Magnetic field affects the fluorescence yield in reaction center preparations from Rhodopseudomonas sphaeroides R-26

Purified photochemical reaction centers from Rhodopseudomonas sphaeroides R-26 were reduced with Na₂S₂O₄ so as to block their photochemical electron-transfer reactions. The magnetic field induced an increase in the emission yield. Our results support the hypothesis that under these conditions, charge recombination in the singlet radical pair composed of the oxidized primary donor and reduced primary acceptor predominantly generates the excited singlet state of the reaction center bacteriochlorophyll.The maximum relative fluorescence change and the value of the magnetic field at which half-saturation of the effect is achieved ($\text{B}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) at room temperature are 5.5% and 75 G, respectively. For the whole cells of Rps. sphaeroides R-26 these parameters are 1.2% and 120 G.The relative fluorescence change at 600 G, $\text{ΔF}\text{F(600)}$, and $\text{B}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ are studied as functions of temperature. The temperature dependencies of $\text{ΔF}\text{F(600)}$ for reaction centers and whole cells of Rps. sphaeroides R-26 are qualitatively the same, with the maximum effect (8% for reaction centers) occurring at 230 K. However, the $\text{B}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ curves for the two preparations are different.     

Cytochrome C553 from Desulfovibrio,vulgaris: Potentiometric characterization by optical and EPR studies

Cytochrome c₅₅₃ is a monohaemic c type cytochrome isolated from the sulfate reducing bacteria $\text{Desulfovibrio}$,$\text{vulgaris}$. Its midpoint potential value, determined by optical, EPR and polarographic studies is significantly lower than the midpoint potentials reported for other monohaemic cytochromes c (+ 10 mV instead of + 290 mV). In an attempt to study correlations between amino acid sequence, haem iron coordination and haem exposure in cytochromes c, cytochrome c₅₅₃ is compared with mitochondrial and bacterial c type cytochromes.     

Electron exchange coupling in a naturally occurring tetramangano cluster in the mineral helvite, (Mn4S)(SiBeO4)3

The mineral helvite, (Mn₄S)(BeSiO₄)₃, contains discrete tetrahedral Mn₄S⁺⁶ clusters in which the S^?2 is tetrahedrally coordinated and each Mn(II) is in a distorted tetrahedron of one S^?2 and three oxygens; the cluster is situated within an encompassing lattice of SiO₄^?4 and BeO₄^?6 tetrahedra. Mn₄S⁺⁶ centers provide an interesting model for comparison to the polynuclear manganese center that is associated with photosynthetic water oxidation. Magnetic susceptibility data between 77 and 298 K have been measured for a natural helvite sample containing principally Mn₄S⁺⁶ centers but with significant contamination from Mn₃FeS⁺⁶ and Mn₃CaS⁺⁶. The data exhibited Curie-Weiss behavior with μ_eff = 5.969 B.M. and θ = 178.3 K. An analysis of the magnetic susceptibility, based on Van Vleck's formalism, demonstrated the presence of antiferromagnetic coupling, with a coupling constant J = ?5.83 cm^?1. Mössbauer spectra of Mn₃FeS centers in helvite and of Fe₄S centers in the related mineral danalite have also been recorded. Isomer shifts show little temperature dependence and lie in the range 1.23–1.43 $\text{mm}\text{sec.}$. This range is typical of tetrahedrally coordinated Fe(II) in several ionic crystals but is significantly above that of Fe(II) in ferredoxins and below that in the [quinone-Fe(II)-quinone] complex of the photosynthetic bacterium,Rhodopseudomonas sphaeroides. Quadrupole splittings are highly temperature dependent, ranging from 2.4 $\text{mm}\text{sec}$ at 4.2 K to less than 0.5 $\text{mm}\text{sec}$ at 248 K.     

