tryptophan 2,3-dioxygenase, and the H55S variant of XcTDO in the absence and in the presence of the substrate l-Trp and a substrate analogue, l-Me-Trp. The results reveal the presence of multiple conformations of the binary ferrous-oxy species of the IDOs. In more populated conformers, most likely a water molecule is within hydrogen-bonding distance of the bound ligand, which favors protonation of a cryogenerated ferric peroxy species at 77 K. In contrast to the binary complexes, cryoreduction of all of the studied ternary [enzyme-O2-Trp] dioxygenase complexes generates a ferric peroxy heme species with very similar EPR and 1H ENDOR spectra in which protonation of the basic peroxy ligand does not occur at 77 K. Parallel studies with l-Me-Trp, in which the proton of the indole nitrogen is replaced with a methyl group, eliminate the possibility that the indole NH group of the substrate acts as a hydrogen bond donor to the bound O2, and we suggest instead that the ammonium group of the substrate hydrogen-bonds to the dioxygen ligand. The present data show that substrate binding, primarily through this H-bond, causes the bound dioxygen to adopt a new conformation, which presumably is oriented for insertion of O2 into the C2−C3 double bond of the substrate. This substrate interaction further helps control the reactivity of the heme-bound dioxygen by “shielding” it from water.

Experimental observation of spin delocalisation onto the aryl-alkynyl ligand in the complexes [Mo(CCAr)(Ph2PCH2CH2PPh2)(η-C7H7)]+ (Ar = C6H5, C6H4-4-F; C7H7 = cycloheptatrienyl): an EPR and ENDOR investigation

The paramagnetic aryl-alkynyl complexes [Mo(C≡CAr)(dppe)(η-C(7)H(7))](+) (dppe = Ph(2)PCH(2)CH(2)PPh(2); Ar = C(6)H(5), [1](+); C(6)D(5), [2](+); C(6)H(4)-4-F, [3](+); C(6)H(4)-4-Me, [5](+)) and [Mo(C≡CBu(t))(dppe)(η-C(7)H(7))](+) [4](+), have been investigated in a combined EPR and ENDOR study. Direct experimental evidence for the delocalisation of unpaired spin density over the framework of an aryl-alkynyl ligand has been obtained. The X-band solution EPR spectrum of the 4-fluoro derivative, [3](+), exhibits resolved hyperfine coupling to the remote para position of the aryl group [a(iso)((19)F) = 4.5 MHz, (1.6 G)] in addition to couplings attributable to (95/97)Mo, (31)P and (1)H of the C(7)H(7) ring. A full analysis of the (1)H ENDOR spectra is restricted by the low g anisotropy of the system which prevents the use of orientation selection. However, inter-comparison of the (1)H cw-ENDOR frozen solution spectra of [1](+), [2](+), [4](+) and [5](+), combined with spectral simulation informed by calculated values derived from DFT investigations, has facilitated estimation of the experimental a(iso)((1)H) hyperfine couplings of [1](+) including the ortho, ±3.7 MHz (±1.3 G) and para, ±3.9 MHz (±1.4 G) positions of the C(6)H(5) substituent of the aryl-alkynyl ligand.

Endor Spectrum of the Protonated Rhodosemiquinone in Bacterial Reaction Centers

A crucial intermediate state in the reduction of QB to QBH2 involves the high energy intermediate QBH. state.(1) Direct observation of this state was achieved upon replacing the native ubiquinone with the low potential analog rhodoquinone (RQ) (2). Here we report the ENDOR spectrum of rhodosemiquinone at low and high pH measured in a mutant RC designed to reduce QB via B-branch electron transfer without interference from QA-. (3, 4). The peaks at 50.2 MHz and 51.7 MHz (dashed lines) are shifted from the proton Larmor frequency (∼53.2 MHz) and are assigned to hydrogen-bonded protons due to their line shape and position and their absence upon exchange in D2O (4). Upon shift to lower pH, these peaks diminish in amplitude and a new proton peak arises near 49.4 MHz (solid line). We attribute these changes to the protonation of rhodosemiquinone at lower pH. (Supported by NIH)(1) Graige et al (1996) J. Am. Chem Soc. 118, 9005-9016(2) Graige et al (1999) Biochemistry38, 11465-11473.(3) Paddock et al (2005) Biochemistry 44, 6920-6928.(4) Paddock et al (2009) Appl. Magn. Reson., in press.View Large Image | View Hi-Res Image | Download PowerPoint Slide

