Comparative ENDOR study at 34 GHz of the triplet state of the primary donor in bacterial reaction centers of Rb. sphaeroides and Bl. viridis

The primary electron donor (P) in the photosynthetic bacterial reaction center of Rhodobacter sphaeroides and Blastochloris viridis consists of a dimer of bacteriochlorophyll a and b cofactors, respectively. Its photoexcited triplet state in frozen solution has been investigated by time resolved ENDOR spectroscopy at 34 GHz. The observed ENDOR spectra for ³P₈₆₅ and ³P₉₆₀ are essentially the same, indicating very similar spin density distributions. Exceptions are the ethylidene groups unique to the bacteriochlorophyll b dimer in ³P₉₆₀. Strikingly, the observed hyperfine coupling constants of the ethylidene groups are larger than in the monomer, which speaks for an asymmetrically delocalized wave function over both monomer halves in the dimer. The latter observation corroborates previous findings of the spin density in the radical cation states P ₈₆₅ ^?+ (Lendzian et al. in Biochim Biophys Acta 1183:139–160, 1993) and P ₉₆₀ ^?+ (Lendzian et al. in Chem Phys Lett 148:377–385, 1988). As compared to the bacteriochlorophyll monomer, the hyperfine coupling constants of the methyl groups 2¹ and 12¹ are reduced by at least a factor of two, and quantitative analysis of these couplings gives rise to a ratio of approximately 3:1 for the spin density on the halves P_L:P_M. Our findings are discussed in light of the large difference in photosynthetic activity of the two branches of cofactors present in the bacterial reaction center proteins.     

Identification of the Intermediate Charge-Separated State PβL in a Leucine M214 to Histidine Mutant of the Rhodobacter sphaeroides Reaction Center Using Femtosecond Midinfrared Spectroscopy

Energy and electron transfer in a Leu M214 to His (LM214H) mutant of the Rhodobacter sphaeroides reaction center (RC) were investigated by applying time-resolved visible pump/midinfrared probe spectroscopy at room temperature. This mutant replacement of the Leu at position M214 resulted in the incorporation of a bacteriochlorophyll (BChl) in place of the native bacteriopheophytin in the L-branch of cofactors (denoted β_L). Purified LM214H RCs were excited at 600 nm (unselective excitation), at 800 nm (direct excitation of the monomeric BChl cofactors B_L and B_M), and at 860 nm (direct excitation of the primary donor (P) BChl pair (P_L/P_M)). Absorption changes associated with carbonyl (C=O) stretch vibrational modes (9-keto, 10a-ester, and 2a-acetyl) of the cofactors and of the protein were recorded in the region between 1600 cm⁻¹ and 1770 cm⁻¹, and the data were subjected to both a sequential analysis and a simultaneous target analysis. After photoexcitation of the LM214H RC, P^∗ decayed on a timescale of ∼6.3 ps to P⁺B_L⁻. The decay of P⁺B_L⁻ occurred with a lifetime of ∼2 ps, ∼3 times slower than that observed in wild-type and R-26 RCs (∼0.7 ps). Further electron transfer to the β_L BChl resulted in formation of the P⁺β_L⁻ state, and its infrared absorbance difference spectrum is reported for the first time, to our knowledge. The fs midinfrared spectra of P⁺B_L⁻ and P⁺β_L⁻ showed clear differences related to the different environments of the two BChls in the mutant RC.     