The conformation and orientation of copper(II)-bleomycin intercalated with DNA

EPR data are used to describe the conformation and identity of the atoms coordinated to Cu(II) in Cu(II)-bleomycin bound to oriented DNA fibers. The fibers were slowly drawn from viscous solutions of Cu(II)-bleomycin-DNA containing one Cu(II)-bleomycin to 200 basepairs. EPR measurements were made at room temperature and 90 K for different orientations of the external magnetic field with respect to the helical axes of the fibers. The g-values ($\text{g}{}_{\mathrm{}}\text{=2.21}$, g_⊥ =2.04) and the hyperfine constant ($\text{A}{}_{\mathrm{}}\text{=175}\text{G}$) are consistent with values expected for Cu(II) chelated to a square planar array of ligands. In the oriented fibers, the square planar arrays do not all have the same orientations with respect to the fiber axes. At room temperature the chelated ions have rotational freedom in which the normal to the planar array has almost complete freedom of rotation about axes perpendicular to the DNA fiber axes. The normal maintains an angle of 75° with respect to the axis, in the plane of the basepair, about which it rotates. Nine superhyperfine peaks on the high field side of the EPR spectrum were partially resolved. The number and splitting (12 G) of these superhyperfine peaks indicate that four nitrogen atoms are chelated to Cu(II) in a square planar array. These data on Cu(II)-bleomycin bound to DNA give information on the orientation of the metal-containing portion of bleomycin which lies outside the double helix.     

The type,amount, location,and energy transfer properties of the carotenoid in reaction centers from Rhodopseudomonas sphaeroides

Analysis of photosynthetic reaction centers from Rhodopseudomonas sphaeroides strains 2.4.1 and Ga shows that each contains approx. 1 mol of a specific carotenoid per mol of reaction center. In strain 2.4.1. the carotenoid is spheroidene (1-methoxy-3,4-didehydro-1,2,7′,8′-tetrahydro-ψ,ψ-carotene); in strain Ga, it is chloroxanthin (1-hydroxy-1,2,7′,8′-tetrahydro-ψ,ψ-carotene). The carotenoid is bound to the same pair of proteins as are the bacteriochlorophylls and bacteriopheophytins of the reaction center. This binding induces strong circular dichroism in the absorption bands of the carotenoid. The carotenoid is close enough to the other pigments of the reaction center so that light energy transfers efficiently from the carotenoid to the bacteriochlorophyll, sensitizing bacteriochlorophyll fluorescence. The fluorescence polarization spectrum of the reaction centers shows that the transition vectors for the visible absorption bands of the carotenoid lie approximately parallel to the 600 nm ($\text{Q}{}_{\mathrm{<mtext>x</mtext>}}$) transition of the bacteriochlorophyll complex.     

Hydrophobic ion probe studies of membrane dipole potentials

Hydrophobic anions of dipicrylamine and of sodium tetraphenylborate have been employed as probes of interfacial dipole potential variations in lipid bilayer membranes. Systematic variation of dipole potentials has been achieved by introduction of compounds incorporating N⁺ and B^? charge centers. Distribution of hydrophilic and and hydrophobic groups relative to these charge centers has been shown to control the orientation in the membrane/solution interface of the electric dipole moment formed by these centers. Thus triphenyl-[4-trimethylphenylammonium] borate orients with the B^? center, surrounded by phenyl groups, embedded in the membrane, while the smaller methylated N⁺ center is directed toward the aqueous phases. This orientation has been confirmed using dipicrylamine probe ions. Results obtained in this system have been interpreted quantitatively using a previously developed model incorporating discrete charge effects. A second class of compounds, $\text{tri}\text{-n-}\text{alkylamine}$ borane (T_nAB) complexes having the generic formula (C_nH_{$\text{2n+1}$})₃N⁺B^?H₃, have also been synthesized for this study, using even-carbon alkyls ranging from ethyl to decyl. Molecular orientation of the complex is with the N⁺ center and its associated alkyl groups directed into the membranes, while the protonated B^? center is directed toward the aqueous phases, as confirmed by use of tetraphenylborate ions as probes.     