EPR and ENDOR spectroscopic study of the reactions of aromatic azides with gallium trichloride

The reactions of gallium trichloride with phenyl and deuterio-phenyl azides, as well as with 4-methoxyphenyl azide and deuterium isotopomers, were examined by product analysis, CW EPR spectroscopy and pulsed ENDOR spectroscopy. The products included the corresponding anilines together with 4-aminodiphenylamine type dimers, and polyanilines. Complex CW EPR spectra of the radical cations of the dimers [ArNHC(6)H(4)NH(2)](+)˙ and trimers [ArNHC(6)H(4)NHC(6)H(4)NH(2)](+)˙ were obtained. These EPR spectra were analysed with the help of data from the deuterium-substituted analogues as well as the pulse Davies ENDOR spectra. DFT computations of the radical cations provided corroborating evidence and suggested the unpaired electrons were accommodated in extensive π-delocalised orbitals. A mechanism to account for the reductive conversion of aromatic azides to the corresponding anilines and thence to the dimers and trimers is proposed.

Simulating Suppression Effects in Pulsed ENDOR, and the ‘Hole in the Middle’ of Mims and Davies ENDOR Spectra

All pulsed ENDOR techniques, and in particular the Mims and Davies sequences, suffer from detectability biases ('blindspots') that are directly correlated to the size of the hyperfine interactions of coupled nuclei. Our efforts at ENDOR 'crystallography' and 'mechanism determination' with these techniques has led our group to refine our simulations of pulsed ENDOR spectra to take into account these biases, and we here describe the process and illustrate it with several examples. We first focus on an issue whose major significance is not widely appreciated, the 'hole in the middle' of pulsed ENDOR spectra caused by the n = 0 suppression hole in Mims ENDOR and by the analogous A→0 suppression in Davies ENDOR (Section I). This section discusses the issue for nuclei with I = ½ and also for (2)H (I = 1), using the treatment of Section II. In Section II we discuss the general treatment of suppression effects for I = 1, illustrating it with a treatment of Mims suppression for (14)N (I = 1) (Section II).

EPR, ESE and pulsed ENDOR study of the nitrogen donor pairs on quasi-cubic lattice sites in 6H SiC

Abstract D-band EPR, X-band FS ESE and pulsed ENDOR studies of the additional nitrogen (N) related centers observed in highly compensated n-type 6H-SiC wafers are presented. The D-band EPR and X-band FS ESE spectrum consists of the 14N hyperfine (hf) triplet lines of the N donors incorporated at the two quasi-cubic (Nc1, Nc2) sites with the intensity ratio of INc1/INc2=0.7 and three additional triplet lines. X-band pulsed ENDOR spectra have shown intense 14N ENDOR signals of Nc1, Nc2 centers and new 14N lines due to the N related centers N1, N2, N3 with the isotropic hf splitting: 21.04, 26.43, 29.77 MHz, respectively. It was found that the g-tensors of the N2 and N3 triplet lines coincide with those of N on quasi-cubic Nc2 site. The g-tensor of the third N1 triplet lines corresponds to the average value of the Nc1 and Nc2 spectrum: g ( N 1 ) ≈ 1 2 [ g ( N c 1 ) + g ( N c 2 ) ] . It was suggested that N1 center with Aiso=21.04 MHz is due to the spin coupling between N on two quasi-cubic Nc1 and Nc2 sites while two others are tentatively attributed to the N pairs formed between N atoms on one quasi-cubic Nc2 site.

