Absorption changes induced by the binding of triazines to the QB pocket in reaction centers of Rhodobacter capsulatus

Inhibitors which block electron transfer from the primary (Q(A)) to the secondary (Q(B)) quinone of the bacterial reaction center are competing with the pool ubiquinones for binding at the Q(B) pocket. Due to the much greater stability of the semiquinone state Q(B)(-) compared with fully oxidized or reduced quinone, a displacement of the inhibitors takes place after one flash from state Q(A)(-)I to state Q(A)Q(B)(-). This process can be monitored from near-IR absorption changes which reflect local absorption shifts specific to Q(A)(-) and Q(B)(-). An anomalous behavior was observed when using triazines in chromatophores of R. capsulatus: the IR absorption change reflecting the formation of Q(B)(-) after one flash was absent. A normal transient decay of this signal was, however, triggered by a second flash, followed by a rapid return to the baseline. We show that this phenomenon is due to an absorption change induced by inhibitor binding (thus present in the dark baseline), with a spectrum close to that of Q(B)(-), so that the Q(B)(-) changes are canceled out during the inhibitor displacement process. On the second flash, one monitors the destruction of the semiquinone, leading transiently to the Q(A)Q(B) state, followed by inhibitor rebinding. This allows a direct measurement of the binding kinetics. This behavior was observed both in chromatophores and in isolated reaction centers from R. capsulatus, but not in R. sphaeroides.     

Proton and electron transfer in bacterial reaction centers

The bacterial reaction center couples light-induced electron transfer to proton pumping across the membrane by reactions of a quinone molecule Q(B) that binds two electrons and two protons at the active site. This article reviews recent experimental work on the mechanism of the proton-coupled electron transfer and the pathways for proton transfer to the Q(B) site. The mechanism of the first electron transfer, k((1))(AB), Q(-)(A)Q(B)-->Q(A)Q(-)(B), was shown to be rate limited by conformational gating. The mechanism of the second electron transfer, k((2))(AB), was shown to involve rapid reversible proton transfer to the semiquinone followed by rate-limiting electron transfer, H(+)+Q(-)(A)Q(-)(B) ifQ(-)(A)Q(B)H-->Q(A)(Q(B)H)(-). The pathways for transfer of the first and second protons were elucidated by high-resolution X-ray crystallography as well as kinetic studies showing changes in the rate of proton transfer due to site directed mutations and metal ion binding.     

Efficiencies of singlet oxygen production and rate constants for oxygen quenching in the S1 state of dicyanonaphthalenes and related compounds.

The quantum yield of singlet oxygen ((1)O(2) ((1)Delta(g))) production (Phi(Delta)) in the oxygen quenching of photoexcited states for 1,2-dicyanonaphthalene (1,2-DCNN), 1,4-dicyanonaphthalene (1,4-DCNN) and 2,3-dicyanonaphthalene (2,3-DCNN) in cyclohexane, benzene, and acetonitrile was measured using a time-resolved thermal lens (TRTL) technique, in order to determine the efficiency of singlet oxygen ((1)Delta(g)) production in the first excited singlet state (S(1)), (f(Delta)(S)). The efficiencies of singlet oxygen ((1)Delta(g)) production from the lowest triplet state (T(1)), (f(Delta)(T)), were nearly unity for all DCNNs in all the solvents. The values of f(Delta)(S) were fairly large for 1,2-DCNN (0.33-0.57) and 1,4-DCNN (0.33-0.66), but were close to zero for 2,3-DCNN. Rate constants for oxygen quenching in the S(1) state (k(q)(S)) obtained for these compounds were significantly smaller than diffusion-controlled rate constants. The kinetics for processes leading to production and no production of singlet oxygen is discussed on the basis of the values of f(Delta)(S) and k(q)(S). The results obtained regarding phenanthrene (PH), 9-cyanophenanthrene (9-CNPH), pyrene (PY) and 1-cyanopyrene (1-CNPY) are also discussed.     