The location of redox centers in the profile structure of a reconstituted membrane containing a photosynthetic reaction center-cytochrome c complex by resonance X-ray diffraction

The technique of resonance X-ray diffraction (Blasie, J.K. and Stamatoff, J. (1981) Annu. Rev. Biophys. Bioeng. 10, 451–452) utilizing synchrotron radiation was used to determine the locations of the cytochrome c heme iron atom and the photosynthetic reaction center iron atom within the profile of a reconstituted membrane. The accuracy of these determinations was better than ±2 ?. The cytochrome c heme iron atom → reaction center iron atom vector was determined to have a magnitude of approx. 44 ? projected onto the membrane profile and to span most of the lipid hydrocarbon core of the membrane profile. Since the reaction center iron atom interacts magnetically with the primary quinone electron acceptor Q_I over a distance of less than 10 ?, the primary light-induced electron-transfer reactions for this system generate the electric charge separation between oxidized cytochrome c⁺ and Fe-Q^?_I across most (approx. $\text{2}\text{3}$) of the membrane profile including most or all of the lipid hydrocarbon core of the membrane.     

Oxidation-reduction properties of the electron acceptors of Photosystem II. II. Redox titration at various pH values of the flash-induced formation of C550 in Chlamydomonas Photosystem II particles lacking the secondary quinone electron acceptor

Redox titrations of the flash-induced formation of C550 (a linear indicator of Q^?) were performed between pH 5.9 and 8.3 in Chlamydomonas Photosystem II particles lacking the secondary electron acceptor, B. One-third of the reaction centers show a pH-dependent midpoint potential (E_m,7.5) = ? 30 mV) for redox couple $\text{Q}\text{Q}{}^{\mathrm{?}}$, which varies by ?60 mV/pH unit. Two-thirds of the centers show a pH-independent midpoint potential (E_mm = + 10 mV) for this couple. The elevated pH-independent E_m suggests that in the latter centers the environment of Q has been modified such as to stabilize the semiquinone anion, Q^?. The midpoint potentials of the centers having a pH-dependent E_m are within 20 mV of those observed in chloroplasts having a secondary electron acceptor. It appears therefore that the secondary electron acceptor exerts little influence on the E_m of $\text{Q}\text{Q}{}^{\mathrm{?}}$. An EPR signal at g 1.82 has recently been attributed to a semiquinone-iron complex which comprises Q^?. The similar redox behavior reported here for C550 and reported by others (Evans, M.C.W., Nugent, J.H.A., Tilling, L.A. and Atkinson, Y.E. (1982) FEBS Lett. 145, 176–178) for the g 1.82 signal in similar Photosystem II particles confirm the assignment of this EPR signal to Q^?. At below ?200 mV, illumination of the Photosystem II particles produces an accumulation of reduced pheophytin (Ph^?). At ?420 mV Ph^? appears with a quantum yield of 0.006–0.01 which in this material implies a lifetime of 30–100 ns for the radical pair P-680⁺Ph^?.     

Inhibitory effect of iron on the uptake of lead by erythrocytes.

It is well known that more than 90% of the lead found in blood is associated with the erythrocytes. The present $\text{in vitro}$ experiments show that the uptake of lead-203 by rabbit erythrocytes is inhibited by the presence of non-radioactive lead or iron or by reduction of the incubation temperature. The inhibitory effect of iron on radioactive lead uptake by erythrocytes is also demonstrable $\text{in vivo}$.When lead-203 is incorporated into erythrocytes $\text{in vitro}$, about 10% of the radioactivity is attached to the membrane and the remainder is found in the cytoplasm associated with hemoglobin and an unidentified low molecular weight intracellular component. In the presence of 25 μg/ml of added iron (Fe⁺⁺⁺) the uptake of radioactive lead by erythrocytes is reduced to 21.7±5.1% and membrane binding accounts for approximately 5% of this total. Chromatographic analyses of hemolysates show that the reduction in cytoplasmic labeling is directly related to decreased lead binding to the low molecular weight component, since hemoglobin binding remains unchanged.This work suggests that in addition to the interaction between iron and lead which occurs during the biosynthesis of heme, these metals may directly compete for specific erythrocyte binding sites.     

