Nitrogenase. III. Nitrogenaseless mutants of Azotobacter vinelandii: Activities,cross-reactions and epr spectra

Mutant strains of Azotobacter vinelandii that are unable to fix nitrogen were analyzed for their ability to reduce acetylene and oxidize dithionite. The activities of Components I (Fe-Mo-protein) and II (Fe-protein), the presence of antibody cross-reacting material to each of the components and the electron paramagnetic resonance (EPR) intensities at g = 3.65 also were examined in these strains. All mutant strains so far studied that are unable to reduce nitrogen, are also incapable of reducing acetylene or oxidizing dithionite. Representatives of various nitrogenaseless mutants have been characterized. Based on activity measurements they fall into three classes: those lacking both components (I^?II^?), those lacking Component I (I^?II⁺) and those lacking Component II (I⁺II^?). Many strains have extremely low levels of activity for either component, but in some of these strains, cross-reacting material is made for one or both of the components. The EPR at g = 3.65 correlates well with the activity for Component I in several of these mutant strains, but in four of the mutants there appears to be 10-20-fold higher amounts of paramagnetic center than the nitrogen-fixing activity in in vitro tests would indicate.     

The role of an iron-sulfur center and siroheme in spinach nitrite reductase.

Purified spinach nitrite reductase, a protein that contains siroheme, is characterized by absorption maxima in the visible region at 385 and 573 nm. On addition of the substrate nitrite, the bands shift to 360 and 570 nm. Dithionite also causes shifts in the maxima of the visible absorption region. Electron paramagnetic resonance studies show that the untreated enzyme contains a high-spin Fe³⁺ heme and that the addition of cyanide, an inhibitor that is competitive with nitrite, results in a spin-state change of the heme. Electron paramagnetic resonance analysis of the enzyme in the presence of dithionite or dithionite plus cyanide indicates the presence of a reduced iron-sulfur center with rhombic symmetry (g-values of 2.03, 1.94, and 1.91). In contrast, when the enzyme is treated with dithionite plus nitrite, the EPR spectrum of an NO-heme complex (g-values of 2.07 and 2.00) is observed. The presence of an iron-sulfur center has also been confirmed by chemical analyses of the nonheme iron and acid-labile sulfide in nitrite reductase. These results are discussed in terms of a mechanism for nitrite reduction that involves electron transfer between the iron-sulfur center and siroheme.     

Spectroscopic studies of cobalt-substituted ferredoxin fromClostridium pasteurianum

Summary Ferredoxin fromClostridium pasteurianum substituted with two Co atoms did not give any cobalt EPR signal at 8 K as isolated, but upon reduction with sodium dithionite, a broad signal appeared withg values that indicate highspin (S=3/2) Co(II). These signals were distinct from Co(II)-dithiothreitol signals, and disappeared upon reoxidation with air. Under anaerobic incubation of apoferredoxin with Co(II), a green derivative showed a visible spectrum typical of tetrahedral Co(Il)-thiolate coordination, which shifted dramatically upon exposure to air. The¹H-NMR spectrum of the aerobically isolated protein is reported at 300 MHz; magnetic susceptibility measurements were indicative of a diamagnetic species. These spectroscopic studies indicate that Co(II)-substituted ferredoxin is oxidized to low-spin Co(III)-ferredoxin in the presence of sulfide and oxygen. The diamagnetic Co(III) state could reversibly be reduced to highspin Co(II) by sodium dithionite.     

Purification, characterization and redox properties of hydrogenase from Methanosarcina barkeri (DSM 800)

