Quenching of chlorophyll fluorescence in the major light-harvesting complex of photosystem II: a systematic study of the effect of carotenoid structure

The role of carotenoids in quenching of chlorophyll fluorescence in the major light-harvesting complex of photosystem II has been studied with a view to understanding the molecular basis of the control of photoprotective nonradiative energy dissipation by the xanthophyll cycle in vivo. The control of chlorophyll fluorescence quenching in the isolated complex has been investigated in terms of the number of the conjugated double bonds for a series of carotenoids ranging from n = 5-19, giving an estimated first excited singlet state energy from 20,700 cm-1 to 10,120 cm-1. At pH 7.8 the addition of exogenous carotenoids with >=10 conjugated double bonds (including zeaxanthin) stimulated fluorescence quenching relative to the control with no added carotenoid, whereas those with n 相似文献   

Localization of different photosystems in separate regions of chloroplast membranes

The stoichiometric amounts and the photoactivity kinetics of photosystem I (PSI) and of the α and β components of photosystem II (PSII_α and PSII_β) were compared in spinach chloroplast membrane (thylakoid) fractions derived from appressed and nonappressed regions. Stroma-exposed thylakoid fractions from the nonappressed regions were isolated by differential centrifugation following a mechanical press treatment of the chloroplasts. Thylakoid vesicles derived mainly from the appressed membranes of grana were isolated by the aqueous polymer two-phase partition method. Stroma-exposed thylakoids were found to have a chlorophyll a/chlorophyll b ratio of 6.0 and a PSII_β/PSI reaction center ratio of 0.3. Kinetic analysis of system II photoactivity revealed the absence of PSII_α from stroma-exposed thylakoids. The photoactivity of system I in stroma-exposed thylakoids showed a single kinetic component identical to that of unfractionated chloroplasts, suggesting that PSI does not receive excitation energy from the PSII-chlorophyll ab light-harvesting complex. Thus, stroma-exposed thylakoids are significantly enriched in both PSI and PSII_β. Inside-out vesicles from the appressed membranes of grana-partition regions had a chlorophyll a/chlorophyll b ratio of 2.0 and a PSII/PSI reaction center ratio of 10.0. The photoactivity of system II showed the membranes of the grana-partition regions to be significantly enriched in PSII_α. We conclude that PSII_α is exclusively located in the membranes of the grana partitions while PSII_β and PSI are located in stroma-exposed thylakoids. The low PSI reaction center (P700) content of vesicles derived from grana partitions and the kinetic homogeneity of the PSI complex suggest total exclusion of P700 as a functional component in the membrane of the grana-partition region.     

Magnetic field-induced increase in chlorophyll a delayed fluorescence of photosystem II: A 100- to 200-ns component between 4.2 and 300 K

At room temperature the delayed fluorescence (luminescence) of spinach chloroplasts, in which the acceptor Q is prereduced, consists of a component with a lifetime of 0.7 μs and a more rapid component, presumably with a lifetime of 100-200 ns and about the same integrated intensity as the 0.7- μs component. Between 4.2 and 200 K only a 100- to 200-ns luminescence component was found, with an integrated intensity appreciably larger than that at room temperature. At 77 K the 150-ns component approached 63% of saturation at roughly the same energy as the variable fluorescence of photosystem II at room temperature. At 77 K the emission spectra of prompt fluorescence but not that of the 150-ns luminescence had a preponderant additional band at about 735 nm. The 150-ns emission also occurred in the photosystem I-lacking mutant FL5 of Chlamydomonas. These experiments indicate that the 150-ns component originates from photosystem II. At room temperature a magnetic field of 0.22 T stimulated the 0.7-μs delayed fluorescence by about 10%. At 77 K the field-induced increase of the 150-ns component amounted to 40-50%, being responsible for the observed ≈2% increase of the total emission; the magnetic field increased the lifetime about 20%. In order to explain these phenomena a scheme for photosystem II is presented with an intermediary acceptor W between Q and the primary donor chlorophyll P-680; recombination of P-680⁺ and W^- causes the fast luminescence. The magnetic field effect on this emission is discussed in terms of the radical pair mechanism.     