Remarkably stereoselective photoinduced electron-transfer reaction between zinc myoglobin and optically active binaphthyl bisviologen

We have designed and synthesized new optically active bisviologens ([BNMV](4+)) containing a binaphthyl moiety to examine the stereoselective photoinduced electron-transfer (ET) reactions with zinc-substituted myoglobin (ZnMb) by flash photolysis. The photoexcited triplet state of ZnMb, (3)(ZnMb)*, was successfully quenched by [BNMV](4+) ions to form the radical pair of a ZnMb cation (ZnMb(.+)) and a reduced viologen ([BNMV](.3+)), followed by a thermal ET reaction to the ground state. The rate constants ( k(q)) for the ET quenching at 25 degrees C were obtained as k(q)( R)=(2.9+/-0.2)x10(7) M(-1) s(-1) and k(q)( S)=(2.2+/-0.2)x10(7) M(-1) s(-1), respectively. The ratio of k(q)( R)/ k(q)( S)=1.3 indicates that the ( R)-isomer of the chiral viologen preferentially quenches (3)(ZnMb)*. On the other hand, the rate constants ( k) for the thermal ET reaction from [BNMV](.3+) to ZnMb(-+) at 25 degrees C were k( R)=(1.2+/-0.1)x10(8) M(-1) s(-1) and k( S)=(0.47+/-0.03)x10(8) M(-1) s(-1), respectively, and the ratio remarkably increased to k( R)/ k( S)=2.6. The activation parameters, Delta H(not equal) and Delta S(not equal), were determined from the kinetic measurements at various temperatures (10-30 degrees C) to understand the ET mechanisms. In the quenching reaction, the energy differences of Delta Delta H*(R- S) and T Delta Delta S*( R- S) at 25 degrees C were calculated to be -3.9+/-1.6 and -3.3+/-0.2 kJ mol(-1), respectively, whereas Delta Delta H*( R-S)=7.7+/-1.9 kJ mol(-1 )and T Delta Delta S*( R-S)=9.9+/-0.5 kJ mol(-1 )were found for the thermal ET reaction. Therefore, the thermal ET reaction to the ground state was proved to be dominated by the entropy term, and the large stereoselectivity may arise from the decrease in charge repulsion between donor and acceptor.     

Two-electron reduction of quinones by Enterobacter cloacae NAD(P)H:nitroreductase: quantitative structure-activity relationships

Enterobacter cloacae NAD(P)H:nitroreductase (NR; EC 1.6.99.7) catalyzes two-electron reduction of a series of quinoidal compounds according to a "ping-pong" scheme, with marked substrate inhibition by quinones. The steady-state catalytic constants (k(cat)) range from 0.1 to 1600s(-1), and bimolecular rate constants (k(cat)/K(m)) range from 10(3) to 10(8)M(-1)s(-1). Quinones, nitroaromatic compounds and competitive to NADH inhibitor dicumarol, quench the flavin mononucleotide (FMN) fluorescence of nitroreductase. The reactivity of NR with single-electron acceptors is consistent with an "outer-sphere" electron transfer model, taking into account high potential of FMN semiquinone/FMNH(-) couple and good solvent accessibility of FMN. However, the single-electron acceptor 1,1(')-dibenzyl-4,4(')-bipyridinium was far less reactive than quinones possessing similar single-electron reduction potentials (E(1)(7)). For all quinoidal compounds except 2-hydroxy-1,4-naphthoquinones, there existed parabolic correlations between the log of rate constants of quinone reduction and their E(1)(7) or hydride-transfer potential (E(7)(Q/QH(-))). Based on pH dependence of rate constants, a single-step hydride transfer seems to be a more feasible quinone reduction mechanism. The reactivities of 2-hydroxy-1,4-naphthoquinones were much higher than expected from their reduction potential. Most probably, their enhanced reactivity was determined by their binding at or close to the binding site of NADH and dicumarol, whereas other quinones used the alternative, currently unidentified binding site.     