A soluble hydrogenase from the methanogenic bacterium, Methanosarcina barkeri (DSM 800) has been purified to apparent electrophoretic homogeneity, with an overall 550-fold purification, a 45% yield and a final specific activity of 270 mumol H2 evolved min-1 (mg protein)-1. The hydrogenase has a high molecular mass of approximately equal to 800 kDa and subunits with molecular masses of approximately equal to 60 kDa. The enzyme is stable to heating at 65 degrees C and to exposure to air at 4 degrees C in the oxidized state for periods up to a week. The overall stability of this enzyme is compared with other hydrogenase isolated from strict anaerobic sulfate-reducing bacteria. Ms. barkeri hydrogenase shows an absorption spectrum typical of a non-heme iron protein with maxima at 275 nm, 380 nm and 405 nm. A flavin component, identified as FMN or riboflavin was extracted under acidic conditions and quantified to approximately one flavin molecule per subunit. In addition to this component, 8-10 iron atoms and 0.6-0.8 nickel atom were also detected per subunit. The electron paramagnetic resonance (EPR) spectrum of the native enzyme shows a rhombic signal with g values at 2.24, 2.20 and approximately equal to 2.0. probably due to nickel which is optimally measured at 40 K but still detectable at 77 K. In the reduced state, using dithionite or molecular hydrogen as reductants, at least two types of g = 1.94 EPR signals, due to iron-sulfur centers, could be detected and differentiated on the basis of power and temperature dependence. Center I has g values at 2.04, 1.90 and 1.86, while center II has g values at 2.08, 1.93 and 1.85. When the hydrogenase is reduced by hydrogen or dithionite the rhombic EPR species disappears and is replaced by other EPR-active species with g values at 2.33, 2.23, 2.12, 2.09, 2.04 and 2.00. These complex signals may represent different nickel species and are only observable at temperatures higher than 20 K. In the native preparation, at high temperatures (T greater than 35 K) or in partially reduced samples, a free radical due to the flavin moiety is observed. The EPR spectrum of reduced hydrogenase in 80% Me2SO presents an axial type of spectrum only detectable below 30 K.     

CO-binding pigments and the functional terminal oxidase of the trypanosomatid hemoflagellate Crithidia fasciculata

CO-difference absorbance spectra of both intact cells and of mitochondrial preparations isolated from Crithidia fasciculata were obtained after anaerobiosis was attained either with substrates or with dithionite. Under both sets of conditions, the CO-difference spectrum of cytochrome a₃, with difference absorbance maxima at 430 and 589 nm and minima at 443 and 612 nm, was readily identified in both the intact cells and in the mitochondria. In addition to the difference absorbance bands of cytochrome a₃-CO, three difference absorbance maxima at 417, 538 and 570 nm and a minimum at 556 nm were observed. The magnitude of the maximum at 570 nm relative to the maximum of cytochrome a₃-CO at 589 nm was less for mitochondria rendered anaerobic with substrate than for mitochondria rendered anaerobic with dithionite. This difference was taken to define operationally two groups of mitochondrial CO-binding pigments: Group I is that group observed on anaerobiosis with substrate: Group II is the additional group observed on anaerobiosis with dithionite. The Group I CO-binding pigments were virtually absent from submitochondrial particles derived by sonication, but the Group II pigments remained.Photochemical action spectra were obtained with isolated mitochondria and intact cells to ascertain if cytochrome o was present. These action spectra, obtained in CO plus O₂ atmospheres, had maxima only at 432, 550 and 588 nm, attributable to the photodissociation of cytochrome a₃-CO. Even after suppression of cytochrome a₃ activity to 10% of the normal value, no contribution of cytochrome o activity to the photochemical action spectrum was observed. Cytochrome a₃ is therefore the only functional terminal oxidase present in the mitochondria of Crithidia fasciculata.     

EPR and optical changes of the photosystem II reaction center produced by low temperature illumination

Electron paramagnetic resonance (EPR) and absorption spectroscopy have been used to study the low temperature photochemical behavior of the Photosystem II D-1/D-2/ cytochrome b559 reaction center complex. The reaction center displays large triplet state EPR signals which are attenuated after actinic illumination at low temperatures in the presence of sodium dithionite. Concomitant with the triplet attenuation is the buildup of a structured radical signal with an effective g value of 2.0046 and a peak-to-peak width of 11.9 G. The structure in the signal is suggestive of it being comprised in part of the anion radical of pheophytin a. This assignment is corroborated by low temperature optical absorbance measurements carried out after actinic illumination at the low temperatures which show absorption bleachings at 681 nm, 544 nm and 422 nm and an absorbance buildup at 446 nm indicating the formation of reduced pheophytin.Abbreviations EPR electron paramagnetic resonance     

UV optical difference spectrum associated with the reduction of electron acceptor A1 in photosystem I of higher plants

Photosystem I particles containing I P700 per 32 chlorophyll molecules were illuminated at cryogenic temperatures in the presence of sodium dithionite. Under conditions which specifically led to reduction of acceptor a₁ (as shown by its characteristic EPR spectrum) optical absorbance changes were detected between 240 and 325 nm. The appearance of these changes correlated closely with the increase in amplitude of the ai EPR signal. The possibility that a quinone-like species is associated with, or directly involved in intermediary PS I electron flow is discussed.     