Live-cell imaging of photosystem II antenna dissociation during state transitions

Plants and green algae maintain efficient photosynthesis under changing light environments by adjusting their light-harvesting capacity. It has been suggested that energy redistribution is brought about by shuttling the light-harvesting antenna complex II (LHCII) between photosystem II (PSII) and photosystem I (PSI) (state transitions), but such molecular remodeling has never been demonstrated in vivo. Here, using chlorophyll fluorescence lifetime imaging microscopy, we visualized phospho-LHCII dissociation from PSII in live cells of the green alga Chlamydomonas reinhardtii. Induction of energy redistribution in wild-type cells led to an increase in, and spreading of, a 250-ps lifetime chlorophyll fluorescence component, which was not observed in the stt7 mutant incapable of state transitions. The 250-ps component was also the dominant component in a mutant containing the light-harvesting antenna complexes but no photosystems. The appearance of the 250-ps component was accompanied by activation of LHCII phosphorylation, supporting the visualization of phospho-LHCII dissociation. Possible implications of the unbound phospho-LHCII on energy dissipation are discussed.     

Genetically engineered mutant of the cyanobacterium Synechocystis 6803 lacks the photosystem II chlorophyll-binding protein CP-47

CP-47 is absent in a genetically engineered mutant of cyanobacterium Synechocystis 6803, in which the psbB gene [encoding the chlorophyll-binding photosystem II (PSII) protein CP-47] was interrupted. Another chlorophyll-binding PSII protein, CP-43, is present in the mutant, and functionally inactive PSII-enriched particles can be isolated from mutant thylakoids. We interpret these data as indicating that the PSII core complex of the mutant still assembles in the absence of CP-47. The mutant lacks a 77 K fluorescence emission maximum at 695 nm, suggesting that the PSII reaction center is not functional. The absence of primary photochemistry was indicated by EPR and optical measurements: no chlorophyll triplet originating from charge recombination between P680⁺ and Pheo^- was observed in the mutant, and there were no flash-induced absorption changes at 820 nm attributable to chlorophyll P680 oxidation. These observations lead us to conclude that CP-47 plays an essential role in the activity of the PSII reaction center.     

Fluorescence lifetimes in the bipartite model of the photosynthetic apparatus with alpha, beta heterogeneity in photosystem II

Recent studies of the lifetime of fluorescence after picosecond pulse excitation of photosynthetic organisms revealed relatively complex decay kinetics that indicated a sum of three exponential components with lifetimes spanning the range from about 0.1-2.5 ns. These fluorescence lifetime data were examined in the context of a simple photochemical model for photosystem II that was used previously to account for fluorescence yield data obtained during continuous illumination. The model, which consists of a single fluorescing species of antenna chlorophyll and a reaction center, shows that, in general, the decay kinetics after pulse excitation should consist of the sum of two exponential decays. The model also shows that in going from open to closed reaction centers the lifetime of fluorescence may increase much more than the yield of fluorescence and surprisingly long fluorescence lifetimes can be obtained. However, conditions can be stated where fluorescence will decay essentially as a single component and with lifetime changes that are proportional to the yield changes. A heterogeneity was also introduced to distinguish photosystem II_α units, which can transfer excitation energy among themselves but not the photosystem I, and photosystem II_β units, which can transfer energy to photosystem I but not to other photosystem II units. It is proposed that the rather complex fluorescence lifetime data can be accounted for in large part by the simple photochemical model with the α, β heterogeneity in photosystem II.     

Chloroplast phosphoproteins: regulation of excitation energy transfer by phosphorylation of thylakoid membrane polypeptides.

Incubation of isolated chloroplast thylakoid membranes with [gamma-32P]ATP results in phosphorylation of surface-exposed segments of several membrane proteins. The incorporation of 32P is light dependent, is blocked by 3(3,4-dichlorophenyl)-1,1-dimethylurea (diuron, an inhibitor of electron transport), but is insensitive to uncouplers of photophosphorylation. Polypeptides of the light-harvesting chlorophyll a/b-protein complex are the major phosphorylated membrane proteins. Addition of ATP to isolated chloroplast thylakoid membranes at 20 degrees C results in a time-dependent reduction of chlorophyll fluorescence emission; this is blocked by diuron but not by nigericin. ADP could not substitute for ATP. Chlorophyll fluorescence induction transients showed a decrease in the variable component after incubation of the membranes with ATP. Chlorophyll fluorescence at 77 K of phosphorylated thylakoid membranes showed an increase in long-wavelength emission compared with dephosphorylated controls. We conclude that a membrane-bound protein kinase can phosphorylate surface-exposed segments of the light-harvesting pigment-protein complex, altering the properties of its interaction with the two photosystems such that the distribution of absorbed excitation energy increasingly favors photosystem I.     

