Theory of proton hyperfine interactions in bacterial photosynthetic systems.: Bacteriopheophytin cation and anion

The electronic structures of the cation and anion of bacteriopheophytin a monomer are investigated by the self-consistent charge extended Hückel procedure including both π and σ electrons in the molecule. The calculated electron distributions are tested by comparison of the predicted hyperfine fields at proton sites with experimental data in both the ions and most important, by their ability to explain the observed trend in the hyperfine fields in going from the cation bacteriopheophytin⁺a to the anion, a trend that is similar in many respects to the corresponding observed trend for the bacteriochlorophyll a cation and anion. Good agreement is obtained with experiment both for the absolute values of the observed proton hyperfine fields in both bacteriopheophytin a cation and anion as well as the ratio of the corresponding fields for the two systems. In particular, our calculated electron distributions in the two molecules lead, for the cation, to substantially different proton hyperfine fields for the two methyl groups attached to rings I and III, while for the anion, the corresponding fields are much closer to each other, a trend in good agreement with recent data. Also explained are the features of larger methine hyperfine constants in the anion as compared to the cation and the reverse trend for the protons in rings II and IV. Other features of the calculated electron distributions in the cation and anion are discussed and compared with each other. Possible additional measurements in the two systems that could provide further tests of the theoretically obtained electron distribution will be pointed out.     

Blue light drives B-side electron transfer in bacterial photosynthetic reaction centers.

The core of the photosynthetic reaction center from the purple non-sulfur bacterium Rhodobacter sphaeroides is a quasi-symmetric heterodimer, providing two potential pathways for transmembrane electron transfer. Past measurements have demonstrated that only one of the two pathways (the A-side) is used to any significant extent upon excitation with red or near-infrared light. Here, it is shown that excitation with blue light into the Soret band of the reaction center gives rise to electron transfer along the alternate or B-side pathway, resulting in a charge-separated state involving the anion of the B-side bacteriopheophytin. This electron transfer is much faster than normal A-side transfer, apparently occurring within a few hundred femtoseconds. At low temperatures, the B-side charge-separated state is stable for at least 1 ns, but at room temperature, the B-side bacteriopheophytin anion is short-lived, decaying within approximately 15 ps. One possible physiological role for B-side electron transfer is photoprotection, rapidly quenching higher excited states of the reaction center.     

Radiation-induced free radicals in solid 5-halouracil derivatives: single crystals of 5-iododeoxyuridine

X-irradiation of single crystals of 5-iododeoxyuridine (IUdR) in the temperature range 8-300 K produces mainly four different radicals which have been studied by electron spin resonance (e.s.r.) and electron nuclear double resonance (ENDOR)-spectroscopy. At low temperatures, a pi-anion is formed which shows predominantly an interaction of the unpaired electron with a proton at carbon C6 of the base (-11.8 G, -23.9 G, -4.6 G). Above 10-20 K, the anion protonates at C6 to yield a RC-I(CH2)-R' radical comprising alpha-iodo and beta-methylene proton hyperfine interactions. The primary oxidation product is an O5'-situated alkoxy radical RCH2O which shows inequivalent beta-proton couplings of about 100 G and 35 G together with a highly anisotropic g-tensor. Upon warming to 265 K, a C2'-located radical on the deoxyribose is formed which is stable at room temperature. A detailed account of its spectral features as obtained by ENDOR exhibits three different alpha-type couplings, two small beta-protons and a dipolar interaction. Other radicals, not reproducibly observed, involve a C5'-hydroxyalkyl radical and a species related to the base cation at low temperatures.     

Density functional calculations on model tyrosyl radicals.

A gradient-corrected density functional theory approach (PWP86) has been applied, together with large basis sets (IGLO-III), to investigate the structure and hyperfine properties of model tyrosyl free radicals. In nature, these radicals are observed in, e.g., the charge transfer pathways in photosystem II (PSII) and in ribonucleotide reductases (RNRs). By comparing spin density distributions and proton hyperfine couplings with experimental data, it is confirmed that the tyrosyl radicals present in the proteins are neutral. It is shown that hydrogen bonding to the phenoxyl oxygen atom, when present, causes a reduction in spin density on O and a corresponding increase on C4. Calculated proton hyperfine coupling constants for the beta-protons show that the alpha-carbon is rotated 75-80 degrees out of the plane of the ring in PSII and Salmonella typhimurium RNR, but only 20-30 degrees in, e.g., Escherichia coli, mouse, herpes simplex, and bacteriophage T4-induced RNRs. Furthermore, based on the present calculations, we have revised the empirical parameters used in the experimental determination of the oxygen spin density in the tyrosyl radical in E. coli RNR and of the ring carbon spin densities, from measured hyperfine coupling constants.     