Octahedral metal coordination in the active site of glyoxalase I as evidenced by the properties of Co(II)-glyoxalase I

Co(II)-glyoxalase I has been prepared by reactivation of apoenzyme from human erythrocytes with Co2+. The visible absorption spectrum showed maxima at 493 and 515 nm and shoulders at 465 and 615 nm. The absorption coefficients at 493 and 515 nm were 35 and 33 M-1 cm-1/cobalt ion, respectively; i.e. 70 and 66 M-1 cm-1 for the dimeric metalloprotein. The product of the enzymatic reaction, S-D-lactoylglutathione, although binding to Co(II)-glyoxalase I, had no demonstrable effect on the visible absorption spectrum, indicating binding outside the first coordination sphere of the metal. The EPR spectrum at 3.9 K was characterized by g1 approximately 6.6, g2 approximately 3.0, and g3 approximately 2.5, and eight hyperfine lines with A1 = 0.025 cm-1. Binding of the strong competitive inhibitor S-p-bromobenzylglutathione to Co(II)-glyoxalase I gave three g values: 6.3, 3.4, and 2.5, indicating a conformational change affecting the environment of the metal ion. Both optical and EPR spectra strongly suggest a high spin Co2+ with octahedral coordination in the active site of the enzyme. The similarities in kinetic properties between native Zn(II)-glyoxalase I and enzyme substituted with Mg2+, Mn2+, or Co2+ is consistent with the view that these enzyme forms have the same metal coordination in the protein.     

The photolytic release of copper from the copper penicillamine complex [Cu(II)6Cu(I)8(D-penicillamine)12Cl]5−

The light sensitivity of CuPen ([Cu(II)₆Cu(I)₈(D-penicillamine)₁₂Cl]⁵⁻ was examined. The wavelength range of the photolytic activity was determined in the visible and near-ultraviolet region of the electromagnetic spectrum. No photolytic activity was observed above 450 nm. The quantum yield of copper release was measured between 450 nm and 250 nm and was found to increase from 0 to 0.1. A shoulder around 400 nm corresponding to electronic absorption and CD features was observed in the photo-action spectrum. Inhibition of formazan formation from nitroblue tetrazolium mediated by xanthine oxidase-generated superoxide was used to quantify the copper release as a result of the photolytic decomposition of CuPen. The influence of oxygen on the photolytic reaction was investigated by EPR and electronic absorption spectroscopy. In the absence of oxygen, visible light induces almost total bleaching. However, EPR reveals only slight changes in the spin concentration. Upon introduction of aerobic EDTA all of the copper is immediately oxidised to Cu(II).     

Light-induced red shift of the Qx absorption band of light-harvesting bacteriochlorophyll in Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides

In chromatophores from Rhodopseudomonas sphaeroides and Rhodopseudomonas capsulata, the Q_x band(s) of the light-harvesting bacteriochlorophyll (BChl) (λ_max ~590 nm) shifts to the red in response to a light-induced membrane potential, as indicated by the characteristics of the light-minus-dark difference spectrum. In green strains, containing light-harvesting complexes I and II, and one or more of neurosporene, methoxyneurosporene, and hydroxyneurosporene as carotenoids, the absorption changes due to the BChl and carotenoid responses to membrane potential in the spectral region 540–610 nm are comparable in magnitude and overlap with cytochrome and reaction center absorption changes in coupled chromatophores. In strains lacking carotenoid and light-harvesting complex II, the BChl shift absorption change is relatively smaller, due in part to the lower BChl/reaction center ratio.In the carotenoid-containing strains, the peak-to-trough absorption change in the BChl difference spectrum is 5–8% of the peak-to-trough change due to the shift of the longest-wavelength carotenoid band, although the absorption of the BChl band is 25–40% of that of the carotenoid band. The responding BChl band(s) does not appear to be significantly red-shifted in the dark in comparison to the total BChl Q_x band absorption.     

