Effect of low temperatures on photochemical activity of PS1 reaction centers from <Emphasis Type="Italic">Synechocystis</Emphasis> sp. frozen under illumination

After cooling of Synechocystis sp. photosystem 1 (PS1) reaction centers (RC) to 160 K under illumination most of the photoactive pigment is fixed for a long time in the oxidized state. The same effect is observed in purple bacteria RC. The dark reduction kinetics of PS1 P700 chlorophyll, which still retains its photochemical activity, in these samples was similar to that in samples cooled in the dark. We suggest that the photoinduced charge separation in PS1 RC, as well as in purple bacteria RC, is accompanied by conformational changes that can be fixed in samples cooled under illumination. As a result, the electrons photomobilized in RC cooled under illumination are unable to return backward the process of electron transfer to P700(+) after cessation of actinic illumination. Such irreversible trapping of electrons can take place in different parts of the PS1 RC electron acceptor chain.     

Probing light-induced conformational transitions in bacterial photosynthetic reaction centers embedded in trehalose-water amorphous matrices

The coupling between electron transfer and protein dynamics has been studied in photosynthetic reaction centers (RC) from Rhodobacter sphaeroides by embedding the protein into room temperature solid trehalose-water matrices. Electron transfer kinetics from the primary quinone acceptor (Q(A)(-)) to the photoxidized donor (P(+)) were measured as a function of the duration of photoexcitation from 20 ns (laser flash) to more than 1 min. Decreasing the water content of the matrix down to approximately 5x10(3) water molecules per RC causes a reversible four-times acceleration of P(+)Q(A)(-) recombination after the laser pulse. By comparing the broadly distributed kinetics observed under these conditions with the ones measured in glycerol-water mixtures at cryogenic temperatures, we conclude that RC relaxation from the dark-adapted to the light-adapted state and thermal fluctuations among conformational substates are hindered in the room temperature matrix over the time scale of tens of milliseconds. When the duration of photoexcitation is increased from a few milliseconds to the second time scale, recombination kinetics of P(+)Q(A)(-) slows down progressively and becomes less distributed, indicating that even in the driest matrices, during continuous illumination, the RC is gaining a limited conformational freedom that results in partial stabilization of P(+)Q(A)(-). This behavior is consistent with a tight structural and dynamical coupling between the protein surface and the trehalose-water matrix.     

ESR signal of the iron-sulfur center F(X) and its function in the homodimeric reaction center of Heliobacterium modesticaldum

Electron transfer in the membranes and the type I reaction center (RC) core protein complex isolated from Heliobacterium modesticaldum was studied by optical and ESR spectroscopy. The RC is a homodimer of PshA proteins. In the isolated membranes, illumination at 14 K led to accumulation of a stable ESR signal of the reduced iron-sulfur center F(B)(-) in the presence of dithiothreitol, and an additional 20 min illumination at 230 K induced the spin-interacting F(A)(-)/F(B)(-) signal at 14 K. During illumination at 5 K in the presence of dithionite, we detected a new transient signal with the following values: g(z)= 2.040, g(y)= 1.911, and g(x)= 1.896. The signal decayed rapidly with a 10 ms time constant after the flash excitation at 5 K and was attributed to the F(X)(-)-type center, although the signal shape was more symmetrical than that of F(X)(-) in photosystem I. In the purified RC core protein, laser excitation induced the absorption change of a special pair, P800. The flash-induced P800(+) signal recovered with a fast 2-5 ms time constant below 150 K, suggesting charge recombination with F(X)(-). Partial destruction of the RC core protein complex by a brief exposure to air increased the level of the P800(+)A(0)(-) state that gave a lifetime (t(1/2)) of 100 ns at 77 K. The reactions of F(X) and quinone were discussed on the basis of the three-dimensional structural model of RC that predicts the conserved F(X)-binding site and the quinone-binding site, which is more hydrophilic than that in the photosystem I RC.     