Recruitment of a foreign quinone into the A1 site of photosystem I. Altered kinetics of electron transfer in phylloquinone biosynthetic pathway mutants studied by time-resolved optical, EPR, and electrometric techniques

Interruption of the menA or menB gene in Synechocystis sp. PCC 6803 results in the incorporation of a foreign quinone, termed Q, into the A(1) site of photosystem I with a number of experimental indicators identifying Q as plastoquinone-9. A global multiexponential analysis of time-resolved optical spectra in the blue region shows the following three kinetic components: 1) a 3-ms lifetime in the absence of methyl viologen that represents charge recombination between P700(+) and an FeS(-) cluster; 2) a 750-microseconds lifetime that represents electron donation from an FeS(-) cluster to methyl viologen; and 3) an approximately 15-microseconds lifetime that represents an electrochromic shift of a carotenoid pigment. Room temperature direct detection transient EPR studies of forward electron transfer show a spectrum of P700(+) Q(-) during the lifetime of the spin polarization and give no evidence of a significant population of P700(+) FeS(-) for t 相似文献   

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.     

Primary charge separation within P870* in wild type and heterodimer mutants in femtosecond time domain

Primary charge separation dynamics in the reaction center (RC) of purple bacterium Rhodobacter sphaeroides and its P870 heterodimer mutants have been studied using femtosecond time-resolved spectroscopy with 20 and 40fs excitation at 870nm at 293K. Absorbance increase in the 1060-1130nm region that is presumably attributed to P(A)(δ+) cation radical molecule as a part of mixed state with a charge transfer character P*(P(A)(δ+)P(B)(δ-)) was found. This state appears at 120-180fs time delay in the wild type RC and even faster in H(L173)L and H(M202)L heterodimer mutants and precedes electron transfer (ET) to B(A) bacteriochlorophyll with absorption band at 1020nm in WT. The formation of the P(A)(δ+)B(A)(δ-) state is a result of the electron transfer from P*(P(A)(δ+)P(B)(δ-)) to the primary electron acceptor B(A) (still mixed with P*) with the apparent time delay of ~1.1ps. Next step of ET is accompanied by the 3-ps appearance of bacteriopheophytin a(-) (H(A)(-)) band at 960nm. The study of the wave packet formation upon 20-fs illumination has shown that the vibration energy of the wave packet promotes reversible overcoming of an energy barrier between two potential energy surfaces P* and P*(P(A)(δ+)B(A)(δ-)) at ~500fs. For longer excitation pulses (40fs) this promotion is absent and tunneling through an energy barrier takes about 3ps. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Electron paramagnetic resonance studies of zinc-substituted reaction centers from Rhodopseudomonas viridis.

The primary quinone acceptor radical anion Q(A)(-)(*) (a menaquinone-9) is studied in reaction centers (RCs) of Rhodopseudomonas viridis in which the high-spin non-heme Fe(2+) is replaced by diamagnetic Zn(2+). The procedure for the iron substitution, which follows the work of Debus et al. [Debus, R. J., Feher, G., and Okamura, M. Y. (1986) Biochemistry 25, 2276-2287], is described. In Rps. viridisan exchange rate of the iron of approximately 50% +/- 10% is achieved. Time-resolved optical spectroscopy shows that the ZnRCs are fully competent in charge separation and that the charge recombination times are similar to those of native RCs. The g tensor of Q(A)(-)(*) in the ZnRCs is determined by a simulation of the EPR at 34 GHz yielding g(x) = 2.00597 (5), g(y) = 2.00492 (5), and g(z) = 2.00216 (5). Comparison with a menaquinone anion radical (MQ(4)(-)(*)) dissolved in 2-propanol identifies Q(A)(-)(*) as a naphthoquinone and shows that only one tensor component (g(x)) is predominantly changed in the RC. This is attributed to interaction with the protein environment. Electron-nuclear double resonance (ENDOR) experiments at 9 GHz reveal a shift of the spin density distribution of Q(A)(-)(*) in the RC as compared with MQ(4)(-)(*) in alcoholic solution. This is ascribed to an asymmetry of the Q(A) binding site. Furthermore, a hyperfine coupling constant from an exchangeable proton is deduced and assigned to a proton in a hydrogen bond between the quinone oxygen and surrounding amino acid residues. By electron spin-echo envelope modulation (ESEEM) techniques performed on Q(A)(-)(*) in the ZnRCs, two (14)N nuclear quadrupole tensors are determined that arise from the surrounding amino acids. One nitrogen coupling is assigned to a N(delta)((1))-H of a histidine and the other to a polypeptide backbone N-H by comparison with the nuclear quadrupole couplings of respective model systems. Inspection of the X-ray structure of Rps. viridis RCs shows that His(M217) and Ala(M258) are likely candidates for the respective amino acids. The quinone should therefore be bound by two H bonds to the protein that could, however, be of different strength. An asymmetric H-bond situation has also been found for Q(A)(-)(*) in the RC of Rhodobacter sphaeroides. Time-resolved electron paramagnetic resonance (EPR) experiments are performed on the radical pair state P(960)(+) (*)Q(A)(-)(*) in ZnRCs of Rps. viridis that were treated with o-phenanthroline to block electron transfer to Q(B). The orientations of the two radicals in the radical pair obtained from transient EPR and their distance deduced from pulsed EPR (out-of-phase ESEEM) are very similar to the geometry observed for the ground state P(960)Q(A) in the X-ray structure [Lancaster, R., Michel, H. (1997) Structure 5, 1339].     