Defining the Far-red Limit of Photosystem I: THE PRIMARY CHARGE SEPARATION IS FUNCTIONAL TO 840 nm*

The far-red limit of photosystem I (PS I) photochemistry was studied by EPR spectroscopy using laser flashes between 730 and 850 nm. In manganese-depleted spinach thylakoid membranes, the primary donor in PS I, P₇₀₀, was oxidized simultaneously with tyrosine Z, the secondary donor in PS II. It was found that at 295 K PS I photochemistry, observed as P₇₀₀⁺ formation, was functional up to 840 nm. This is 30 nm further to the red region than was reported for PS II photochemistry (Thapper, A., Mamedov, F., Mokvist, F., Hammarström, L., and Styring, S. (2009) Plant Cell 21, 2391–2401). The same far-red limit for the P₇₀₀⁺ formation was observed in a PS I reaction center core preparation from Nostoc punctiforme. The reduction of the acceptor side of PS I, observed as reduction of the iron-sulfur centers F_A and F_B by low temperature EPR measurements, was also functional at 15 K with light up to >830 nm. Taken together, these results, obtained from both plants and cyanobacteria, most likely rule out involvement of the red-absorbing antenna chlorophylls in this reaction. Instead we propose the existence of weak charge transfer bands absorbing in the far-red region in the ensemble of excitonically coupled chlorophyll a molecules around P₇₀₀ similar to what has been found in the reaction center of PS II. These charge transfer bands could be responsible for the far-red light absorption leading to PS I photochemistry at wavelengths up to 840 nm.     

Evidence that the intermediate electron acceptor,A2, in Photosystem I is a bound iron-sulfur protein

Absorption changes accompanying the formation of light-induced P-700⁺ were investigated in a highly enriched Photosystem I preparation where an intermediate electron acceptor preceding P-430 could be detected. In an enriched Photosystem I particle, light-induced reversible absorption changes observed at 700 nm in the presence of dithionite resembled those previously seen at 703 nm and 820 nm [9], thus indicating the presence of a backreaction between P-700⁺ and A^?₂. After this same Photosystem I particle was treated to denature the bound iron-sulfur centers, the photochemical changes that could be attributed to P-700 A₂ were completely lost. These results provide evidence that the intermediate electron acceptor, A₂, is a bound iron-sulfur protein. Additional studies in the 400–500 nm region with Photosystem I particles prepared by sonication indicate that the spectrum of A₂ is different from that of P-430.     

Regulation of Nitrogenase Synthesis by Oxygen in Klebsiella pneumoniae

Klebsiella pneumoniae does not fix N₂ under aerobic conditions. The two protein components required for nitrogenase activity were studied during aeration of cells in nitrogen-free media. Component II of nitrogenase was inactivated more slowly in vivo than component I during aeration. The rate of loss of component II was less than the rate of component II synthesis during derepression. No inactive components were detected in cells that had been growing on NH₄⁺ and then aerated in nitrogen-free medium. This supports the hypothesis that O₂ somehow represses the formation of nitrogenase.     

Chlorophyll composition of Photosystems IIα, IIβ and I in tobacco chloroplasts

The antenna composition of the Photosystems II_α, II_β and I was studied in tobacco chloroplasts. Absorbance spectra, recorded at 4 K, were analyzed for the wild type and the mutants Su/su and Su/su var. Aurea, containing higher concentrations of the photosystems. With chloroplasts of Su/su we measured the action spectra of the three photosystems from 625 to 690 nm. Above 675 nm absorption by Photosystem I dominated. This sytem had a maximum at 678 nm and a shoulder at 660 nm. Of the long-wavelength chlorophyll a forms, absorbing at 690, 697 and 705 nm at 4 K, which are generally assigned to Photosystem I, the 697 nm form occurred in an amount of four molecules per reaction center of Photosystem I in each type of chloroplast. The Photosystem II_α spectrum was characterized by maxima at 650 and 672 nm, showing clearly the participation of the chlorophyll a and b containing light-harvesting complex. In the mutants the light-harvesting complex has a chlorophyll a to chlorophyll b ratio of more than 1; the amount of the 672 nm chlorophyll a was normal, whereas the amount of chlorophyll b was markedly decreased in the mutants relative to the wild type. The Photosystem II_β spectrum mainly consisted of a band at 683 nm.     

Orientation of pigments in the thylakoid membrane and in the isolated chlorophyll-protein complexes of higher plants. II. Linear dichroism spectra of isolated pigment-protein complexes oriented in polyacrylamide gels at 300 and 100 K

The absorption and linear dichroism (LD) spectra (380–780 nm) of isolated light-harvesting complex (LHC), Photosystem I (PS I), Photosystem II (PS II), as well as intact thylakoids have been determined at 300 and 100 K. The samples were oriented in squeezed polyacrylamide gel. The low-temperature spectra of LHC and PS I present LD signals which are characteristic enough to be recognized in the LD spectrum of thylakoids. Tentative assignments of the various features of the LD spectra to the major photosynthetic pigments are discussed. A shoulder in the low-temperature absorption spectra is observed at about 673 nm in all the systems under investigation. The absence of an associated LD signal suggests that this ubiquitous chlorophyll (Chl) a form is non-dichroic. Furthermore, in the three isolated chlorophyll-protein complexes described in this study the sign of the LD signal indicates that both the Q_y transition of the Chl a and the carotenoid molecules are preferentially oriented parallel to the largest dimension(s) of the particles.     