Studies of Electron Transport Dynamics in Photosynthetic Reaction Centers Using Fast Temperature Changes

Rates of thermoinduced conformational transitions of reaction center (RC) complexes providing effective electron transport were studied in chromatophores and isolated RC preparations of various photosynthesizing purple bacteria using methods of fast freezing and laser-induced temperature jump. Reactions of electron transfer from the primary to secondary quinone acceptors and from the multiheme cytochrome c subunit to photoactive bacteriochlorophyll dimer were used as probes of electron transport efficiency. The thermoinduced transition of the acceptor complex to the conformational state facilitating electron transfer to the secondary quinone acceptor was studied. It was shown that neither the characteristic time of the thermoinduced transition within the temperature range 233-253 K nor the characteristic time of spontaneous decay of this state at 253 K exceeded several tens of milliseconds. In contrast to the quinone complex, the thermoinduced transition of the macromolecular RC complex to the state providing effective electron transport from the multiheme cytochrome c to the photoactive bacteriochlorophyll dimer within the temperature range 220-280 K accounts for tens of seconds. This transition is thought to be mediated by large-scale conformational dynamics of the macromolecular RC complex.     

An electron spin-polarized signal of the P800+A1(Q)- state in the homodimeric reaction center core complex of Heliobacterium modesticaldum

The function of menaquinone as electron acceptor A 1 was identified by EPR in the purified type 1 homodimeric reaction center core complex (RC core) of an anoxygenic photosynthetic bacterium, Heliobacterium modesticaldum. After illumination of the RC core at 210 K in the absence and presence of dithionite, we detected the radical of a special pair of bacteriochlorophyll g molecules (P800 (+)) at g = 2.0033 and a quinone-type radical at g = 2.0062, respectively, at 14 K. Flash excitation of the dark-frozen RC core at 14 K induced two types of transient EPR signals, i.e., the P800 (+) radical that decayed with a time constant of 3.7 ms and a much faster decay component that showed the electron spin polarization (ESP) pattern of E/A (E, emission; A, absorption). The latter one was assigned to the P800 (+)F X (-) radical pair state. A new ESP signal that had an apparent A/E/A/E pattern extended to the lower-magnetic-field side was transiently induced by the flash excitation in the RC core that was preilluminated at 210 K in the presence of ascorbate and subsequently cooled to 14 K in the light. The 210 K preillumination of the RC core in the presence of dithionite led to accumulation of the dark stable semiquinone-type signal at g = 2.0062 and increased the intensity of the light-induced P800 triplet signal. Flash excitation at 14 K induced the smaller A/E/A/E-type signal that had the greater contribution of the lower-magnetic-field envelope. This ESP signal could thus be ascribed to the P800 (+)A 1 (-) radical pair. The kinetics and spectral shape of this ESP signal suggest that menaquinone serves as secondary electron acceptor A 1 with the molecular orientation of its ring being somewhat different from that of phylloquinone in photosystem I.     

Electron transport dynamics at the quinone acceptor site of bacterial photosynthetic reaction centers as probed using fast temperature changes

Methods of laser-induced temperature jumps and fast freezing were used for testing the rates of thermoinduced conformational transitions of reaction center (RC) complexes in chromatophores and isolated RC preparations of various photosynthesizing purple bacteria. An electron transfer reaction from primary to secondary quinone acceptors was used as a probe of electron transport efficiency. The thermoinduced transition of the acceptor complex to the conformational state facilitating electron transfer to the secondary quinone acceptor was studied. To investigate the dynamics of spontaneous decay of the RC state induced by the thermal pulse, the thermal pulse was applied either before or during photoinduced activation of electron transport reactions in the RC acceptor complex. The maximum effect was observed if the thermal pulse was applied against the background of steady-state photoactivation of the RC. It was shown that neither the characteristic time of the thermoinduced transition within the temperature range 233-253 K nor the characteristic time of spontaneous decay of this state at 253 K exceeded several tens of milliseconds. Independent support of the estimates was obtained from experiments with varied cooling rates of the samples tested.     