Antenna size dependence of fluorescence decay in the core antenna of photosystem I: estimates of charge separation and energy transfer rates.

We have examined the photophysics of energy migration and trapping in photosystem I by investigating the spectral and temporal properties of the fluorescence from the core antenna chlorophylls as a function of the antenna size. Time-correlated single photon counting was used to determine the fluorescence lifetimes in the isolated P700 chlorophyll a-protein complex and in a mutant of Chlamydomonas reinhardtii that lacks the photosystem II reaction center complex. The fluorescence decay in both types of sample is dominated by a fast (15-45 psec) component that is attributed to the lifetime of excitations in the photosystem I core antenna. These excitations decay primarily by an efficient photochemical quenching on P700. The measured lifetimes show a linear relationship to the core antenna size. A linear dependence of the excitation lifetime on antenna size was predicted previously in a lattice model for excitation migration and trapping in arrays of photosynthetic pigments [Pearlstein, R.M. (1982) Photochem. Photobiol. 35, 835-844]. Based on this model, our data predict a time constant for photochemical charge separation in the photosystem I reaction center of 2.8 +/- 0.7 or 3.4 +/- 0.7 psec, assuming monomeric or dimeric P700, respectively. The predicted average single-step transfer time for excitation transfer between core antenna pigments is 0.21 +/- 0.04 psec. Under these conditions, excitation migration in photosystem I is near the diffusion limit, with each excitation making an average of 2.4 visits to the reaction center before photoconversion.     

Fluorescence lifetime snapshots reveal two rapidly reversible mechanisms of photoprotection in live cells of Chlamydomonas reinhardtii

Photosynthetic organisms avoid photodamage to photosystem II (PSII) in variable light conditions via a suite of photoprotective mechanisms called nonphotochemical quenching (NPQ), in which excess absorbed light is dissipated harmlessly. To quantify the contributions of different quenching mechanisms to NPQ, we have devised a technique to measure the changes in chlorophyll fluorescence lifetime as photosynthetic organisms adapt to varying light conditions. We applied this technique to measure the fluorescence lifetimes responsible for the predominant, rapidly reversible component of NPQ, qE, in living cells of Chlamydomonas reinhardtii. Application of high light to dark-adapted cells of C. reinhardtii led to an increase in the amplitudes of 65 ps and 305 ps chlorophyll fluorescence lifetime components that was reversed after the high light was turned off. Removal of the pH gradient across the thylakoid membrane linked the changes in the amplitudes of the two components to qE quenching. The rise times of the amplitudes of the two components were significantly different, suggesting that the changes are due to two different qE mechanisms. We tentatively suggest that the changes in the 65 ps component are due to charge-transfer quenching in the minor light-harvesting complexes and that the changes in the 305 ps component are due to aggregated light-harvesting complex II trimers that have detached from PSII. We anticipate that this technique will be useful for resolving the various mechanisms of NPQ and for quantifying the timescales associated with these mechanisms.     

Variable thermal emission and chlorophyll fluorescence in photosystem II particles.

In photosynthetic systems, the absorbed light energy is used to generate electron transport or it is lost in the form of fluorescence and thermal emission. While fluorescence can be readily measured, the detection of thermal deactivation processes can be achieved by the photoacoustic technique. In that case, the pressure wave generated by the thermal deactivations in a sample irradiated with modulated light is detected by a sensitive microphone. The relationships between the yield of fluorescence and thermal emissions measured simultaneously were analyzed by using a spinach photosystem II (PSII)-enriched preparation. It is shown that the quenching of fluorescence due to the photochemical activity of the preparations (photochemical quenching) increases in proportion to the fraction of thermal deactivations that is not immediately released as heat but is stored in photochemical intermediates (energy-storage yield) as the intensity of the photoacoustic modulated measuring beam (35 Hz) is decreased. Maximal levels of fluorescence and thermal emissions were both decreased in similar proportions upon photoreduction of pheophytin (Pheo), the primary acceptor of PSII. The variable components of fluorescence and thermal emissions were strongly decreased upon depletion of Mn from the Mn complex that catalyzes water oxidation and were recovered proportionally during reconstitution with Mn2+ at various Mn2+/reaction center ratios. Finally, depletion of Mn from the Mn complex together with the Fe of the QA-Fe-QB complex that is composed of the secondary quinone acceptors of PSII resulted in an increased initial level of fluorescence Fo and in the loss of the variable components of fluorescence and thermal emissions. The initial Fo and the variable components could be partially recovered by reconstitution of both donor and acceptor sides with Mn2+, Co2+, HCO3- and plastoquinone. It is concluded that the photochemical fluorescence quenching is correlated with a simultaneous "quenching" of a variable component of thermal emission. It is proposed that the measured component of variable thermal emission is related to the decay of the pair [P680+ Pheo-]. The suggestion is also made that a bicarbonate-induced protonation of reduced QA or QB or conformational change in the PSII complex, or both, adds an additional entropic factor to the variable thermal emission component.     