Comparative study of the bimolecular electron transfer of fullerenes (C60/C70) and 9,9-disubstituted fluorenes by laser flash photolysis.

Photoinduced bimolecular electron transfer processes of fullerenes (C60/C70) with fluorene derivatives, namely 9,9-bis(4-amino-3-methylphenyl)fluorene (BAMF), 9,9-bis(4-amino-3-fluorophenyl)fluorene (BAFF) and 9,9-bis(3-amino-4-hydroxyphenyl)fluorene (BAHF) have been comparatively studied in benzonitrile by nanosecond laser photolysis technique in the visible/near-IR regions. By the selective excitation of C60/C70 using 532 nm laser light, it has been proved that electron transfer takes place from the ground state fluorenes to the triplet excited state of C60/C70. The observed rates and efficiencies of the electron transfer processes are found to be significantly associated with the substitution patterns on the 9,9-bisphenylfluorenes, and to be correlative with the free energy changes on the basis of the Rehm-Weller relation.     

Modeling binding kinetics at the Q(A) site in bacterial reaction centers

Bacterial reaction centers (RCs) catalyze a series of electron-transfer reactions reducing a neutral quinone to a bound, anionic semiquinone. The dissociation constants and association rates of 13 tailless neutral and anionic benzo- and naphthoquinones for the Q(A) site were measured and compared. The K(d) values for these quinones range from 0.08 to 90 microM. For the eight neutral quinones, including duroquinone (DQ) and 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ(0)), the quinone concentration and solvent viscosity dependence of the association rate indicate a second-order rate-determining step. The association rate constants (k(on)) range from 10(5) to 10(7) M(-)(1) s(-)(1). Association and dissociation rate constants were determined at pH values above the hydroxyl pK(a) for five hydroxyl naphthoquinones. These negatively charged compounds are competitive inhibitors for the Q(A) site. While the neutral quinones reach equilibrium in milliseconds, anionic hydroxyl quinones with similar K(d) values take minutes to bind or dissociate. These slow rates are independent of ionic strength, solvent viscosity, and quinone concentration, indicating a first-order rate-limiting step. The anionic semiquinone, formed by forward electron transfer at the Q(A) site, also dissociates slowly. It is not possible to measure the association rate of the unstable semiquinone. However, as the protein creates kinetic barriers for binding and releasing anionic hydroxyl quinones without greatly increasing the affinity relative to neutral quinones, it is suggested that the Q(A) site may do the same for anionic semiquinone. Thus, the slow semiquinone dissociation may not indicate significant thermodynamic stabilization of the reduced species in the Q(A) site.     