Structure and redox properties of radicals derived from one-electron oxidised methylxanthines

The repair of the one electron oxidised form of a xanthine derivative by another xanthine derivative was studied by reacting aqueous binary mixtures of xanthine derivatives with sulphate radical anion (SO(4)( -)) and following the concentration of both compounds as a function of time. The relative behaviour observed enabled the establishment of a qualitative order of antioxidant capacities for the several xanthines studied in acidic and neutral media. The same order was confirmed quantitatively by measuring the reduction potentials of the xanthines by cyclic voltammetry. Theoretical DFT calculations were used to calculate the relative stabilities of the tautomers of each xanthine neutral radical. It was also demonstrated that the deprotonation of a xanthine radical cation never occurs from N1, unless no other possibility is available. At high pH values, it was possible to obtain the ESR spectra of the radical anions derived from 1-methylxanthine, 3-methylxanthine and xanthosine. The theoretical calculations also enabled the assignment of the ESR hyperfine coupling constants of the spectra of these radical anions. The coupling constants calculated are in good agreement with the experimental values.     

Photochemical trapping of a bacteriopheophytin anion in site-specific reaction-center mutants from the photosynthetic bacterium Rhodobacter sphaeroides.

The mutant YY in the reaction center of Rhodobacter sphaeroides, in which Phe181 on the L chain has been replaced by Tyr, and the double mutant FY, with Tyr210 on the M chain replaced by Phe and Phe181 on the L chain replaced by Tyr, have been constructed by site-directed mutagenesis. The studies described here were performed to complement a previous mutational analysis of mutant FF with Tyr210 replaced by Phe. Both new strains grow photoheterotrophically. The optical absorption spectra of reaction centers isolated from these mutants have band shifts attributable to the monomer bacteriochlorophylls in the vicinity of the substitutions. Photochemical trapping of the bacteriopheophytin anion (I-) indicates that the bacteriopheophytin on the B branch is reduced to a much greater extent in FF and FY as compared to YY and wild-type YF. Low temperature (77 K) absorption spectra clearly show that in the wild-type (YF) and YY reaction centers only the 545-nm-absorbing bacteriopheophytin is reduced while in the FF and FY reaction centers both the 535-nm and 545-nm-absorbing bacteriopheophytins are reduced. A simple kinetic analysis is used to explain these results. This analysis suggests that, in order for the observed trapping results to occur, a decrease in the 'cycling' time must take place, that is changes in the rate(s) of charge recombination must accompany the already known decrease in the forward electron transfer rate.     

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.     

X-ray structure analysis of a membrane protein complex. Electron density map at 3 A resolution and a model of the chromophores of the photosynthetic reaction center from Rhodopseudomonas viridis

X-ray analysis of three-dimensional crystals of the photosynthetic reaction center from the purple bacterium Rhodopseudomonas viridis led to an electron density distribution at 3 A resolution calculated with phases from multiple isomorphous replacement. The protein subunits of the complex were identified. An atomic model of the prosthetic groups of the reaction center complex (4 bacteriochlorophyll b, 2 bacteriopheophytin b. 1 non-heme iron, 1 menaquinone, 4 heme groups) was built. The arrangement of the ring systems of the bacteriochlorophyll b and bacteriopheophytin b molecules shows a local 2-fold rotation symmetry; two bacteriochlorophyll b form a closely associated, non-covalently linked dimer ("special pair"). A different local 2-fold symmetry axis is observed for the heme groups of the cytochrome part.     

Proton nuclear magnetic resonance studies of hemoglobin Malm?: implications of mutations at homologous positions of the alpha and beta chains.