Charge separation in photosynthesis via a spin exchange coupling mechanism

A new mechanism for the primary photoinduced charge separation in photosynthesis is proposed. It involves as a real intermediate between the excited special pair state P^* and the primary charge separated state P⁺ H_L ^– a trip-trip-singlet B^T B_L ^T, which consists of a triplet on the dimer P and a further triplet on the monomer B_L. Both combine to a singlet. The electron transfer is caused by spin exchange couplings. The transient spectrum of the short lived intermediate, formerly taken as evidence for the charge transfer state P⁺ B_L ^–, is reinterpreted as a transient excitation of this trip-trip singlet. Received: 2 June 1997 / Accepted: 18 July 1997     

Orientation-selected ENDOR of the active center in Chromatium vinosum [NiFe] hydrogenase in the oxidized "ready" state

 Electron nuclear double resonance (ENDOR) was applied to study the active site of the oxidized "ready" state, Ni_r, in the [NiFe] hydrogenase of Chromatium vinosum. The magnetic field dependence of the EPR was used to select specific subsets of molecules contributing to the ENDOR response by stepping through the EPR envelope. Three hyperfine couplings could be clearly followed over the complete field range. Two protons, H1 and H2, display a very similar large isotropic coupling of 12.5 and 12.6 MHz, respectively. Their dipolar coupling is small (2.1 and 1.4 MHz, respectively). A third proton, H3, exhibits a small isotropic coupling of 0.5 MHz and a larger anisotropic contribution of 3.5 MHz. Based on a comparison with structural data obtained from X-ray crystallography of single crystals of hydrogenases from Desulfovibrio gigas and D. vulgaris and the known g-tensor orientation of Ni_r, an assignment of the ¹H hyperfine couplings could be achieved. H1 and H2 were assigned to the β-CH₂ protons of the bridging cysteine Cys533 and H3 could belong to a β-CH₂ proton of Cys68 or to a protonated cysteine (-SH) of Cys68 or Cys530. Received: 26 November 1998 / Accepted: 1 April 1999     

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.     

Measurement of the extent of electron transfer to the bacteriopheophytin in the M-subunit in reaction centers of Rhodopseudomonas viridis

We have measured the extent of flash-induced electron transfer from the bacteriochlorophyll dimer, P, to the bacteriopheophytin in the M-subunit, H_M, in reaction centers of Rhodopseudomonas viridis. This has been done by measuring the transient states produced by excitation of reaction centers trapped in the PH_L ^–H_M state at 90 K. Under these conditions the normal forward electron transfer to the bacteriopheophytin in the L-subunit, H_L, is blocked and the yield of transient P⁺H_M ^– can be estimated with respect to the lifetime of P^*. Under these conditions flash induced absorbance decreases of the bacteriochlorophyll dimer 990 nm band suggest that a transient P⁺ state is formed with a quantum yield of 0.09±0.06 compared to that formed during normal photochemistry. These transient measurements provide an upper limited on the yield of a transient P⁺ H_M ^– state. An estimate of 0.09 as the yield of the P⁺ H_M ^– state is consistent with all current observations. This estimate and the lifetime of P^* suggest that the electron transfer rate from P^* to H_M, k_M, is about 5 × 10⁹ sec^–1 (_M = 200ps). These measurements suggest that the a branching ratio k_L/k_M is on the order of 200. The large value of the branching ratio is remarkable in view of the structural symmetry of the reaction center. This measurement should be useful for electron transfer calculations based upon the reaction center structure.     

EPR and ENDOR studies of the water oxidizing complex of Photosystem II

A comparative study of X-band EPR and ENDOR of the S₂ state of photosystem II membrane fragments and core complexes in the frozen state is presented. The S₂ state was generated either by continuous illumination at T=200 K or by a single turn-over light flash at T=273 K yielding entirely the same S₂ state EPR signals at 10 K. In membrane fragments and core complex preparations both the multiline and the g=4.1 signals were detected with comparable relative intensity. The absence of the 17 and 23 kDa proteins in the core complex preparation has no effect on the appearance of the EPR signals. ¹H-ENDOR experiments performed at two different field positions of the S₂ state multiline signal of core complexes permitted the resolution of four hyperfine (hf) splittings. The hf coupling constants obtained are 4.0, 2.3, 1.1 and 0.6 MHz, in good agreement with results that were previously reported (Tang et al. (1993) J Am Chem Soc 115: 2382–2389). The intensities of all four line pairs belonging to these hf couplings are diminished in D₂O. A novel model is presented and on the basis of the two largest hfc's distances between the manganese ions and the exchangeable protons are deduced. The interpretation of the ENDOR data indicates that these hf couplings might arise from water which is directly ligated to the manganese of the water oxidizing complex in redox state S₂.Abbreviations cw continuous wave - ENDOR electron nuclear double resonance - EPR electron paramagnetic resonance - hf hyperfine - hfc hyperfine coupling - MLS multiline signal - PS II Photosystem II - rf radio frequency - WOC water oxidizing complex     