Residual water modulates QA- -to-QB electron transfer in bacterial reaction centers embedded in trehalose amorphous matrices

The role of protein dynamics in the electron transfer from the reduced primary quinone, Q(A)(-), to the secondary quinone, Q(B), was studied at room temperature in isolated reaction centers (RC) from the photosynthetic bacterium Rhodobacter sphaeroides by incorporating the protein in trehalose water systems of different trehalose/water ratios. The effects of dehydration on the reaction kinetics were examined by analyzing charge recombination after different regimes of RC photoexcitation (single laser pulse, double flash, and continuous light) as well as by monitoring flash-induced electrochromic effects in the near infrared spectral region. Independent approaches show that dehydration of RC-containing matrices causes reversible, inhomogeneous inhibition of Q(A)(-)-to-Q(B) electron transfer, involving two subpopulations of RCs. In one of these populations (i.e., active), the electron transfer to Q(B) is slowed but still successfully competing with P(+)Q(A)(-) recombination, even in the driest samples; in the other (i.e., inactive), electron transfer to Q(B) after a laser pulse is hindered, inasmuch as only recombination of the P(+)Q(A)(-) state is observed. Small residual water variations ( approximately 7 wt %) modulate fully the relative fraction of the two populations, with the active one decreasing to zero in the driest samples. Analysis of charge recombination after continuous illumination indicates that, in the inactive subpopulation, the conformational changes that rate-limit electron transfer can be slowed by >4 orders of magnitude. The reported effects are consistent with conformational gating of the reaction and demonstrate that the conformational dynamics controlling electron transfer to Q(B) is strongly enslaved to the structure and dynamics of the surrounding medium. Comparing the effects of dehydration on P(+)Q(A)(-)-->PQ(A) recombination and Q(A)(-)Q(B)-->Q(A)Q(B)(-) electron transfer suggests that conformational changes gating the latter process are distinct from those stabilizing the primary charge-separated state.     

Transient optical spectroscopy of single crystals of the reaction center from Rhodobacter sphaeroides wild-type 2.4.1

The photoactivity of the crystallized reaction centers from Rhodobacter sphaeroides wild-type strain 2.4.1 has been examined by light-induced absorption spectral changes associated with charge separation and triplet state formation in the reaction center. Upon excitation of a crystal at ambient redox potential, the primary donor 865 nm band bleaches reversibly. The kinetics of its recovery were found to be biphasic with rate constants 11.5 +/- 1.3 s-1 and 0.9 +/- 0.4 s-1 which correspond to lifetimes of 87.0 +/- 9.0 ms and 1.0 +/- 0.7 s, respectively. The ratio of the fast-to-slow component preexponential terms was 3.5 +/- 1.1 suggesting that the majority (78.9 +/- 13.0%) of the reaction centers in the crystals lack the secondary quinone, QB. The addition of sodium ascorbate to the crystals attenuates the 865 nm absorption change, and gives rise to strong carotenoid triplet-triplet absorption changes at 547 nm. These data indicate that the reaction center-bound carotenoid in the crystals is capable of accepting triplet energy from the primary donor triplet.     

Secondary quinone in photosystem II of Thermosynechococcus elongatus: semiquinone-iron EPR signals and temperature dependence of electron transfer

The secondary quinone acceptor, Q(B), has been studied in photosystem II (PSII) isolated from Thermosynechococcus (T.) elongatus. Thermoluminescence indicated that Q(B) was present in this preparation. An EPR signal observed at low temperature at g = 1.9 was attributed to Fe2+ Q(B)- on the basis of the characteristic period-of-two variations in its intensity depending on the number of laser flashes given at 20 degrees C. When samples showing the Fe2+ Q(B)- signal were illuminated at 77 K, an EPR signal at g = 1.66 appeared with an amplitude proportional to that of the Fe2+ Q(B)- signal. This signal is attributed to the Q(A)- Fe2+ Q(B)- state. While these attributions have been made previously in PSII from other origins, they have remained relatively tentative since the characteristic period-of-two oscillations of Q(B) had not previously been observed. The flash experiments indicated that more than one exchangeable plastoquinone is associated with the isolated PSII. The g = 1.66 signal from the Q(A)- Fe2+ Q(B)- state was used to study the temperature dependence of electron transfer between the two quinones. Electron transfer occurred in half of the centers (after 30 s incubation) at -28 degrees C for Q(A)- to Q(B) but at -58 degrees C for Q(A)- to Q(B)-. This marked difference for the two electron transfer reactions indicates different types of rate-limiting reactions. In the better studied but homologous system, the purple bacterial reaction center, the Q(A)- to Q(B) step is limited by a gating process, while the Q(A)- to Q(B)- step is limited by protonation events. Similar reactions in PSII could give rise to the observed temperature dependence.     