The abnormal human hemoglobin Malm? (beta97FG4 His leads to Gln) has been studied and its properties are compared with those of normal adult hemoglobin A. The data presented here show that the ring-current shifted proton resonances of both HbCO and HbO2 Malm? are very different from the corresponding forms of Hb A. The hyperfine shifted proton resonances of deoxy-Hb Malm? do not differ drastically from those of deoxy-Hb A. This result, together with the finding that the exchangeable proton resonances of the deoxy form of the two hemoglobins are similar, suggests that unliganded Hb Malm? can assume a deoxy-like quaternary structure both in the absence and presence of organic phosphates We have also compared the properties of Hb Malm? with those of Hb Chesapeake (alpha92FG4 Arg leads to Leu). This allows us to study the properties of two abnormal human hemoglobins with mutations at homologous positions of the alpha and beta chains in the three-dimenstional structure of the hemoglobin molecule. Our present results suggest that the mutaion at betaFG4 has its greatest effect on the teritiary structure of the heme pocket of the liganded forms of the hemoglobin while the mutation at alphaFG4 alters the deoxy structure of the hemoglogin molecule but does not alter the teriary structure of the heme pockets of the liganded form of the hemoglobin molecule. Both hemoglobins undergo a transition from the deoxy (T) to the oxy (R) quaternary structure upon ligation. The abnormally high oxygen affinities and low cooperativities of these two hemoglobins must therefore be due to either the structural differences which we have observed and/or to an altered transition between the T and R structures.     

Proton hyperfine resonance assignments using the nuclear Overhauser effect for ferric forms of horse and tuna cytochrome c.

Proton hyperfine resonance assignments for cytochromes c from several species are currently being successfully pursued by several laboratories. These efforts focus mostly on the ferrous forms. In contrast to that work, we have pursued assignments of the proton hyperfine shifted resonances for horse and tuna ferricytochromes c. Our results indicate that assignments are nearly identical in those two proteins. Using the pre-steady state nuclear Overhauser effect, several additional assignments have been made for the tuna protein, whereas for the horse protein, the following protons have been assigned: heme 7, alpha CH2; heme 7, beta CH2; histidine 18, beta CH2 and alpha CH; and the methionine 80, beta CH2.     

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

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

Evidence for more than one Ca2+ transport mechanism in mitochondria.

The active transport and internal binding of the Ca2+ analogue Mn2+ by rat liver mitochondria were monitored with electron paramagnetic resonance. The binding of transported Mn2+ depended strongly on internal pH over the range 7.7-8.9. Gradients of free Mn2+ were compared with K+ gradients measured on valinomycin-treated samples. In the steady state, the electrochemical Mn2+ activity was larger outside than inside the mitochondria. The observed gradients of free Mn2+ and of H+ could not be explained by a single "passive" uniport or antiport mechanism of divalent cation transport. This conclusion was further substantiated by observed changes in steady-state Ca2+ and Mn2+ distributions induced by La3+ and ruthenium red. Ruthenium red reduced total Ca2+ or Mn2+ uptake, and both inhibitors caused release of divalent cation from preloaded mitochondria. A model is proposed in which divalent cations are transported by at least two mechanisms: (1) a passive uniport and (2) and active pump, cation antiport or anion symport. The former is more sensitive to La3+ and ruthenium red. Under energized steady-state conditions, the net flux of Ca2+ or Mn2+ is inward over (1) and outward over (2). The need for more than one transport system inregulating cytoplasmic Ca2+ is discussed.     

Primary and secondary electron transfer in hexane-solubilized proteolipid complexes of Rhodopseudomonas sphaeroides R-26

Primary electron transfer in hexane-solubilized reaction center proteolipid complexes is similar to that in detergent-solubilized reaction centers or chromatophores when diaminodurene is electron donor. Approximate values for the extinction coefficients of ubisemiquinone and the diaminodurene cation can be calculated. The primary and secondary quinone sites are dissimilar and results in only the transient formation of a semiquinone anion pair after two photochemical turnovers. One of the semiquinone anions decays rapidly, the remaining one and the diaminodurene cation have long lifetimes. Disproportionation between the semiquinone anions does not occur.     

Electron-spin-resonance studies of a chlorpromazine derivative bound to DNA fibres.