FID detection of EPR and ENDOR spectra at high microwave frequencies

High-frequency pulsed EPR spectroscopy allows FID detection of EPR spectra owing to the short dead time that can be achieved. This FID detection is particularly attractive for EPR and ENDOR spectroscopy of paramagnetic species that exhibit inhomogeneously broadened EPR lines and short dephasing times. Experiments are reported for the metalloprotein azurin at 275 GHz.

Spin Density Distribution of the Excited Triplet State of Bacteriochlorophylls. Pulsed ENDOR and DFT Studies

The photoexcited triplet states of bacteriochlorophyll a and bacteriochlorophyll b have been investigated by ENDOR spectroscopy at 34 GHz in frozen solution and by DFT calculations. The spin density distributions in the triplet state were found to be similar for bacteriochlorophyll a and bacteriochlorophyll b except for the presence of spin density on carbon 8(1) in bacteriochlorophyll b. Judging from a comparison with ENDOR experiments for the radical cation and anion, the triplet state in bacteriochlorophylls cannot be explained as being a simple HOMO-->LUMO excitation of Gouterman orbitals. Rather, it must be described as a mixture of HOMO-->LUMO and HOMO-1-->LUMO excitations for a satisfactory explanation of the observed hyperfine interactions. The observed hyperfine couplings in the ENDOR spectra were assigned, and the field dependence of the signals was found to be fully compatible with the orientation of the zero-field splitting tensor determined from magnetophotoselection studies.

ENDOR Spectroscopy Shows that QA Remains in the Same Orientation Upon Reduction in Reaction Centers from Rhodobacter Sphaeroides

It has been, recently, proposed that QA moves and rotates ∼60° upon its reduction.1 Here we investigated possible changes using ENDOR spectroscopy that provides a very sensitive (to ∼0.01 A) probe for the binding site of QA•-.2 Identical RC samples were made - (a) one frozen in the dark (ground state) and then illuminated generating D•+QA•- and (b) one frozen under illumination (excited state) which trapped D•+QA•- in ∼70% of the RCs at 80 K. Figure 1 shows the resultant 1H ENDOR spectra of QA•-. The peaks labeled L1, L2 and L3 correspond to the two H-bonds to QA•-.2,3 Essentially no differences in the ENDOR spectra were observed indicating that the interactions of QA•- with the protein are the same in the ground state as in the excited state. These results are irreconcilable with the proposed rotation.1 Thus, QA is preset in an environment that favors its reduction.1 Heinent et al. (2007), J. Am. Chem. Soc. 129, 15935. 2 Flores et al. (2007), Biophys. J. 92, 671. 3 Sinnecker et al. (2006), Phys. Chem. Chem. Phys. 8, 5659.View Large Image | View Hi-Res Image | Download PowerPoint Slide

The reaction mechanism of the molybdenum hydroxylase xanthine oxidoreductase: evidence against the formation of intermediates having metal-carbon bonds.

ENDOR spectra of the catalytically relevant "very rapid" Mo(V) species generated in the course of the reaction of xanthine oxidoreductase with substrate have been examined by two different groups. While the data themselves are virtually identical, the analysis has been variously interpreted as supporting or refuting the existence of a molybdenum-carbon bond in the signal-giving species. While the basis for this difference in interpretation has now been generally agreed upon - the Mo-C distance in the signal-giving species is now understood to be too long to represent a direct Mo-C bond - independent information concerning the structure of the signal-giving species is highly desirable. Recently, several X-ray crystal structures of catalytically relevant complexes of the enzyme with several substrates and inhibitors have been reported. Taken together, these structures strongly and unambiguously support the interpretation that the intermediate giving rise to the "very rapid" EPR signal, as well as the Mo(IV) intermediate that precedes it in the reaction mechanism, has product coordinated to the active site molybdenum via the catalytically introduced hydroxyl group in a simple "end-on" fashion, with no metal-carbon bond character to the complex. The manner in which product is bound and its orientation within the active site provide important clues as to the specific catalytic roles of active sites in accelerating the reaction rate.