Generation and quenching of singlet molecular oxygen by aggregated bacteriochlorophyll d in model systems and chlorosomes

Both photogeneration and quenching of singlet oxygen by monomeric and aggregated (dimeric and oligomeric) molecules of bacteriochlorophyll (BChl) d have been studied in solution and in chlorosomes isolated from the green photosynthetic bacterium Chlorobium vibrioforme f. thiosulfatophilum. The yield of singlet-oxygen photogeneration by pigment dimers was about 6 times less than for monomers. Singlet oxygen formation was not observed in oligomer-containing solutions or in chlorosomes. To estimate the efficiency of singlet oxygen quenching an effective rate constant for ¹O₂ quenching by BChl molecules (k_q ^M) was determined using the Stern-Volmer equation and the total concentration of BChl d in the samples. In solutions containing only monomeric BChl, the k_q ^M values coincide with the real values for ¹O₂ quenching rate constants by BChl molecules. Aggregation weakly influenced the k_q ^M values in pigment solutions. In chlorosomes (which contain both BChl and carotenoids) the k_q ^M value was less than in solutions of BChl alone and much less than in acetone extracts from chlorosomes. Thus ¹O₂ quenching by BChl and carotenoids is much less efficient in chlorosomes than in solution and is likely caused primarily by BChl molecules which are close to the surface of the large chlorosome particles. The data allow a general conclusion that monomeric and dimeric chlorophyll molecules are the most likely sources of ¹O₂ formation in photosynthetic systems and excitation energy trapping by the long wavelength aggregates as well as ¹O₂ physical quenching by monomeric and aggregated chlorophyll can be considered as parts of the protective system against singlet oxygen formation.Abbreviations BChl bacteriochlorophyll - MBpd methyl bacteriopheophorbide - Chl chlorophyll - TPP meso-tetraphenylporphyrin - TPPS meso-tetra (p-sulfophenyl) porphyrin     

Electric-field dependence of the quantum yield in reaction centers of photosynthetic bacteria

Multilayer Langmuir-Blodgett films of reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides have been fabricated with partial net orientation. The films showed substantial electrical response under pulsed illumination. From measurements of the light-induced voltage generated across the Langmuir-Blodgett film, we have succeeded in quantitating the electric-field dependence of the quantum yield of charge separation in photosynthesis. The results presented here are compared with our previous determination of the field effect on quantum yield, in which flash-activated charge separation as a function of the applied field was assayed by the extent of bacteriochlorophyll dimer, (BChl)₂, oxidation measured optically at 860 nm. The two methods provided consistent dependencies of quantum yield on applied electric field. Analysis of the data reveals that the quantum yield of (BChl)^⨥₂BPhQ^⨪_A formation decreases from a value of 0.96 at zero applied field to about 0.75 for a field of 120 mV/nm vectorially directed to hinder light-activated electron transfer. For oppositely applied fields, the quantum yield saturates at unity. The source of the effects is considered to reside in the electric field dependence of the free-energy difference between the energy levels that are involved in the initial charge separation between the (BChl)₂ in the first singlet excited state, (BChl)1₂, through the bacteriopheophytin, BPh, to the primary ubiquinone, Q_A. Possible contributions to the field-induced loss of quantum yield of (BChl)^⨥₂BPhQ^⨪_A formation are: (1) a decrease in the free-energy gap between the states (BChl)1₂ and (BChl)^⨥₂BPh^⨪Q_A, leading to an increased rate of decay via the excited singlet state back to the ground state; (2) a stimulated return from (BChl)^⨥₂BPh^⨪Q_A directly or via the (BChl)₂ triplet state to the ground state and (3) an impeded electron transfer from (BChl)^⨥₂BPh^⨪Q_A to (BChl)^⨥₂BPhQ^⨪_A. These possibilities are discussed. Correlation of the electrical response with measurements of the photo-induced absorbance change allows determination of the projection of the electron-transfer distance on the normal to the plane of the film, which is in good agreement with previous measurements using different techniques.     