Time-resolved picosecond fluorescence spectra of the antenna chlorophylls in Chlorella vulgaris. Resolution of Photosystem I fluorescence

The time-resolved fluorescence emission and excitation spectra of Chlorella vulgaris cells have been measured by single-photon timing with picosecond resolution. In a three-exponential analysis the time-resolved excitation spectra recorded at 685 and 706 nm emission wavelength with closed PS II reaction centers show large variations of the preexponential factors of the different decay components as a function of wavelength. At λ_em = 685 nm the major contribution to the fluorescence decay originates from two components with life-times of 2.1–2.4 and 1.2–1.3 ns. A short-lived component with life-times of 0.1–0.16 ns of relatively small amplitude is also found. When the emission is detected at 706 nm, the short-lived component with a life-time of less than 0.1 ns predominates. Time-resolved emission spectra using λ_exc = 630 or λ_exc = 652 nm show a spectral peak of the two longer-lived components at about 680–685 nm, whereas the fast component is red-shifted as compared to the others and shows a maximum at about 690 nm. The emission spectrum observed upon excitation at 696 nm with closed PS II reaction centers shows a large increase in the amplitude of the fast component with a lifetime of 80–100 ps as compared to that at 630 nm excitation. At almost open Photosystem II (PS II) reaction centers (F₀), the life-time of the fast component decreased from 150–160 ps at 682 nm to less than 100 ps at 720 nm emission wavelength. We conclude that at least two pigment pools contribute to the fast component. One is attributed to PS II and the other to Photosystem I (PS I). They have life-times of approx. 180 ps and 80 ps, respectively. The 80 ps (PS I) contribution has a spectral maximum slightly below 700 nm, whereas the 180 ps (PS II) spectrum peaks at 680–685 nm. The spectra of the middle decay component τ_m and its sensitivity to inhibitors of PS II suggest that this component is not preferentially related to LHC II but arises mainly from Chl a pigments probably associated with a second type of PS II centers. The amplitudes of the fast (180 ps, PS II) component and the long-lived decay show an opposite dependence on the state of the PS II centers and confirm our earlier conclusion that the contribution of PS II to the fast component probably disappears at the F_max state (Haehnel W., Holzwarth, A.R. and Wendler, J. (1983) Photochem. Photobiol. 34, 435–443). Our data are discussed in terms of α,β-heterogeneity in PS II centers.     

Evidence for manganese(III) binding to the mangano superoxide dismutase from a higher plant (Pisum sativum L.)

Homogenous preparations of a manganese superoxide dismutase from a higher plant (Pisum sativum L.) were studied by epr and optical spectroscopies. The visible spectrum of manganese superoxide dismutase shows a weak and broad band in the range 350–700 nm with two shoulders at about 480 and 600 nm. Reduction with dithionite brought about a considerable disappearance of the visible component of the spectrum. The epr spectra of the native and dithionite-treated enzyme did not show any signal attributable to Mn(II) that only was visible after acid hydrolysis of the protein. The lack of epr signal both in the native and reduced superoxide dismutase can be attributed to the presence of Mn(III) in the former and of Mn(II) strongly bound to the protein in the latter. The results obtained with the manganese superoxide dismutase from leaves of the higher plant Pisum sativum are consistent with the general catalytic mechanism of action postulated for superoxide dismutases from other sources studied so far.     

Kinetic studies on cytochrome c oxidase by combined EPR and reflectance spectroscopy after rapid freezing