The detection of a bound ferredoxin in the photosynthetic lamellae of blue-green algae and other oxygen evolving photosynthetic organisms

The presence of an electron transport component with an EPR spectrum similar to that of a ferredoxin has been demonstrated in the blue-green alga $\text{Anabaena}$$\text{cylindrica}$, the green alga $\text{Euglena}$$\text{gracilis}$, and in chloroplasts from sorghum ($\text{Sorghum}$$\text{bicolour}$) and beans ($\text{Phaseolus}$$\text{vulgaris}$). The component is photoreduced at 77°K and is very similar to that previously reported in spinach. It seems likely that this component is a primary electron acceptor in photosynthesis in all of these organisms.     

The existence of a high photochemical turnover rate at the reaction centers of System II in Tris-washed chloroplasts

In Tris-washed chloroplasts the kinetics of the primary electron acceptor X 320 of reaction center II has been investigated by fast repetitive flash spectroscopy with a time resolution of $\text{≈ 1 μs}$. It has been found that X 320 is reduced by a flash in $\text{? 1 μs}$. The subsequent reoxidation in the dark occurs mainly by a reaction with a 100–200 μs kinetics. The light-induced difference spectrum confirms X 320 to be the reactive species. From these results it is concluded that in Tris-washed chloroplasts the reaction centers of System II are characterized by a high photochemical turnover rate mediated either via rapid direct charge recombination or via fast cyclic electron flow.     

The role of the Rieske iron-sulfur center as the electron donor to ferricytochrome c2 in Rhodopseudomonas sphaeroides

The Rieske iron-sulfur center in the photosynthetic bacterium Rhodopseudomonas sphaeroides appears to be the direct electron donor to ferricytochrome c₂, reducing the cytochrome on a submillisecond timescale which is slower than the rapid phase of cytochrome oxidation ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{3–5 μ}\text{s}$). The reduction of the ferricytochrome by the Rieske center is inhibited by 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) but not by antimycin. The slower (1–2 ms) antimycin-sensitive phase of ferricytochrome c₂ reduction, attributed to a specific ubiquinone-10 molecule (Q_z), and the associated carotenoid spectral response to membrane potential formation are also inhibited by UHDBT. Since the light-induced oxidation of the Rieske center is only observed in the presence of antimycin, it seems likely that the reduced form of Q_z (Q_zH₂) reduces the Rieske center in an antimycin-sensitive reaction. From the extent of the UHDBT-sensitive ferricytochrome c₂ reduction we estimate that there are 0.7 Rieske iron-sulfur centers per reaction center.UHDBT shifts the EPR derivative absorption spectrum of the Rieske center from g_y 1.90 to g_y 1.89, and shifts the E_m,7 from 280 to 350 mV. While this latter shift may account for the subsequent failure of the iron-sulfur center to reduce ferricytochrome c₂, it is not clear how this can explain the other effects of the inhibitor, such as the prevention of cytochrome b reduction and the elimination of the uptake of H⁺_II; these may reflect additional sites of action of the inhibitor.     

The primary photoreactions in the complex cytochrome-P-890 · P-760 (bacteriopheophytin760) of Chromatium minutissimum at low redox potentials