Cumulant analysis of charge recombination kinetics in bacterial reaction centers reconstituted into lipid vesicles

The kinetics of charge recombination between the primary photoxidized donor (P(+)) and the secondary reduced quinone acceptor (Q(B)(-)) have been studied in reaction centers (RCs) from the purple photosynthetic bacterium Rhodobacter sphaeroides incorporated into lecithin vesicles containing large ubiquinone pools over the temperature range 275 K = (50 +/- 15) nm). Following these premises, we describe the kinetics of P(+)Q(B)(-) recombination with a truncated cumulant expansion and relate it to P(Q) and to the free energy changes for Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron transfer (DeltaG(AB)(o)) and for quinone binding (DeltaG(bind)(o)) at Q(B). The model accounts well for the temperature and quinone dependence of the charge recombination kinetics, yielding DeltaG(AB)(o) = -7.67 +/- 0.05 kJ mol(-1) and DeltaG(bind)(o) = -14.6 +/- 0.6 kJ mol(-1) at 298 K.     

-deltaG(AB) and pH dependence of the electron transfer from P(+)Q(A)(-)Q(B) toP(+)Q(A)Q(B)(-) in Rhodobacter sphaeroides reaction centers

The electron transfer from the reduced primary quinone (Q(A)(-)) to the secondary quinone (Q(B)) can occur in two phases with a well-characterized 100 micros component (tau(2)) and a faster process occurring in less than 10 micros (tau(1)). The fast reaction is clearly seen when the native ubiquinone-10 at Q(A) is replaced with naphthoquinones. The dependence of tau(1) on the free-energy difference between the P(+)Q(A)(-)Q(B) and P(+)Q(A)Q(B)(-) states (-) and on the pH was measured using naphthoquinones with different electrochemical midpoint potentials as Q(A) in Rhodobacter sphaeroides reaction centers (RCs) and in RCs where - is changed by mutation of M265 in the Q(A) site from Ile to Thr (M265IT). Q(B) was ubiquinone (UQ(B)) in all cases. Electron transfer was measured by using the absorption differences of the naphthosemiquinone at Q(A) and the ubisemiquinone at Q(B) between 390 and 500 nm. As - was changed from -90 to -250 meV tau(1) decreased from 29 to 0.2 micros. The free-energy dependence of tau(1) provides a reorganization energy of 850 +/- 100 meV for the electron transfer from Q(A)(-) to Q(B). The slower reaction at tau(2) is free-energy independent, so processes other than electron transfer determine the observed rate. The fraction of the reaction at tau(1) increases with increasing driving force and is 100% of the reaction when - is approximately 100 meV more favorable than in the native RCs with ubiquinone as Q(A). The fast phase, tau(1), is pH independent from pH 6 to 11 while tau(2) slows above pH 9. As the Q(A) isoprene tail length is increased from 2 to 10 isoprene units the fraction at tau(1) decreases. However, tau(1), tau(2), and the fraction of the reaction in each phase are independent of the tail length of UQ(B).     

Insertional inactivation of the menG gene, encoding 2-phytyl-1,4-naphthoquinone methyltransferase of Synechocystis sp. PCC 6803, results in the incorporation of 2-phytyl-1,4-naphthoquinone into the A(1) site and alteration of the equilibrium constant between A(1) and F(X) in photosystem I.