1. Techniques and experiments are described concerned with the millisecond kinetics of EPR-detectable changes brought about in cytochrome c oxidase by reduced cytochrome c and, after reduction with various agents, by reoxidation with O₂ or ferricyanide. Some experiments in the presence of ligands are also reported. Light absorption was monitored by low-temperature reflectance spectroscopy.2. In the rapid phase of reduction of cytochrome c oxidase by cytochrome c (< 50 ms) approx. 0.5 electron equivalent per hame a is transferred mainly to the low-spin heme component of cytochrome c oxidase and partly to the EPR-detectable copper. In a slow phase (> 1 s) the copper is reoxidized and high-spin ferric heme signals appear with a predominant rhombic component. Simultaneously the absorption band at 655 nm decreases and the Soret band at 444 nm appears between the split Soret band (442 and 447 nm) of reduced cytochrome a.3. On reoxidation of reduced enzyme by oxygen all EPR and optical features are restored within 6 ms. On reoxidation by O₂ in the presence of an excess of reduced cytochrome c, states can be observed where the low-spin heme and copper signals are largely absent but the absorption at 655 nm is maximal, indicating that the low-spin heme and copper components are at the substrate side and the component(s) represented in the 655 nm absorption at the O₂ side of the system. On reoxidation with ferricyanide the 655 nm absorption is not readily restored but a ferric high-spin heme, represented by a strong rhombic signal, accumulates.4. On reoxidation of partly reduced enzyme by oxygen, the rhombic high-spin signals disappear within 6 ms, whereas the axial signals disappear more slowly, indicating that these species are not in rapid equilibrium. Similar observations are made when partly reduced enzyme is mixed with CO.5. The results of this and the accompanying paper are discussed and on this basis an assignment of the major EPR signals and of the 655 nm absorption is proposed, which in essence is that published previously (Hartzell, C. R., Hansen, R. E. and Beinert, H. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2477–2481). Both the low-spin (g = 3; 2.2; 1.5) and slowly appearing high-spin (g = 6; 2) signals are attributed to ferric cytochrome a, whereas the 655 nm absorption is thought to arise from ferric cytochrome a₃, when it is present in a state of interaction with EPR-undetectable copper. Alternative possibilities and possible inconsistencies with this proposal are discussed.     

Spectral study of ascorbate oxidase

The electronic, CD and EPR spectra of ascorbate oxidase isolated from the green zucchini squash (Cucurbita pepo medullosa) in 0.1 M phosphate buffer (pH 6.8) have been investigated. The visible absorption bands are clearly resolved in the CD spectrum, where the extrema occur at 735, 610, 550, 475 and 330 nm, while weak additional CD activity possibly occurs near 420 nm. The near-UV spectrum is dominated by the absorption of the aromatic amino acid residues centered at 280 nm, while resolved CD bands occur at 296, 291, 283, 265 and 240 nm. In the far-UV region the protein CD spectrum reflects its secondary structure: a single negative maximum at 218 nm suggests a predominant anti-parallel β conformation for ascorbate oxidase. The frozen solution EPR spectrum of the protein has been fitted according to a new computer simulation procedure. The following parameters were obtained: for the type 1 copper g_z = 2.222, g_x = 2.032, g_y = 2.056, A_z = 59 G, A_x = 11 G, and A_y = 5 G; for the type 2 copper g ? = 2.240, g_⊥ = 2.057, A? = 179 G and A_⊥ = 1 G. Of the eight copper atoms present in the protein four are EPR-detectable: three of type 1 and one of type 2, as shown by computer simulation of the EPR spectrum. Ascorbate oxidase is a rather unstable protein when purified and it is sensitive to a number of environmental factors. Aging of the protein leads to a decrease in the ratio between the type 1 and type 2 coppers. A new species formed at the early stages of the aging process, that has been spectrally characterized, suggests that the loss of the type 1 copper is preceded by a change in the symmetry of the original type 1 site from pseudotetrahedral to pseudotetragonal.     

Probing the kinetics of Photosystem I and Photosystem II fluorescence in pea chloroplasts on a picosecond pulse fluorometer

Picosecond fluorescence kinetics of pea chloroplasts have been investigated at room temperature using a pulse fluorometer with a resolution time of 10^?11 s. Fluorescence has been excited by both a ruby and neodymium-glass mode-locked laser and has been recorded within the 650 to 800 nm spectral region.We have found three-component kinetics of fluorescence from pea chloroplasts with lifetimes of 80, 300 and 4500 ps, respectively. The observed time dependency of the fluorescence of different components on the functional state of the photosynthetic mechanism as well as their spectra enabled us to conclude that Photosystem I fluoresces with a lifetime of 80 ps (τ_I) and Photosystem II fluoresces with a lifetime of 300 ps (τ_II). Fluorescence with a lifetime of 4500 ps (τ_III) may be interpreted as originating from chlorophyll monomeric forms which are not involved in photosynthesis.It was determined that the rise time of Photosystem I and Photosystem II fluorescence after 530 nm photoexcitation is 200 ps, which corresponds to the time of energy migration to them from carotenoids.