55Mn-ENDOR of the S2-state multiline signal of Photosystem II from Thermosynechococcus elongatus

Pulse EPR and 55Mn-ENDOR (electron nuclear double resonance) experiments at 34 GHz (Q-band) were performed on the S2 state of the OEC (oxygen-evolving complex) in PSII (Photosystem II) from Thermosynechococcus elongatus. The ENDOR spectra are discussed with respect to earlier data obtained from X- (9 GHz) and Q-band experiments on PSII preparations from spinach with and without the addition of methanol.

EPR and ENDOR Studies of Cryoreduced Compounds II of Peroxidases and Myoglobin. Proton-Coupled Electron Transfer and Protonation Status of Ferryl Hemes

The nature of the [Fe(IV)-O] center in hemoprotein Compounds II has recently received considerable attention, as several experimental and theoretical investigations have suggested that this group is not necessarily the traditionally assumed ferryl ion, [Fe(IV)=O]2+, but can be the protonated ferryl, [Fe(IV)-OH]3+. We show here that cryoreduction of the EPR-silent Compound II by gamma-irradiation at 77 K produces Fe(III) species retaining the structure of the precursor [Fe(IV)=O]2+ or [Fe(IV)-OH]3+, and that the properties of the cryogenerated species provide a report on structural features and the protonation state of the parent Compound II when studied by EPR and 1H and 14N ENDOR spectroscopies. To give the broadest view of the properties of Compounds II we have carried out such measurements on cryoreduced Compounds II of HRP, Mb, DHP and CPO and on CCP Compound ES. EPR and ENDOR spectra of cryoreduced HRP II, CPO II and CCP ES are characteristic of low-spin hydroxy-Fe(III) heme species. In contrast, cryoreduced "globins", Mb II, Hb II, and DHP II, show EPR spectra having lower rhombicity. In addition the cryogenerated ferric "globin" species display strongly coupled exchangeable (1)H ENDOR signals, with A max approximately 20 MHz and a iso approximately 14 MHz, both substantially greater than for hydroxide/water ligand protons. Upon annealing at T > 180 K the cryoreduced globin compounds II relax to the low-spin hydroxy-ferric form with a solvent kinetic isotope effect, KIE > 6. The results presented here together with published resonance Raman and Mossbauer data suggest that the high-valent iron center of globin and HRP compounds II, as well as of CCP ES, is [Fe(IV)=O]2+, and that its cryoreduction produces [Fe(III)-O]+. Instead, as proposed by Green and co-workers, CPO II contains [Fe(IV)-OH]3+ which forms [Fe(III)-OH]2+ upon radiolysis. The [Fe(III)-O]+ generated by cryoreduction of HRP II and CCP ES protonate at 77 K, presumably because the heme is linked to a distal-pocket hydrogen bonding/proton-delivery network through an H-bond to the "oxide" ligand. The data also indicate that Mb and HRP compounds II exist as two major conformational substates.

Identification, Characterization, and Structure/Function Analysis of a Corrin Reductase Involved in Adenosylcobalamin Biosynthesis

Vitamin B(12), the antipernicious anemia factor, is the cyano derivative of adenosylcobalamin, which is one of nature's most complex coenzymes. Adenosylcobalamin is made along one of two similar yet distinct metabolic pathways, which are referred to as the aerobic and anaerobic routes. The aerobic pathway for cobalamin biosynthesis proceeds via cobalt insertion into a ring-contracted macrocycle, which is closely followed by adenosylation of the cobalt ion. An important prerequisite for adenosylation is the reduction of the centrally chelated metal from Co(II) to a highly nucleophilic Co(I) form. We have cloned a gene, cobR, encoding a biosynthetic enzyme with this co(II)rrin reductase activity from Brucella melitensis. The protein has been overproduced, and the resulting flavoprotein has been purified, characterized, and crystallized and its structure determined to 1.6A resolution. Kinetic and EPR analysis reveals that the enzyme proceeds via a semiquinone form. It is proposed that CobR may interact with the adenosyltransferase to overcome the large thermodynamic barrier required for co(II)rrin reduction.