Low-frequency resonance Raman studies of the H(M202)G cavity mutant of bacterial photosynthetic reaction centers

Low-frequency (90–435 cm⁻¹) NIR-excitation (875–900 nm) resonance Raman (RR) studies are reported for the H(M202)G cavity mutant of bacterial photosynthetic reaction centers (RCs) from Rb. sphaeroides that was first described by Goldsmith et al. [(1996) Biochemistry 35: 2421–2428]. In this mutant, the His residue that axially ligates the Mg ion of the M-side bacteriochlorophyll (BChl) of the special pair primary donor (P) is replaced by a non-ligating Gly residue. Regardless, the Mg ion of P_M in the H(M202)G RCs remains pentacoordinates and is presumably ligated by a water molecule, although this axial ligand has not been definitively identified. The low-frequency RR studies of the H(M202)G RCs are accompanied by studies of RCs exchanged with D₂O and incubated with imidazole (Im). The RR studies of the cavity mutant RCs reveal the following: (1) The structure of P_M in the H(M202)G RCs is different from that of the wild-type, consistent with an altered BChl core. (2) A water ligand for P_M in the H(M202)G RCs is generally consistent with the low-frequency RR spectra. The Mg-OH₂ stretching vibration is tentatively assigned to a band at 318 cm⁻¹, a frequency higher than that of the Mg-His stretch of the native pigment (∼ ∼235 cm⁻¹). (3) The BChl core structure of P_M in the cavity mutant is rendered similar (but not identical) to that of the wild-type when the adventitious water axial ligand is replaced by Im. (4) Exchange with D₂O results in more global structural changes, likely involving the protein, which in turn affect the structure of the BChls in P. (5) Assignment of the low-frequency vibrational spectrum of P is generally more complex than originally suggested.     

Radicals associated with the catalytic intermediates of bovine cytochrome c oxidase

Two radicals have been detected previously by electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies in bovine cytochrome oxidase after reaction with hydrogen peroxide, but no correlation could be made with predicted levels of optically detectable intermediates (P_M, F and F) that are formed. This work has been extended by optical quantitation of intermediates in the EPR/ENDOR sample tubes, and by comparison with an analysis of intermediates formed by reaction with carbon monoxide in the presence of oxygen. The narrow radical, attributed previously to a porphyrin cation, is detectable at low levels even in untreated oxidase and increases with hydrogen peroxide treatments generally. It is presumed to arise from a side-reaction unrelated to the catalytic intermediates. The broad radical, attributed previously to a tryptophan radical, is observed only in samples with a significant level of F but when F is generated with hydrogen peroxide, is always accompanied by the narrow radical. When P_M is produced at high pH with CO/O₂, no EPR-detectable radicals are formed. Conversion of the CO/O₂-generated P_M into F when pH is lowered is accompanied by the appearance of a broad radical whose ENDOR spectrum corresponds to a tryptophan cation. Quantitation of its EPR intensity indicates that it is around 3% of the level of F determined optically. It is concluded that low pH causes a change of protonation pattern in P_M which induces partial electron redistribution and tryptophan cation radical formation in F. These protonation changes may mimic a key step of the proton translocation process.     