Temperature dependence of protein globule polarization and electron transport in preparations of photosynthetic reaction centers from Rhodopseudomonas sphaeroides

Differential "light-minus-dark" spectra were obtained for reaction center (RC) preparations cooled in the light and in the dark at 77 K. The two types of preparations were found to display different spectral features in the spectral regions 760-770, 790-810, 880-990 nm. Differences in the spectra of the two types were found to exist in preparations cooled to temperatures below 120-100 K, whereas at temperatures above 130 K such differences were not observed. The observed spectral changes may be associated with the polarization processes occurring in the RC globule. Samples cooled in the light and in the dark show different temperature dependencies of the efficiency of electron transfer to the secondary quinone acceptor. The differences are irreversible after cooling to temperatures below 170 K, and reversible after cooling to 180-200 K. It is postulated that the observed kinetic changes are reflections of changes in the configuration of the acceptor complex of the RC. The possible existence of a correlation between the polarization processes in the protein globule and the structural configurations of the RC is discussed.     

Orientated binding of photosynthetic reaction centers on gold using Ni-NTA self-assembled monolayers

Coupling of photosynthetic reaction centers (RCs) with inorganic surfaces is attractive for the identification of the mechanisms of interprotein electron transfer (ET) and for possible applications in construction of photo- and chemosensors. Here we show that RCs from Rhodobacter sphaeroides can be immobilized on gold surfaces with the RC primary donor looking towards the substrate by using a genetically engineered poly-histidine tag (His₇) at the C-terminal end of the M-subunit and a Ni---NTA terminated self-assembled monolayer (SAM). In the presence of an electron acceptor, ubiquinone-10, illumination of this RC electrode generates a cathodic photocurrent. The action spectrum of the photocurrent coincides with the absorption spectrum of RC and the photocurrent decreases in response to the herbicide, atrazine, confirming that the RC is the primary source of the photoresponse. Disruption of the Ni---NTA---RC bond by imidazole leads to about 80% reduction of the photocurrent indicating that most of the photoactive protein is specifically bound to the electrode through the linker.     

Temperature dependence of biphasic forward electron transfer from the phylloquinone(s) A1 in photosystem I: only the slower phase is activated

The temperature dependence of the biphasic electron transfer (ET) from the secondary acceptor A1 (phylloquinone) to iron-sulfur cluster F(X) was investigated by flash absorption spectroscopy in photosystem I (PS I) isolated from Synechocystis sp. PCC 6803. While the slower phase (tau=340 ns at 295 K) slowed upon cooling according to an activation energy of 110 meV, the time constant of the faster phase (tau=11 ns at 295 K) was virtually independent of temperature. Following a suggestion in the literature that the two phases arise from bidirectional ET involving two symmetrically arranged phylloquinones, Q(K)-A and Q(K)-B, it is concluded that energetic parameters (most likely the driving forces) rather than the electronic couplings are different for ET from Q(K)-A to F(X) and from Q(K)-B to F(X). Two alternative schemes of ET in PS I are presented and discussed.     

Low-temperature electron transfer in photosystem II: a tyrosyl radical and semiquinone charge pair

The states induced by illumination at 7 K in the oxygen-evolving enzyme (PSII) from Thermosynechococcus elongatus were studied by EPR. In the S(0) and S(1) redox states, two g approximately 2 EPR signals, a split signal and a g = 2.03 signal, respectively, were generated by illumination with visible light. These signals were comparable to those already reported in plant PSII in terms of their g value, shape, and stability at low temperatures. We report that the formation and decay of these signals correlate with EPR signals from the semiquinone of the first quinone electron acceptor, Q(A)(-). The light-induced EPR signals from oxidized side-path electron donors (Cyt b(559), Car, and Chl(Z)) were also measured, and from these and the signals from Q(A)(-), estimates were made of the proportion of centers involved in the formation of the g approximately 2 signals (approximately 50% in S(0) and 40% in S(1)). Comparisons with the signals generated in plant PSII indicated approximately similar yields for the S(0) split signal. A single laser flash at 7 K induced more than 75% of the maximum split and g = 2.03 EPR signal observed by continuous illumination, with no detectable oxidation of side-path donors. The matching electron acceptor side reactions, the high quantum yield, and the relatively large proportion of centers involved support earlier suggestions that the state being monitored is Tyr(Z)(*)Q(A)(-), with the g approximately 2 EPR signals arising from Tyr(Z)(*) interacting magnetically with the Mn complex. The current picture of the photochemical reactions occurring in PSII at low temperatures is reassessed.     