Electronic structure of [Ga 2(tren) 2(CA sq,cat)](BPh 4) 2(BF 4): An EPR, ENDOR, and density functional study

The bimetallic [M 1M 2(tren) 2(CA n− )] m+ series, where M = Ga III or Cr III and CA is the chloranilate ligand which can take on diamagnetic (CA cat,cat) 4− or paramagnetic (CA sq,cat) 3− forms, comprises an electronically diverse series of compounds ranging from the closed-shell [Ga 2(tren) 2(CA cat,cat)] 2+ to the S = 5/2 ground state of [Cr 2(tren) 2(CA sq,cat)] 3+. This report deals with the interpretation of the EPR and ENDOR spectra of [Ga 2(tren) 2(CA sq,cat)](BPh 4) 2(BF 4) ( 2) and the related derivative [Ga 2(tren) 2(DHBQ)](BPh 4) 2(BF 4) ( 2a) (where DHBQ is the fully deprotonated trianionic form of 2,5-dihydroxy-1,4-benzoquinone) in an effort to further characterize the electronic structure of this radical species. The X-band (∼9.5 GHz) EPR spectrum of complex 2 acquired in a butyronitrile/propionitrile glass at 4 K reveals a rhombic g-tensor with g xx = 2.0100, g yy = 2.0097, and g zz = 2.0060 with hyperfine interactions due to spin delocalization onto the two Ga nuclei ( a xx = 4.902 G, a yy = 4.124 G, a zz = 3.167 G); the origin of the hyperfine coupling was confirmed by analysis of the room temperature spectra of complexes 2 and 2a. The low-temperature spectrum of complex 2 also indicates the presence of a triplet electronic state characterized by a g-value of 2.009 and axial zero-field splitting of D = 150 G (0.012 cm −1) as determined from measurements carried out at both X- and W-band (∼95 GHz) frequencies. This triplet state is believed to arise due to a weak intermolecular Heisenberg exchange interaction between two aggregating complexes. ENDOR measurements on complex 2a at 20 K allowed for a determination of the magnitude of hyperfine coupling to the protons associated with the radical bridge as well as providing a rare example of an ENDOR signal arising from coupling to a gallium nucleus. Finally, these results were combined with literature data on the free semiquinone form of the bridging ligand in order to assess the extent to which density functional theory can predict unpaired spin density distribution in a complex molecule of this type. Although differences between theory and experiment were noted, DFT was able to provide a reasonably accurate picture of the electronic structure of this system as well as provide insight into the spin polarization mechanism(s) responsible for the observed hyperfine interactions.

Pulse ENDOR and density functional theory on the peridinin triplet state involved in the photo-protective mechanism in the peridinin–chlorophyll a–protein from Amphidinium carterae

The photoexcited triplet state of the carotenoid peridinin in the Peridinin–chlorophyll a–protein of the dinoflagellate Amphidinium carterae has been investigated by pulse EPR and pulse ENDOR spectroscopies at variable temperatures. This is the first time that the ENDOR spectra of a carotenoid triplet in a naturally occurring light-harvesting complex, populated by energy transfer from the chlorophyll a triplet state, have been reported. From the electron spin echo experiments we have obtained the information on the electron spin polarization dynamics and from Mims ENDOR experiments we have derived the triplet state hyperfine couplings of the α- and β-protons of the peridinin conjugated chain. Assignments of β-protons belonging to two different methyl groups, with a iso = 7.0 MHz and a iso = 10.6 MHz respectively, have been made by comparison with the values predicted from density functional theory. Calculations provide a complete picture of the triplet spin density on the peridinin molecule, showing that the triplet spins are delocalized over the whole π-conjugated system with an alternate pattern, which is lost in the central region of the polyene chain. The ENDOR investigation strongly supports the hypothesis of localization of the triplet state on one peridinin in each subcluster of the PCP complex, as proposed in [Di Valentin et al. Biochim. Biophys. Acta 1777 (2008) 186–195]. High spin density has been found specifically at the carbon atom at position 12 (see Fig. 1B), which for the peridinin involved in the photo-protective mechanism is in close contact with the water ligand to the chlorophyll a pigment. We suggest that this ligated water molecule, placed at the interface between the chlorophyll–peridinin pair, is functioning as a bridge in the triplet–triplet energy transfer between the two pigments.