Microevolutionary trends in the dentition of the Red fox (Vulpes vulpes)

Microevolutionary trends in dental traits were studied in a Polish population of the Red fox, Vulpes vulpes (Linnaeus, 1758). Changes in qualitative and quantitative traits over a 70‐year interval were analysed in 1453 museum specimens collected between 1927 and 1996. Over that period, there were qualitative trends towards increasing complication of occlusal crown surface in posterior premolars (i.e. P⁴, P₃, P₄) and I³. Other cheek teeth did not undergo directional change. Changes in trait correlations were assessed using samples from the 1960s and 1990s. The correlations between C¹–C₁ and M¹–M² increased, while correlation values in the incisor region (I₁–I₂, I¹–I₁, I¹–I₂, I³–I₂), carnassial region (P₄–M₁, P⁴–M₁ and M₁–M₁) and in P²–P₁ decreased. These changes may be related to increasing dietary opportunism of the Red fox during the 20th century.     

Engineered biosynthesis of bacteriochlorophyll gF in Rhodobacter sphaeroides

Engineering photosynthetic bacteria to utilize a heterologous reaction center that contains a different (bacterio) chlorophyll could improve solar energy conversion efficiency by allowing cells to absorb a broader range of the solar spectrum. One promising candidate is the homodimeric type I reaction center from Heliobacterium modesticaldum. It is the simplest known reaction center and uses bacteriochlorophyll (BChl) g, which absorbs in the near-infrared region of the spectrum. Like the more common BChls a and b, BChl g is a true bacteriochlorin. It carries characteristic C3-vinyl and C8-ethylidene groups, the latter shared with BChl b. The purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides was chosen as the platform into which the engineered production of BChl g_F, where F is farnesyl, was attempted. Using a strain of Rba. sphaeroides that produces BChl b_P, where P is phytyl, rather than the native BChl a_P, we deleted bchF, a gene that encodes an enzyme responsible for the hydration of the C3-vinyl group of a precursor of BChls. This led to the production of BChl g_P. Next, the crtE gene was deleted, thereby producing BChl g carrying a THF (tetrahydrofarnesol) moiety. Additionally, the bchG^Rs gene from Rba. sphaeroides was replaced with bchG^Hm from Hba. modesticaldum. To prevent reduction of the tail, bchP was deleted, which yielded BChl g_F. The construction of a strain producing BChl g_F validates the biosynthetic pathway established for its synthesis and satisfies a precondition for assembling the simplest reaction center in a heterologous organism, namely the biosynthesis of its native pigment, BChl g_F.     

α-2,6-Difluorophenyl-N-Tert-Butylnitrone: A Spin Trap for Distinguishing Different Types of Alkyl Radicals Based on Long-Range Fluorine Hyperfine Splitting

α-2.6-Difluorophenyl-N-tert-butylnitrone (F₂PBN) was synthesized and evaluated. A number of alkyl adducts of F₂PBN were studied by ESR/ENDOR. An additional hyperfine splitting (a triplet of doublets of doublets) is reported. The existence of two (one large, one small) F-hfsc's from the ortho-fluorine atoms in the phenyl ring of most alkyl adducts was confirmed by ENDOR spectroscopy.     

Spectroscopic properties of the peridinins involved in chlorophyll triplet quenching in high-salt peridinin-chlorophyll a-protein from Amphidinium carterae as revealed by optically detected magnetic resonance, pulse EPR and pulse ENDOR spectroscopies