An intermediate state of the gamma-aminobutyric acid transporter GAT1 revealed by simultaneous voltage clamp and fluorescence

The rat gamma-aminobutyric acid transporter GAT1 expressed in Xenopus oocytes was labeled at Cys74, and at one or more other sites, by tetramethylrhodamine-5-maleimide, without significantly altering GAT1 function. Voltage-jump relaxation analysis showed that fluorescence increased slightly and monotonically with hyperpolarization; the fluorescence at -140 mV was approximately 0. 8% greater than at +60 mV. The time course of the fluorescence relaxations was mostly described by a single exponential with voltage-dependent but history-independent time constants ranging from approximately 20 ms at +60 mV to approximately 150 ms at -140 mV. The fluorescence did not saturate at the most negative potentials tested, and the midpoint of the fluorescence-voltage relation was at least 50 mV more negative than the midpoint of the charge-voltage relation previously identified with Na(+) binding to GAT1. The presence of gamma-aminobutyric acid did not noticeably affect the fluorescence waveforms. The fluorescence signal depended on Na(+) concentration with a Hill coefficient approaching 2. Increasing Cl(-) concentration modestly increased and accelerated the fluorescence relaxations for hyperpolarizing jumps. The fluorescence change was blocked by the GAT1 inhibitor, NO-711. For the W68L mutant of GAT1, the fluorescence relaxations occurred only during jumps to high positive potentials, in agreement with previous suggestions that this mutant is trapped in one conformational state except at these potentials. These observations suggest that the fluorescence signals monitor a novel state of GAT1, intermediate between the E*(out) and E(out) states of Hilgemann, D.W., and C.-C. Lu (1999. J. Gen. Physiol. 114:459-476). Therefore, the study provides verification that conformational changes occur during GAT1 function.     

Metabolism of adrenic acid to vasodilatory 1alpha,1beta-dihomo-epoxyeicosatrienoic acids by bovine coronary arteries

Adrenic acid (docosatetraenoic acid), an abundant fatty acid in the vasculature, is produced by a two-carbon chain elongation of arachidonic acid. Despite its abundance and similarity to arachidonic acid, little is known about its role in the regulation of vascular tone. Gas chromatography/mass spectrometric analysis of bovine coronary artery and endothelial cell lysates revealed arachidonic acid concentrations of 2.06 +/- 0.01 and 6.18 +/- 0.60 microg/mg protein and adrenic acid concentrations of 0.29 +/- 0.01 and 1.56 +/- 0.16 microg/mg protein, respectively. In bovine coronary arterial rings preconstricted with the thromboxane mimetic U-46619, adrenic acid (10(-9)-10(-5) M) induced concentration-related relaxations (maximal relaxation = 83 +/- 4%) that were similar to arachidonic acid relaxations. Adrenic acid relaxations were blocked by endothelium removal and the K(+) channel inhibitor, iberiotoxin (100 nM), and inhibited by the cyclooxygenase inhibitor, indomethacin (10 microM, maximal relaxation = 53 +/- 4%), and the cytochrome P-450 inhibitor, miconazole (10 microM, maximal relaxation = 52 +/- 5%). Reverse-phase HPLC and liquid chromatography/mass spectrometry isolated and identified numerous adrenic acid metabolites from coronary arteries including dihomo (DH)-epoxyeicosatrienoic acids (EETs) and DH-prostaglandins. DH-EET [16,17-, 13,14-, 10,11-, and 7,8- (10(-9)-10(-5) M)] induced similar concentration-related relaxations (maximal relaxations averaged 83 +/- 3%). Adrenic acid (10(-6) M) and DH-16,17-EET (10(-6) M) hyperpolarized coronary arterial smooth muscle. DH-16,17-EET (10(-8)-10(-6) M) activated iberiotoxin-sensitive, whole cell K(+) currents of isolated smooth muscle cells. Thus, in bovine coronary arteries, adrenic acid causes endothelium-dependent relaxations that are mediated by cyclooxygenase and cytochrome P-450 metabolites. The adrenic acid metabolite, DH-16,17-EET, activates smooth muscle K(+) channels to cause hyperpolarization and relaxation. Our results suggest a role of adrenic acid metabolites, specifically, DH-EETs as endothelium-derived hyperpolarizing factors in the coronary circulation.     