Low-temperature-induced accumulation of xanthophylls and its structural consequences in the photosynthetic membranes of the cyanobacterium Cylindrospermopsis raciborskii: an FTIR spectroscopic study

The effects of the growth temperature on the lipids and carotenoids of a filamentous cyanobacterium, Cylindrospermopsis raciborskii, were studied., The relative amounts of polyunsaturated glycerolipids and myxoxanthophylls in the thylakoid membranes increased markedly when this cyanobacterium was grown at 25 degrees C instead of 35 degrees C. Fourier transform infrared spectroscopy was used to analyze the low-temperature-induced structural alterations in the thylakoid membranes. Despite the higher amount of unsaturated lipids there, conventional analysis of the v(sym)CH(2) band (characteristic of the lipid disorder) revealed more tightly arranged fatty-acyl chains for the thylakoids in the cells grown at 25 degrees C as compared with those grown at 35 degrees C. This apparent controversy was resolved by a two-component analysis of the v(sym)CH(2) band, which demonstrated very rigid, myxoxanthophyll-related lipids in the thylakoid membranes. When this rigid component was excluded from the analysis of the thermotropic responses of the v(sym)CH(2) bands, the expected higher fatty-acyl disorder was observed for the thylakoids prepared from cells grown at 25 degrees C as compared with those grown at 35 degrees C. Both the carotenoid composition and this rigid component in the thylakoid membranes were only growth temperature-dependent; the intensity of the illuminating light during cultivation had no apparent effect on these parameters. We propose that, besides their well-known protective functions, the polar carotenoids in particular may have structural effects on the thylakoid membranes. These effects should be exerted locally--by forming protective patches, in-membrane barriers of low dynamics--to prevent the access of reactive radicals generated in either enzymatic or photosynthetic processes to sensitive spots of the membranes.     

Tripartite model for the photochemical apparatus of green plant photosynthesis.

Equations for fluorescence and the rates of photochemistry of photosystem I and photosystem II are derived from a photochemical model for the photosynthetic apparatus that includes the various interactions of the light-harvesting chlorophyll a/b complex with photosystem I and photosystem II as specific photochemical rate constants. The degree of coupling between photosystem II and the chlorophyll a/b complex which is expressed as a product of two probability terms plays a central role in this three-pigment system. The cycling of excitation energy back and forth between photosystem II and the chlorophyll a/b complex increases the exciton density in both arrays of chlorophyll according to a simple analytical expression in the equations. These equations of the tripartite model provide new and credible insights into the photochemical apparatus of photosynthesis.     

A M(r) 95,000 polypeptide in Porphyridium cruentum phycobilisomes and thylakoids: Possible function in linkage of phycobilisomes to thylakoids and in energy transfer

Two pigmented polypeptides with the same molecular weight (M_r 95,000) were isolated from the photosynthetic apparatus of Porphyridium cruentum by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A blue polypeptide from phycobilisomes had absorption and fluorescence emission spectra similar to those of allophycocyanin. A green-pigmented polypeptide from photosynthetic membranes (free of phycobilisomes) contained chlorophyll a. Several properties were common to the M_r 95,000 polypeptides from both sources: (i) identical molecular weights, (ii) identical gel electrophoresis patterns after limited protease digestion, and (iii) immunological crossreactivity with an IgG fraction directed against the M_r 95,000 polypeptide from phycobilisomes. On the basis of this evidence, a common polypeptide exists in phycobilisomes and thylakoids, and it probably anchors the phycobilisome to the thylakoid membrane. The fluorescence emission overlap of the blue and green polypeptides suggests that they are involved in the transfer of energy from phycobilisomes to thylakoids.     