Solutions of DNA, spin-labelled with the radical cation of chlorpromazine, were used to produce oriented species of fibres pulled from a gel obtained by ultracentrifugation. The electron spin resonance spectra, recorded at X and Q band frequencies, are given for both gel and fibres; 14N hyperfine coupling parameters were obtained by computer fitting. The spectra are explained in terms of a strongly immobilized label having one principal hyperfine tensor axis parallel to the axis of the DNA helix, and the preferential orientation of the chlorpromazine ions with their planes perpendicular to the DNA helical axis.     

Reorientation of the acetyl group of the photoactive bacteriopheophytin in reaction centers of Rhodobacter sphaeroides: an ENDOR/TRIPLE resonance study

The freeze-trapped bacteriopheophytin alpha radical anion phi(*)A- has been investigated by 1H-ENDOR/Special TRIPLE resonance spectroscopy in photosynthetic reaction centers of Rhodobacter sphaeroides, in which the Tyr at position M210 had been replaced by either Phe, Leu, His or Trp. In the wild type reaction center and the mutants YF(M210) and YW(M210) two distinct states of phi(*)A-, denoted I(*)1- and I(*)2-, can be stabilized below 200 K. The state I(*)1 is metastable and relaxes to I(*)2- as the temperature is raised from 135 K to 180 K. The difference in the electronic structure of phi(*)A- between the two states is interpreted in terms of a conformational change of phiA after freeze-trapping, involving a reorientation of the 3-acetyl group with respect to the macrocycle of the bacteriopheophytin. This interpretation is supported by the results of RHF-INDO/SP calculations. In the YH(M210) reaction center only one phiA- state is obtained that is distinct from I(*)1- and I(*)2, and the observed electronic structure indicates an almost in-plane orientation of the 3-acetyl group. This is consistent with the proposal that a hydrogen bond is formed between His M210 and the 3(1)-keto oxygen of phiA that impedes the reorientation of the acetyl group. Only one phi(*)A- state is observed in the YL(M210) reaction center, which is similar to the metastable state I(*)1 in the wild type complex. This result is interpreted in terms of a steric hindrance of the reorientation of the 3-acetyl group that is exerted by the side chain of Leu at position M210. Possible implications of these findings for the mechanism of electron transfer in bacterial reaction centers are discussed.     

The [NiFe] hydrogenase from Allochromatium vinosum studied in EPR-detectable states: H/D exchange experiments that yield new information about the structure of the active site

In this study we report on thus-far unobserved proton hyperfine couplings in the well-known EPR signals of [NiFe] hydrogenases. The preparation of the enzyme in several highly homogeneous states allowed us to carefully re-examine the Ni(u)*, Ni(r)*, Ni(a)-C* and Ni(a)-L* EPR signals which are present in most [NiFe] hydrogenases. At high resolution (modulation amplitude 0.57 G), clear indications for hyperfine interactions were observed in the g(z) line of the Ni(r)* EPR signal. The hyperfine pattern became more pronounced in 2H2O. Simulations of the spectra suggested the interaction of the Ni-based unpaired electron with two equivalent, non-exchangeable protons (A1,2=13.2 MHz) and one exchangeable proton (A3=6.6 MHz) in the Ni(r)* state. Interaction with an exchangeable proton could not be observed in the Ni(u)* EPR signal. The identity of the three protons is discussed and correlated to available ENDOR data. It is concluded that the NiFe centre in the Ni(r)* state contains a hydroxide ligand bound to the nickel, which is pointing towards the gas channel rather than to iron.     

Quantitative pressure injection of picoliter volumes into Limulus ventral photoreceptors.

The conformation of the 14 amino acid peptide hormone somatostatin in aqueous solution was investigated through a proton magnetic resonance (PMR) scalar coupling analysis. Experiments were performed at two fields, 270 and 600 MHz, and included double and triple resonance difference scalar decoupling, resolution enhancement and computer simulation. The agreement between simulated and observed spectra at both fields provided support for the correctness of the analysis. The resultant scalar coupling constants, 3J alpha H-NH and 3J alpha B, gave information on the backbone (phi) and side chain (chi 1) torsional angles, respectively, which eliminated either of the proposed conformations of somatostatin as describing a predominant conformer of the molecule in solution under our conditions.     