Trapping conformational intermediate states in the reaction center protein from photosynthetic bacteria

In protein, conformational changes are often crucial for function but not easy to observe. Two functionally relevant conformational intermediate states of photosynthetic reaction center protein (RCs) are trapped and characterized at low temperature. RCs frozen in the dark do not allow electron transfer from the reduced primary quinone, Q(A)(-), to the secondary quinone, Q(B). In contrast, RCs frozen under illumination in the product (P(+)Q(A)Q(B)(-)) state, with the oxidized electron donor, P(+), and reduced Q(B)(-), return to the ground state at cryogenic temperature in a conformation that allows a high yield of Q(B) reduction. Thus, RCs frozen under illumination are found to be trapped above the ground state in a conformation that allows product formation. When the temperature is raised above 120 K, the protein relaxes to an inactive conformation which is different from the RCs frozen in the dark. The activation energy for this change is 87 +/- 8 meV, and the active and inactive states differ in energy by only 16 +/- 3 meV. Thus, there are several conformational substates along the reaction coordinate with different transition temperatures. The ground state spectra of the RCs in active and inactive conformations report differences in the intraprotein electrostatic field, demonstrating that the dipole or charge distribution has changed. In addition, the electrochromic shift associated with the Q(A)(-) to Q(B) electron transfer at low temperature was characterized. The electron-transfer rate from Q(B)(-) to P(+) was measured at cryogenic temperature and is similar to the rate at room temperature, as expected for an exothermic, electron tunneling reaction in RCs.     

Effect of Oxygen on Temporary Stabilization of Photoreduced Quinone Acceptors in Rhodobacter sphaeroides Reaction Centers

The effect of molecular oxygen on the photochemical activity of the Rhodobacter sphaeroides reaction centers frozen to 160 K under actinic illumination was investigated by the ESR method. About 90% of initially photochemically active bacteriochlorophyll (P) were fixed at 160 K for a long time in aerobic samples in an inactive form. In anaerobic samples, not more than 65% were fixed in an inactive form under the same conditions. In aerobic preparations, a small portion of photochemically active bacteriochlorophyll (about 10%) that retains its photochemical activity at 160 K after freezing under illumination has dark reduction kinetics similar to that of samples at room temperature after several seconds of actinic illumination. In anaerobic samples frozen under illumination, the remaining photochemically active reaction centers (35%) have the same dark reduction kinetics as samples illuminated at 295 K for 1-2 min. The conclusion is that the irreversible stabilization of bacteriochlorophyll P in the oxidized inactive state formed in the reaction centers frozen under illumination is brought about by light-induced conformational changes fixed under low temperatures.     

Purification and characterization of protein kinase A from liver of the freeze-tolerant wood frog: role in glycogenolysis during freezing

Freeze tolerance by various amphibians includes cryoprotectant production in the form of glucose. Activation of the catalytic subunit of liver cAMP-dependent protein kinase (PKAc) facilitates activation of glycogenolysis, a critical biochemical process necessary for production of glucose. Here, we purified PKAc from Rana sylvatica liver to determine the extent to which cold temperature, which stimulates cryoprotectant production, affected PKAc activity and function. PKAc was purified to greater than 95% homogeneity, with a final specific activity of 71 nmol phosphate transferred/min/mg protein. The molecular weight of frog liver PKAc was 47.6 +/- 1.1 kDa and K(m) values for the phosphate acceptor kemptide and Mg-ATP were 9.0 +/- 0.1 and 51.8 +/- 1.0 microM at 22 degrees C, respectively. K(m) values for both substrates dropped significantly at 5 degrees C. The enzyme was sensitive to specific inhibitors of mammalian PKAc (PKA(i), H89) but was only moderately inhibited by high salt concentrations. Furthermore, salt inhibition was reduced at low temperature. The effect of temperature on enzyme activity indicated a conformational change in PKAc at 10 +/- 2 degrees C, with calculated activation energies of 51 +/- 4 kJ/mol at temperatures above 10 degrees C and 110 +/- 9 kJ/mol below 10 degrees C. PKAc in wood frog liver plays a crucial role in mediating the freeze-induced glycogenolysis that is responsible for the production of 200-300 mM levels of glucose as a cryoprotectant. Differential effects of low temperature on enzyme function, increased substrate affinity and reduced ion inhibition, appear to be central to this role.     