The photoexcited triplet state of the carotenoid peridinin in the high-salt peridinin-chlorophyll a-protein (HSPCP) of the dinoflagellate Amphidinium carterae was investigated by ODMR (optically detected magnetic resonance), pulse EPR and pulse ENDOR spectroscopies. The properties of peridinins associated to the triplet state formation in HSPCP were compared to those of peridinins involved in triplet state population in the main-form peridinin-chlorophyll protein (MFPCP), previously reported. In HSPCP no signals due to the presence of chlorophyll triplet state have been detected, during either steady-state illumination or laser-pulse excitation, meaning that peridinins play the photo-protective role with 100% efficiency as in MFPCP. The general spectroscopic features of the peridinin triplet state are very similar in the two complexes and allow drawing the conclusion that the triplet formation pathway and the triplet localization in one specific peridinin in each subcluster are the same in HSPCP and MFPCP. However some significant differences also emerged from the analysis of the spectra. Zero field splitting parameters of the peridinin triplet states are slightly smaller in HSPCP and small changes are also observed for the hyperfine splittings measured by pulse ENDOR and assigned to the β-protons belonging to one of the two methyl groups present in the conjugated chain, (a_iso = 10.3 MHz in HSPCP vs a_iso = 10.6 MHz in MFPCP). The differences are explained in terms of local distortion of the tails of the conjugated chains of the peridinin molecules, in agreement with the conformational data resulting from the X-ray structures of the two complexes.     

Electronic structure of the primary electron donor of Blastochloris viridis heterodimer mutants: High-field EPR study

High-field electron paramagnetic resonance (HF EPR) has been employed to investigate the primary electron donor electronic structure of Blastochloris viridis heterodimer mutant reaction centers (RCs). In these mutants the amino acid substitution His(M200)Leu or His(L173)Leu eliminates a ligand to the primary electron donor, resulting in the loss of a magnesium in one of the constituent bacteriochlorophylls (BChl). Thus, the native BChl/BChl homodimer primary donor is converted into a BChl/bacteriopheophytin (BPhe) heterodimer. The heterodimer primary donor radical in chemically oxidized RCs exhibits a broadened EPR line indicating a highly asymmetric distribution of the unpaired electron over both dimer constituents. Observed triplet state EPR signals confirm localization of the excitation on the BChl half of the heterodimer primary donor. Theoretical simulation of the triplet EPR lineshapes clearly shows that, in the case of mutants, triplet states are formed by an intersystem crossing mechanism in contrast to the radical pair mechanism in wild type RCs. Photooxidation of the mutant RCs results in formation of a BPhe anion radical within the heterodimer pair. The accumulation of an intradimer BPhe anion is caused by the substantial loss of interaction between constituents of the heterodimer primary donor along with an increase in the reduction potential of the heterodimer primary donor D/D⁺ couple. This allows oxidation of the cytochrome even at cryogenic temperatures and reduction of each constituent of the heterodimer primary donor individually. Despite a low yield of primary donor radicals, the enhancement of the semiquinone-iron pair EPR signals in these mutants indicates the presence of kinetically viable electron donors.     

The primary electron acceptor of green sulfur bacteria,bacteriochlorophyll 663, is chlorophyll a esterified with Δ 2,6-phytadienol

The primary electron acceptor of green sulfur bacteria, bacteriochlorophyll (BChl) 663, was isolated at high purity by an improved purification procedure from a crude reaction center complex, and the molecular structure was determined by fast atom bombardment mass spectroscopy (FAB-mass), ¹H- and ¹³C-NMR spectrometry, double quantum filtered correlation spectroscopy (DQF-COSY), heteronuclear multiple-quantum coherence (HMQC) and heteronuclear multiple-bond correlation (HMBC) spectral measurements. BChl 663 was 2.0 mass units smaller than plant Chl a. The NMR spectra showed that the macrocycle was identical to that of Chl a. In the esterifying alcohol, a singlet P7¹ signal was observed at the high-field side of the singlet P3¹ signal in BChl 663, while a doublet peak of P7¹ overlapped that of P11¹ in Chl a. A signal of P7-proton, seen in Chl a, was lacking, and the P6-proton appeared as a triplet signal near the triplet P2-proton signal in BChl 663. These results indicate the presence in BChl 663 of a C=C double bond between P6 and P7 in addition to that between P2 and P3. The structure of BChl 663 was hence concluded to be Chl a esterified with 2,6-phytadienol instead of phytol. In addition to BChl 663, two molecules of the 13²-epimer of BChl a, BChl a′, were found to be present per reaction center, which may constitute the primary electron donor. This revised version was published online in June 2006 with corrections to the Cover Date.     