W-band31P-ENDOR on the high-affinity Mn2+ binding site in the minimal and tertiary stabilized hammerhead ribozymes

The catalytic activity of the tertiary stabilized hammerhead ribozyme (tsHHRz) is by three orders of magnitude higher than the one of the long-known minimal construct (mHHRz). This gives rise to the question whether the single high-affinity manganese(II) binding site present in both ribozymes is located closer to the cleavage site and the transition state in the tsHHRz than in the mHHRz, which would make a direct involvement of this metal(II) ion in the bond-breaking step more likely. Here, we used W-band31P-Davies-ENDOR (electron-nuclear double resonance) to complement earlier reported14N-ESEEM/HYSCORE (electron spin echo envelope modulation/hyperfine sublevel correlation) studies. The31P-ENDOR spectrum of the mHHRz revealed a doublet with a splitting of 8.4(±0.5) MHz but unresolved hyperfine anisotropy. Such a large splitting indicates an inner-sphere coordination of a phosphate backbone group with a significant amount of spin density on the phosphorous nucleus. This is in good agreement with the31P isotropic hyperfine constant,Aiso(31P), of +7.8 MHz obtained by density functional theory calculations on the structure of the Mn2+ binding site as found in crystals of the same ribozyme. This supports the idea that the structure and location of the binding site in the mHHRz is in frozen buffer similar to that found in the crystal. Since the W-band ENDOR spectrum of the tsHHRz also shows a31P splitting of 8.4(±0.5) MHz, the local structures of both binding sites appear to be similar, which agrees with the coincidence of the14N data. An involvement of the high-affinity Mn2+ ion in the catalytic step seems therefore unlikely.

ENDOR Spectroscopy Reveals Light Induced Movement of the H-Bond from Ser-L223 upon Forming the Semiquinone (QB-•) in Reaction Centers fromRhodobacter sphaeroides

Proton ENDOR spectroscopy was used to monitor local conformational changes in bacterial reaction centers (RC) associated with the electron-transfer reaction DQB --> D+*QB-* using mutant RCs capable of photoreducing QB at cryogenic temperatures. The charge separated state D+*QB-* was studied in mutant RCs formed by either (i) illuminating at low temperature (77 K) a sample frozen in the dark (ground state protein conformation) or (ii) illuminating at room temperature prior to and during freezing (charge separated state protein conformation). The charge recombination rates from the two states differed greatly (>10(6) fold) as shown previously, indicating a structural change (Paddock et al. (2006) Biochemistry 45, 14032-14042). ENDOR spectra of QB-* from both samples (35 GHz, 77 K) showed several H-bond hyperfine couplings that were similar to those for QB-* in native RCs indicating that in all RCs, QB-* was located at the proximal position near the metal site. In contrast, one set of hyperfine couplings were not observed in the dark frozen samples but were observed only in samples frozen under illumination in which the protein can relax prior to freezing. This flexible H-bond was assigned to an interaction between the Ser-L223 hydroxyl and QB-* on the basis of its absence in Ser L223 --> Ala mutant RCs. Thus, part of the protein relaxation, in response to light induced charge separation, involves the formation of an H-bond between the OH group of Ser-L223 and the anionic semiquinone QB-*. These results show the flexibility of the Ser-L223 H-bond, which is essential for its function in proton transfer to reduced QB.