Experimental evidence for electron transfer, photosensitized by bacteriochlorophyll, from cytochrome c to a pigment complex P-760 (involving bacteriopheophytin-760 and also bacteriochlorophyll-800) in the reaction centers of Chromatium minutissimum has been described. This photoreaction occurs between 77 and 293 °K at a redox potential of the medium between ?250 and ?530 mV. Photoreduction of P-760 is accompanied by development of a wide absorption band at 650 nm and of an EPR signal with g = 2.0025±0.0005 and linewidth of 12.5±0.5 G, which are characteristic of the pigment radical anion.It is suggested that the photoreduction of P-760 occurs under the interaction of reduced cytochrome c with the reaction center state P⁺-890 · P^?-760 which is induced by light. The existence of short-lived state P⁺-890 · P^?-760 is indicated by the recombination luminescence with activation energy of 0.12 eV and $\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{< 6}\text{ns}$. This luminescence is excited and emitted by bacteriochlorophyll and disappears when P-760 is reduced.At low redox potentials, the flash-induced absorbance changes related to the formation of the carotenoid triplet state with $\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 6 μ}\text{s}$ at 20 °C are observed. This state is not formed when P-760 is reduced at 293 and 160 °K. It is assumed that this state is formed from the reaction center state P⁺-890 · P^?-760, which appears to be a primary product of light reaction in the bacterial reaction centers and which is probably identical with the state P^F described in recent works.     

The role of the membrane-bound iron-sulphur centres A and B in the Photosystem I reaction centre of spinach chloroplasts

Photosystem I particles prepared from spinach chloroplast using Triton X-100 were frozen in the dark with the bound iron-sulphur Centre A reduced. Illumination at cryogenic temperatures of such samples demonstrated the photoreduction of the second bound iron-sulphur Centre B. Due to electron spin-electron spin interaction between these two bound iron-sulphur centres, it was not possible to quantify amounts of Centre B relative to the other components of the Photosystem I reaction centre by simulating the line-shape of its EPR spectrum. However, by deleting the free radical signal I from the EPR spectra of reduced Centre A alone or both Centres A plus B reduced, it was possible to double integrate these spectra to demonstrate that Centre B is present in the Photosystem I reaction centre in amounts comparable to those of Centre A and thus also signal I ($\text{P-700}$) and X.Oxidation-reduction potential titrations confirmed that Centre A had $\text{E}{}_{\mathrm{<mtext>m</mtext>}}\text{? ?550}\text{mV}$, Centre B had $\text{E}{}_{\mathrm{<mtext>m</mtext>}}\text{? ?585}\text{mV}$. These results, and those presented for the photoreduction of Centre B, place Centre B before Centre A in the sequence of electron transport in Photosystem I particles at cryogenic temperatures. When both A and B are reduced, $\text{P-700}$ photooxidation is reversible at low temperature and coupled to the reduction of the component X. The change from irreversible to reversible $\text{P-700}$ photooxidation and the photoreduction of X showed the same potential dependence as the reduction of Centre B with $\text{E}{}_{\mathrm{<mtext>m</mtext>}}\text{? ?585}$ mV, substantiating the identification of X as the primary electron acceptor of Photosystem I.     

On the purification of nitrite reductase from Thiobacillus denitrificans and its reaction with nitrite under reducing conditions.

Nitrite reductase (cytochrome $\text{cd}$) from $\text{T. denitrificans}$ has been crystallized in high yield in three simple and rapid steps. The spectral absorption ratio at 408 to 280 nm was 1.52. Light absorption spectra in the oxidized and reduced states were virtually identical to those of nitrite reductase from $\text{P. aeruginosa}$. EPR spectroscopy of nitrite reductase at 12° showed a low-spin ferric heme resonance with g-values at 2.52, 2.45 and 1.73 assigned to the d-heme. Reaction of nitrite reductase with nitrite in the presence of the reducing systems [(ascorbate + PMS) or sulfide] resulted in the formation of nitric oxide (confirmed by gas chromatography) which reacted with both $\text{c}$- and $\text{d}$-hemes of nitrite reductase yielding an EPR-detectable enzyme-NO complex with g-values at 2.07, 2.04 and 1.99 and a ¹⁴N hyperfine splitting constant of 22.5 gauss. The amount of nitric oxide produced enzymatically with sulfide as electron donor was only 5% of that found when ascorbate plus PMS served as reductant.To our knowledge the detection of the unique enzyme-NO complex is the first definitive EPR evidence for the mandatory liganding of nitric oxide with pure nitrite reductase during nitrite reduction.     