Fast voltage clamp discloses a new component of presteady-state currents from the Na(+)-glucose cotransporter.

The human Na(+)-glucose cotransporter (hSGLT1) has been shown to generate, in the absence of sugar, presteady-state currents in response to a change in potential, which could be fitted with single exponentials once the voltage had reached a new constant value. By the cut-open oocyte technique (voltage rising-speed approximately 1 mV/microsecond), phlorizin-sensitive transient currents could be detected with a higher time resolution during continuous intracellular perfusion. In the absence of sugar and internal Na+, and with 90 mM external Na+ concentration ([Na+]o), phlorizin-sensitive currents exhibited two relaxation time-constants: tau 1 increased from 2 to 10 ms when Vm decreased from +60 mV to -80 mV and remained at 10 ms for more negative Vm; tau 2 ranged from 0.4 to 0.8 ms in a weakly voltage-dependent manner. According to a previously proposed model, these two time constants could be accounted for by 1) Na+ crossing a fraction of the membrane electrical field to reach its binding site on the carrier and 2) conformational change of the free carrier. To test this hypothesis, the time constants were measured as [Na+]o was progressively reduced to 0 mM. At 30 and 10 mM external Na+, tau 1 reached the same plateau value of 10 ms but at more negative potentials (-120 and -160 mV, respectively). Contrary to the prediction of the model, two time constants continued to be detected in the bilateral absence of Na+ (at pH 8.0). Under these conditions, tau 1 continuously increased through the whole voltage range and did not reach the 10 ms level even when Vm had attained -200 mV while tau 2 remained in the range of 0.4-0.8 ms. These results indicate that 1) conformational change of the free carrier across the membrane must occur in more than one step and 2) Na+ binding/debinding is not responsible for either of the two observed exponential components of transient currents. By use of the simplest kinetic model accounting for the portion of the hSGLT1 transport cycle involving extracellular Na+ binding/debinding and the dual-step conformational change of the free carrier, tau 1 and tau 2 were fitted throughout the voltage range, and a few sets of parameters were found to reproduce the data satisfactorily. This study shows that 1) tau 1 and tau 2 correspond to two steps in the conformational change of the free carrier, 2) Na+ binding/debinding modulates the slow time constant (tau 1) and 3) a voltage-independent slow conformational change of the free carrier accounts for the observed plateau value of 10 ms.     

Turnover of ubiquinone-0 at the acceptor side of photosynthetic reaction center

The steady-state operation of photosynthetic reaction center from Rhodobacter sphaeroides was investigated by measuring the rate of cytochrome photo-oxidation under intensive continuous illumination (808 nm, 5 W cm(-2)). The native quinone UQ(10) in Q(B) binding site of the reaction center was substituted by tailless UQ(0) and the binding parameters and the turnover rate of the UQ(0) was studied to test the recently discovered light-intensity dependent acceptor side effect (Gerencsér and Maróti 2006). The binding parameters of UQ(0) (k (on) = 2.1 x 10(5) M(-1) s(-1) and k (off) = 100 s(-1)) were characteristic to the RC exposed to high light-intensity. The dissociation constant (K (D) = 480 muM) determined under high light intensity is 2-3 times larger than that determined from flash-experiments. The light-intensity dependent acceleration of cytochrome turnover measured on reaction center of inhibited proton binding was independent of the type of the quinone and was sensitive only to the size ("pressure") of the quinone pool. The dissociation constants of different types of semiquinones show similarly high (several orders of magnitude) increase in the modified conformation of the Q(B) binding pocket due to high intensity of illumination. This result indicates the exclusive role of the quinone headgroup in the binding of semiquinone to different conformations of the protein.     