The structure of the heterodimer reaction center from Rhodobacter sphaeroides at 2.55 å resolution

Crystals have been obtained of reaction centers of the heterodimer mutant that has significantly different properties than wild type due to the primary donor being formed from both a bacteriochlorophyll and bacteriopheophytin rather than two bacteriochlorophylls as found for wild type. The crystals belong to the trigonal space group P3(1)21 and the structure has been refined to a resolution limit of 2.55 A with an R factor of 19.0%. The electron density maps confirm that a primary donor does indeed contain a bacteriopheophytin due to the His to Leu substitution at M202 that coordinates the corresponding bacteriochlorophyll in wild-type. Other structural changes compared to wild type are relatively minor with the relative orientation and positioning of the two tetrapyrroles forming the primary donor being unchanged within the error. Compared to wild type, the only significant alterations are small shifts of residues M196 to M206, a rotation of the side chain of Ile M206, and the loss of a bound water molecule that in wild-type is hydrogen-bonded to both His M202 and the bacteriochlorophyll monomer on the active branch. Since hydrogen-bonding interactions strongly influence the energies of tetrapyrroles, the loss of the water molecule should result in changes in the energies of the bacteriochlorophyll monomer that contributes to the observed functional differences with wild-type.     

Theoretical study of model compound I complexes of horseradish peroxidase and catalase.

Theoretical studies of the electronic structure and spectra of models for the ferric resting state and Compound I intermediates of horseradish peroxidase (HRP-I) and catalase (CAT-I) have been performed using the INDO-RHF/CI method. The goals of these studies were twofold: i) to determine whether the axial ligand of HRP is best described as imidazole or imidazolate, and ii) to address the long-standing question of whether HRP-I and CAT-I are a1u and a2u tau cation radicals. Only the imidazolate HRP-I model led to a calculated electronic spectra consistent with the experimentally observed significant reduction in the intensity of the Soret band compared with the ferric resting state. These results provide compelling evidence for significant proton transfer to the conserved Asp residue by the proximal histidine. The origin of the observed reduction of the Soret band intensity in HRP-I and CAT-I spectra has been examined and found to be caused by the mixing of charge transfer transitions into the predominantly porphyrin tau-tau transitions. For both HRP-I and CAT-I, the a1u porphyrin tau cation state is the lowest energy, and it is further stabilized by both the anionic form of the ligand and the porphyrin ring substituents of protoporphyrin-IX. The calculated values of quadrupole-splitting observed in the Mossbauer resonance of HRP-I and CAT-I are similar for the a1u and a2u tau cation radicals. Electronic spectrum of the a1u tau cation radical of HRP-I are more similar to the observed spectra, whereas the spectra of both a1u tau and a2u tau cation radicals of CAT-I resemble the observed spectra. These results also indicate the limitations of using any one observable property to try to distinguish between these states. Taken together, comparison of calculated and observed properties indicate that there is no compelling reason to invoke the higher energy a2u tau cation radical as the favored state in HRP-I and CAT-I. Both ground-state properties and electronic spectra are consistent with the a1u tau cation radical.     

Electron nuclear double resonance differentiates complementary roles for active site histidines in (6-4) photolyase

(6-4) photolyase catalyzes the light-dependent repair of UV-damaged DNA containing (6-4) photoproducts. Blue light excitation of the enzyme generates the neutral FAD radical, FADH., which is believed to be transiently formed during the enzymatic DNA repair. Here (6-4) photolyase has been examined by optical spectroscopy, electron paramagnetic resonance, and pulsed electron nuclear double resonance spectroscopy. Characterization of selected proton hyperfine couplings of FADH., namely those of H(8alpha) and H(1'), yields information on the micropolarity at the site where the DNA substrate is expected to bind. Shifts in the hyperfine couplings as a function of structural modifications induced by point mutations and pH changes distinguish the protonation states of two highly conserved histidines, His(354) and His(358), in Xenopus laevis (6-4) photolyase. These are proposed to catalyze formation of the oxetane intermediate that precedes light-initiated DNA repair. The results show that at pH 9.5, where the enzymatic repair activity is highest, His(358) is deprotonated, whereas His(354) is protonated. Hence, the latter is likely the proton donor that initiates oxetane formation from the (6-4) photoproduct.