Effect of the transmembrane electric field on the photochemical and quenching properties of Photosystem II in vivo

The intermediate phase of fluorescence relaxation (lms-ls) (Joliot, P., Joliot, A., Bouges, B, and Barbieri, G. (1971) Photochem. Photobiol. 14, 287–305), following a single saturating flash, is shown to be controlled by a slow phase of the reoxidation of Q^? by a secondary acceptor and, in vivo, by the transmembrane electric field.The kinetics of reoxidation of Q^? are slowed by lowering the pH. This slowing effect is interpreted in terms of the reversible formation at low pH of QH which is not oxidizable by the secondary acceptor.The electric field transforms Photosystem II centers into a non-quenching photochemically inactive state that cannot be attributed to an accumulation of Q^?. Centers are unequally sensitive to the field. A critical field strength can be defined for each center above which that center is blocked and below which the center is photochemically active. The transformation from the active to inactive state occurs over a narrow range of field strength.Sensitive centers are blocked by the field in less than 1 ms and become active again in less than 10 ms as the field strength falls. Two hypotheses are proposed for the mechanism of blockage of centers by the field: (1) a field induced conformational change in the centers, (2) the formation or suppression of a dipole critical to the function of a center.The activity of the ATP synthetase, determining the rate of relaxation of the field, was controlled by a light-dark treatment or by a chemical method using $\text{p-}\text{benzoquinone}$.     

Resonance Raman scattering of bacteriochlorophyll, bacteriopheophytin and spheroidene in reaction centers of Rhodopseudomonas speroides.

We report and discuss Raman spectra of bacteriochlorophyll $\text{a}$ and of bacteriopheophytin $\text{a}$ obtained $\text{in vitro}$ by resonance effect in their Q_X and Soret electronic bands. Selective excitation of spectra of either of these molecules in reaction centers of $\text{Rhodopseudomonas spheroides}$, strains Y and R 26, was achieved by illumination in their respective Q_X bands. Preliminary interpretation of the spectra yields information about the interactions assumed by these molecules in the reaction centers. Spheroidene bound to reaction centers of strain Y probably affects a conformation different from that assumed by the bulk spheroidene of the chromatophore.     

EPR studies on a Hipip type iron-sulfur center in the succinate dehydrogenase segment of the respiratory chain

In addition to the two species of ferredoxin-type iron-sulfur centers (Centers S-1 and S-2), a Hipip-type iron-sulfur center (Center S-3) has been detected in the reconstitutively active soluble succinate dehydrogenases. E_m7,4 determined in a particulate, antimycin A sensitive succinate-cytochrome $\text{c}$ reductase is +60 ± 15 mV. This center is extremely labile towards oxygen in a manner similar to the reconstitutive activity of the dehydrogenase. Even freshly prepared reconstitutively active enzyme shows a considerably diminished content of Center S-3 relative to flavin and displays a partly modified spectra. All reconstitutively inactive dehydrogenases give rise to a highly modified or no Center S-3 spectra at all. These observations indicate that Center S-3 is a constituent of succinate dehydrogenase and plays a role in the physiological function of the enzyme, $\text{i.e.}$ transferring electrons most probably to ubiquinone.     

Properties of the primary electron acceptor complex of photosystem I in the blue green alga Chlorogloea fritschii.

Photooxidation of P700 at low temperatures in membrane fractions from the blue-green alga $\text{Chlorogloea fritschii}$ may be coupled irreversibly to the reduction of a bound ferredoxin. If this ferredoxin is reduced before freezing, P700 photooxidation at low temperatures becomes reversible. This reversible photooxidation is coupled to the reduction of a component with an EPR signal at g = 2.08, 1.88 and 1.78. A complete spectrum of this component has been obtained for the first time. We propose that as in higher plants this component is the primary electron acceptor of Photosystem I, the bound ferredoxin is a secondary electron acceptor. Using ⁵⁷Fe enriched preparations we have shown that the ERP signals attributed to the bound ferredoxin are due to iron containing centres. This experiment did not show the presence of iron in the primary electron acceptor.