A gene encoding a methyltransferase (menG) was identified in Synechocystis sp. PCC 6803 as responsible for transferring the methyl group to 2-phytyl-1,4-naphthoquinone in the biosynthetic pathway of phylloquinone, the secondary electron acceptor in photosystem I (PS I). Mass spectrometric measurements showed that targeted inactivation of the menG gene prevented the methylation step in the synthesis of phylloquinone and led to the accumulation of 2-phytyl-1,4-naphthoquinone in PS I. Growth rates of the wild-type and the menG mutant strains under photoautotrophic and photomixotrophic conditions were virtually identical. The chlorophyll a content of the menG mutant strain was similar to that of wild type when the cells were grown at a light intensity of 50 microE m(-2) s(-1) but was slightly lower when grown at 300 microE m(-2) s(-1). Chlorophyll fluorescence emission measurements at 77 K showed a larger increase in the ratio of PS II to PS I in the menG mutant strain relative to the wild type as the light intensity was elevated from 50 to 300 microE m(-2) s(-1). CW EPR studies at 34 GHz and transient EPR studies at multiple frequencies showed that the quinone radical in the menG mutant has a similar overall line width as that for the wild type, but consistent with the presence of an aromatic proton at ring position 2, the pattern of hyperfine splittings showed two lines in the low-field region. The spin polarization pattern indicated that 2-phytyl-1,4-naphthoquinone is in the same orientation as phylloquinone, and out-of-phase, spin-echo modulation spectroscopy shows the same P700(+) to Q(-) center-to-center distance as in wild-type PS I. Transient EPR studies indicated that the lifetime for forward electron transfer from Q(-) to F(X) is slowed from 290 ns in the wild type to 600 ns in the menG mutant. The redox potential of 2-phytyl-1,4-naphthoquinone is estimated to be 50 to 60 mV more oxidizing than phylloquinone in the A(1) site, which translates to a lowering of the equilibrium constant between Q(-)/Q and F(X)(-)/F(X) by a factor of ca. 10. The lifetime of the P700(+) [F(A)/F(B)](-) backreaction decreased from 80 ms in the wild type to 20 ms in the menG mutant strain and is evidence for a thermally activated, uphill electron transfer through the quinone rather than a direct charge recombination between [F(A)/F(B)](-) and P700(+).     

Evaluation of selected benzoquinones, naphthoquinones, and anthraquinones as replacements for phylloquinone in the A1 acceptor site of the photosystem I reaction center

Selected substituted 1,4-benzoquinones, 1,4-naphthoquinones, and 9,10-anthraquinones were investigated as possible replacement quinones in spinach photosystem I (PSI) preparations that had been depleted of endogenous phylloquinone by extraction with hexane/methanol. As a criterion for successful biochemical reconstitution, the restoration of electron transfer was determined by measuring P-430 turnover at room temperature from flash-induced absorbance transients. Restoration of complete electron transfer between A0- and P-430 (terminal iron-sulfur centers, FAFB) was demonstrated by using phylloquinone, 2-methyl-3-decyl-1,4-naphthoquinone, 2-methyl-3-(isoprenyl)2-1,4-naphthoquinone, and 2-methyl-3-(isoprenyl)4-1,4-naphthoquinone. All other quinones tested did not restore P-430 turnover but acted as electron acceptors and oxidized A0-. It is concluded that the specificity of the replacement quinone for interaction with the primary acceptor, A0-, is low but additional structural constraints are required for the quinone occupying the A1 site to donate to the iron-sulfur center, Fx. It is suggested that the 3-phytyl side chain of phylloquinone and the 3-alkyl tails of the three naphthoquinones that restored P-430 turnover may be required for interaction with a hydrophobic domain of the A1 site in the PSI core to promote electron transfer to Fx and then to FAFB.     

Kinetics and mechanism of the reduction of horse heart ferricytochrome c by glutathione.