Selective perturbation of the second electron transfer step in mutant bacterial reaction centers

In order to specifically perturb the primary electron acceptor B(A) -- a monomeric bacteriochlorophyll (BChl) a -- involved in bacterial photosynthetic charge separation (CS), the protein environment of B(A) in the reaction center (RC) of Rhodobacter sphaeroides was modified by site-directed mutagenesis. Isolated RCs were characterized by redox titrations, low temperature optical spectroscopy, ENDOR/TRIPLE resonance spectroscopy and femtosecond time-resolved spectroscopy. Two mutations were studied: In the GS(M203) mutant a serine is introduced near the ring E keto group of B(A), while in FY(L146) a phenylalanine near the ring A acetyl group of B(A) is replaced by tyrosine. In all mutations the oxidation potential of the primary electron donor P as well as the electronic structure of both the P(*+) radical cation and the radical anion of the secondary electron acceptor, H(A)(*-), are not significantly altered compared to the wild type (WT), while changes of the optical absorption spectra at 77 K in the BChl Q(X) and Q(Y) regions are observed. The GS(M203) mutation only leads to a minor retardation of the CS reactions at room temperature, whereas for FY(L146) significant deviations from the native electron transfer (ET) rates could be detected: In addition to a faster first (2.9 ps) and a slower second (1 ps) ET step, a new 8-ps time constant was found in the FY(L146) mutant, which can be ascribed to a fraction of RCs with slowed down secondary ET. The results allow us to address the functional role of the acetyl group of B(A) and question the role of the free energy changes as the main determining factor of ET rates in RCs. It is concluded that structural rearrangements alter the electronic coupling between the pigments and thereby influence the rate of fast CS.     

Energy trapping and detrapping in reaction center mutants from Rhodobacter sphaeroides

Time-resolved fluorescence of chromatophores isolated from strains of Rhodobacter sphaeroides containing light harvesting complex I (LHI) and reaction center (RC) (no light harvesting complex II) was measured at several temperatures between 295 K and 10 K. Measurements were performed to investigate energy trapping from LHI to the RC in RC mutants that have a P/P(+) midpoint potential either above or below wild-type (WT). Six different strains were investigated: WT + LHI, four mutants with altered RC P/P(+) midpoint potentials, and an LHI-only strain. In the mutants with the highest P/P(+) midpoint potentials, the electron transfer rate decreases significantly, and at low temperatures it is possible to directly observe energy transfer from LHI to the RC by detecting the fluorescence kinetics from both complexes. In all mutants, fluorescence kinetics are multiexponential. To explain this, RC + LHI fluorescence kinetics were analyzed using target analysis in which specific kinetic models were compared. The kinetics at all temperatures can be well described with a model which accounts for the energy transfer between LHI and the RC and also includes the relaxation of the charge separated state P(+)H(A)(-), created in the RC as a result of the primary charge separation.     

The effects of light-induced reduction of the photosystem II reaction center

Accumulation of reduced pheophytin a (Pheo-D1) in photosystem II reaction center (PSII RC) under illumination at low redox potential is accompanied by changes in absorbance and circular dichroism spectra. The temperature dependences of these spectral changes have the potential to distinguish between changes caused by the excitonic interaction and temperature-dependent processes. We observed a conformational change in the PSII RC protein part and changes in the spatial positions of the PSII RC pigments of the active D1 branch upon reduction of Pheo-D1 only in the case of high temperature (298 K) dynamics. The resulting absorption difference spectra of PSII RC models equilibrated at temperatures of 77 K and 298 K were highly consistent with our previous experiments in which light-induced bleaching of the PSII RC absorbance spectrum was observable only at 298 K. These results support our previous hypothesis that Pheo-D1 does not interact excitonically with the other chlorins of the PSII RC, since the reduced form of Pheo-D1 causes absorption spectra bleaching only due to temperature-dependent processes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.