Xanthophyll cycle-dependent quenching of photosystem II chlorophyll a fluorescence: formation of a quenching complex with a short fluorescence lifetime

Excess light triggers protective nonradiative dissipation of excitation energy in photosystem II through the formation of a trans-thylakoid pH gradient that in turn stimulates formation of zeaxanthin and antheraxanthin. These xanthophylls when combined with protonation of antenna pigment-protein complexes may increase nonradiative dissipation and, thus, quench chlorophyll a fluorescence. Here we measured, in parallel, the chlorophyll a fluorescence lifetime and intensity to understand the mechanism of this process. Increasing the xanthophyll concentration in the presence of a pH gradient (quenched conditions) decreases the fractional intensity of a fluorescence lifetime component centered at approximately 2 ns and increases a component at approximately 0.4 ns. Uncoupling the pH gradient (unquenched conditions) eliminates the 0.4-ns component. Changes in the xanthophyll concentration do not significantly affect the fluorescence lifetimes in either the quenched or unquenched sample conditions. However, there are differences in fluorescence lifetimes between the quenched and unquenched states that are due to pH-related, but nonxanthophyll-related, processes. Quenching of the maximal fluorescence intensity correlates with both the xanthophyll concentration and the fractional intensity of the 0.4-ns component. The unchanged fluorescence lifetimes and the proportional quenching of the maximal and dark-level fluorescence intensities indicate that the xanthophylls act on antenna, not reaction center processes. Further, the fluorescence quenching is interpreted as the combined effect of the pH gradient and xanthophyll concentration, resulting in the formation of a quenching complex with a short (approximately 0.4 ns) fluorescence lifetime.     

Energy transfer and trapping in photosystem I reaction centers from cyanobacteria.

A mutant strain of the cyanobacterium Synechocystis 6803, TolE4B, was constructed by genetic deletion of the protein that links phycobilisomes to thylakoid membranes and of the CP43 and CP47 proteins of photosystem II (PSII), leaving the photosystem I (PSI) center as the sole chromophore in the photosynthetic membranes. Both intact membrane and detergent-isolated samples of PSI were characterized by time-resolved and steady-state fluorescence methods. A decay component of approximately 25 ps dominates (99% of the amplitude) the fluorescence of the membrane sample. This result indicates that an intermediate lifetime is not associated with the intact membrane preparation and the charge separation in PSI is irreversible. The decay time of the detergent-isolated sample is similar. The 600-nm excited steady-state fluorescence spectrum displays a red fluorescence peak at approximately 703 nm at room temperature. The 450-nm excited steady-state fluorescence spectrum is dominated by a single peak around 700 nm without 680-nm "bulk" fluorescence. The experimental results were compared with several computer simulations. Assuming an antenna size of 130 chlorophyll molecules, an apparent charge separation time of approximately 1 ps is estimated. Alternatively, the kinetics could be modeled on the basis of a two-domain antenna for PSI, consistent with the available structural data, each containing approximately 65 chlorophyll a molecules. If excitation can migrate freely within each domain and communication between domains occurs only close to the reaction center, a charge separation time of 3-4 ps is obtained instead.     

Identification of chlorophyll b in extracts of prokaryotic algae by fluorescence spectroscopy.

Solvent extracts of three different prokaryotic algae from three species of didemnid ascidians contained pigments identified, on the basis of their fluorescence excitation (E)and fluorescence emission (F)spectral maxima (measured in nm) at 77K, as chlorophyll a (E 449, F 678) and chlorophyll b (E 478, F 658). The release of algae on cutting or freezing Diplosoma virens was accompanied by a strong unidentified acid that converted these pigments to pheophytins. This unexpected finding provided further confirmation of the identity of the chlorophylls on the basis of the fluorescence spectra at 77K of pheophytin a (E 415, F 669) and pheophytin b (E 439, F 655). There was no evidence for the presence of the fluorescent bilin pigments found in other prokaryotic blue-green algae. Chlorophyll a/b ratios ranged from 2.6 to 12.0 in algae from different ascidians. The photosynthetic membranes were not organized into appressed thylakoids or grana in the algae from any of the three species of ascidians. The relationship between these observations and those in higher eukaryotic organisms is discussed.     