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 alpha- and beta-protons of the peridinin conjugated chain. Assignments of beta-protons belonging to two different methyl groups, with aiso=7.0 MHz and aiso=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 pi-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.     

Electron nuclear double resonance and electron paramagnetic resonance study on the structure of the NO-ligated heme alpha 3 in cytochrome c oxidase

The techniques of EPR and electron nuclear double resonance (ENDOR) were used to probe structure and electronic distribution at the nitric oxide (NO)-ligated heme alpha 3 in the nitrosylferrocytochrome alpha 3 moiety of fully reduced cytochrome c oxidase. Hyperfine and quadrupole couplings to NO (in both 15NO and 14NO forms), to histidine nitrogens, and to protons near the heme site were obtained. Parallel studies were also performed on NO-ligated myoglobin and model NO-heme-imidazole systems. The major findings and interpretations on nitrosylferrocytochrome alpha 3 were: 1) compared to other NO-heme-imidazole systems, the nitrosylferrocytochrome alpha3 gave better resolution of EPR and ENDOR signals; 2) at the maximal g value (gx = 2.09), particularly well resolved NO nitrogen hyperfine and quadrupole couplings and mesoproton hyperfine couplings were seen. These hyperfine and quadrupole couplings gave information on the electronic distribution on the NO, on the orientation of the g tensor with respect to the heme, and possibly on the orientation of the FeNO plane; 3) a combination of experimental EPR-ENDOR results and EPR spectral simulations evidenced a rotation of the NO hyperfine tensor with respect to the electronic g tensor; this implied a bent Fe-NO bond; 4) ENDOR showed a unique proton not seen in the other NO heme systems studied. The magnitude of this proton's hyperfine coupling was consistent with this proton being part of a nearby protein side chain that perturbs an axial ligand like NO or O2.     

Advanced electron paramagnetic resonance on the catalytic iron–sulfur cluster bound to the CCG domain of heterodisulfide reductase and succinate: quinone reductase

Heterodisulfide reductase (Hdr) is a key enzyme in the energy metabolism of methanogenic archaea. The enzyme catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) to the thiol coenzymes M (CoM-SH) and B (CoB-SH). Cleavage of CoM-S-S-CoB at an unusual FeS cluster reveals unique substrate chemistry. The cluster is fixed by cysteines of two cysteine-rich CCG domain sequence motifs (CX_31–39CCX_35–36CXXC) of subunit HdrB of the Methanothermobacter marburgensis HdrABC complex. We report on Q-band (34 GHz) ⁵⁷Fe electron-nuclear double resonance (ENDOR) spectroscopic measurements on the oxidized form of the cluster found in HdrABC and in two other CCG-domain-containing proteins, recombinant HdrB of Hdr from M. marburgensis and recombinant SdhE of succinate: quinone reductase from Sulfolobus solfataricus P2. The spectra at 34 GHz show clearly improved resolution arising from the absence of proton resonances and polarization effects. Systematic spectral simulations of 34 GHz data combined with previous 9 GHz data allowed the unambiguous assignment of four ⁵⁷Fe hyperfine couplings to the cluster in all three proteins. ¹³C Mims ENDOR spectra of labelled CoM-SH were consistent with the attachment of the substrate to the cluster in HdrABC, whereas in the other two proteins no substrate is present. ⁵⁷Fe resonances in all three systems revealed unusually large ⁵⁷Fe ENDOR hyperfine splitting as compared to known systems. The results infer that the cluster’s unique magnetic properties arise from the CCG binding motif.