Isolation of a photosystem II reaction center consisting of D-1 and D-2 polypeptides and cytochrome b-559

A photosystem II reaction center complex consisting of D-1 and D-2 polypeptides and cytochrome b-559 was isolated from spinach grana thylakoids, treated with 4% (wt/vol) Triton X-100, by ion-exchange chromatography using DEAE-Toyopearl 650S. The isolated complex appears to contain five chlorophyll a, two pheophytin a, one β-carotene, and one or two cytochrome b-559 heme(s) (molar ratio) and exhibits a reversible absorbance change attributable to the photochemical accumulation of reduced pheophytin typical for the intermediary electron acceptor of photosystem II reaction center. These results strongly suggest that the site of primary charge separation in photosystem II is located on the heterodimer composed of D-1 and D-2 subunits.     

Insight into the relationship of chlorophyll a fluorescence yield to the concentration of its natural quenchers in oxygenic photosynthesis

Fluorescence of chlorophyll a (Chla) is a noninvasive and very sensitive intrinsic probe of photosynthesis. It monitors the composition and organization of the photosystems, the exciton energy transfer, the photochemistry, and the effects of various types of stress on plants. It is the most used as well as the most abused tool in photosynthesis. Thus, an understanding of its relationship to photosynthesis has been of paramount importance. Both the oxidized primary plastoquinone, QA, and the oxidized primary reaction-center Chla, P680+ (for short, P+), are known to be quenchers of Chla fluorescence yield (phi f) of photosystem II. Flash-number dependence of Chla fluorescence yield shows either a period 4, due to the four-step charge-accumulation process of water oxidation (donor side), or period 2 behavior, due to the two-step reduction of the plastoquinone QB (acceptor side) of photosystem II reaction centers. We provide here a further insight into the relationship of variable Chla fluorescence yield (phi f) to the concentration of the two quenchers. The observed time dependence of the ratio of psi f after flash 3 to that after flash 1 (or flash 5) in spinach thylakoids at pH 6 can be explained if we suggest that 1/psi f approximately equals a[PQA] + c, where a, b, and c are constants. From this it follows that the quenching of Chla fluorescence by P680+ after a flash is dependent on QA: for low [QA] (when most reaction centers are closed, [PQA] is low) the quenching of Chla fluorescence by P680+ predominates, while for high [QA] (when most reaction centers are open), the quenching of Chla fluorescence is due predominantly to the increased concentration of the reduced form of P680 ([P+] is low).     

A simple artificial light-harvesting dyad as a model for excess energy dissipation in oxygenic photosynthesis

Under excess illumination, plant photosystem II dissipates excess energy through the quenching of chlorophyll fluorescence, a process known as nonphotochemical quenching. Activation of nonphotochemical quenching has been linked to the conversion of a carotenoid with a conjugation length of nine double bonds (violaxanthin) into an 11-double-bond carotenoid (zeaxanthin). It has been suggested that the increase in the conjugation length turns the carotenoid from a nonquencher into a quencher of chlorophyll singlet excited states, but unequivocal evidence is lacking. Here, we present a transient absorption spectroscopic study on a model system made up of a zinc phthalocyanine (Pc) molecule covalently linked to carotenoids with 9, 10, or 11 conjugated carbon-carbon double bonds. We show that a carotenoid can act as an acceptor of Pc excitation energy, thereby shortening its singlet excited-state lifetime. The conjugation length of the carotenoid is critical to the quenching process. Remarkably, the addition of only one double bond can turn the carotenoid from a nonquencher into a very strong quencher. By studying the solvent polarity dependence of the quenching using target analysis of the time-resolved data, we show that the quenching proceeds through energy transfer from the excited Pc to the optically forbidden S(1) state of the carotenoid, coupled to an intramolecular charge-transfer state. The mechanism for excess energy dissipation in photosystem II is discussed in view of the insights obtained on this simple model system.     

Molecular electronics of a single photosystem I reaction center: studies with scanning tunneling microscopy and spectroscopy

Thylakoids and photosystem I (PSI) reaction centers were imaged by scanning tunneling microscopy. The thylakoids were isolated from spinach chloroplasts, and PSI reaction centers were extracted from thylakoid membranes. Because thylakoids are relatively thick nonconductors, they were sputter-coated with Pd/Au before imaging. PSI photosynthetic centers and chemically platinized PSI were investigated without sputter-coating. They were mounted on flat gold substrates that had been treated with mercaptoacetic acid to help bind the proteins. With tunneling spectroscopy, the PSI centers displayed a semiconductor-like response with a band gap of 1.8 eV. Lightly platinized (platinized for 1 hr) centers displayed diode-like conduction that resulted in dramatic contrast changes between images taken with opposite bias voltages. The electronic properties of this system were stable under long-term storage.