A detailed investigation of the reduction of cytochrome c by glutathione has shown that the reaction proceeds through several steps. A rapid combination of the reducing agent with the cytochrome leads to the formation of a glutathione-cytochrome intermediate in which the glutathione most likely interacts with the edge of the heme moiety. The electron transfer takes place in a subsequent slower step. Since cytochrome c(III) exists in two conformational forms at neutral pH [Kujundzic, N., & Everse, J. (1978) Biochem. Biophys. Res. Commun. 82, 1211], the reduction of cytochrome c by glutathione may be represented by cyt c(III) + GS- reversible K1 cyt c(III) ... GS- reversible k1 products cyt c*(III) + GS- reversible K2 cyt c*(III) ... GS- reversible k2 products At 25 degrees C, pH 7.5, and an ionic strength of 1.0 (NaCl), k1 = 1.2 X 10(-3) S-1, k2 = 2.0 X 10(-3) S-1, k1 = 2.9 X 10(3) M-1, and K2 = 5.3 X 10(3) M-1. The reaction is catalyzed by trisulfides, and second-order rate constants of 4.55 X 10(3) and 7.14 X 10(3) M-1 S-1 were obtained for methyl trisulfide and cysteine trisulfide, respectively.     

Triplet- vs. singlet-state imposed photochemistry. The role of substituent effects on the photo-Fries and photodissociation reaction of triphenylmethyl silanes.

The photochemistry of three structurally very similar triphenylmethylsilanes 1, 2, 3 [p-X-C(6)H(4)-CPh(2)-SiMe(3): X = PhCO, 1; H, ; Ph(OCH(2)CH(2)O)C, 3] is described by means of 248 and 308 nm nanosecond laser flash photolysis (ns-LFP), femtosecond LFP, EPR spectroscopy, emission spectroscopy (fluorescence, phosphorescence), ns-pulse radiolysis (ns-PR), photoproduct analysis studies in MeCN, and X-ray crystallographic analysis of the two key-compounds 1 and 2. The photochemical behavior of 1, 2 and 3 is discussed and compared with that of a fourth one, 4, bearing on the p-position an amino group (X = Me(2)N) and whose detailed photochemistry we reported earlier (J. Org. Chem., 2000, 65, 4274-4280). Silane 1 undergoes on irradiation with 248 and 308 nm laser light a fast photodissociation of the C-Si bond giving the p-(benzoyl)triphenylmethyl radical (1*) with a rate constant of k(diss)= 3 x 10(7) s(-1). The formation of 1* is a one-quantum process and takes place via the carbonyl triplet excited state with high quantum yield (Phi(rad)= 0.9); the intervention of the triplet state is clearly demonstrated through the phosphorescence spectrum and quenching experiments with ferrocene (k(q)= 9.3 x 10(9) M(-1) s(-1)), Et(3)N (1.1 x 10(9) M(-1) s(-1)), and styrene (3.1 x 10(9) M(-1) s(-1)) giving quenching rate constants very similar to those of benzophenone. For comparative reasons radical 1* was generated independently from p-(benzoyl)triphenylmethyl bromide via pulse radiolysis in THF and its absorption coefficient at lambda(max)= 340 nm was determined ([epsilon]= 27770 M(-1) cm(-1)). We found thus that the p-PhCO-derivative 1 behaves similar to the p-Me(2)N one (the latter giving the p-(dimethylamino)triphenylmethyl radical with Phi(rad)= 0.9), irrespective of their completely different ground state electronic properties. In contrast, compounds 2, 3 that bear only the aromatic chromophore give by laser or lamp irradiation both, (i) radical products [Ph(3)C* and p-Ph(OCH(2)CH(2)O)C-C(6)H(4)-C(*)Ph(2), respectively] after dissociation of the central C-Si bond (Phi(rad)= 0.16), and (ii) persistent photo-Fries rearrangement products (of the type of 5-methylidene-6-trimethylsilyl-1,3-cyclohexadiene) absorbing at 300-450 nm and arising from a 1,3-shift of the SiMe(3) group from the benzylic to the ortho-position of the aromatic ring (Phi approximately 0.85 for 2). Using fs-LFP on 2 we showed that the S(1) state recorded at 100 fs after the pulse decays on a time scale of 500 fs giving Ph(3)C* through C-Si bond dissociation. In a second step and within the next 10 ps trityl radicals either escape from the solvent cage (the quantum yield of Ph(3)C* formation Phi(rad)= 0.16 was measured with ns-LFP), or undergo in-cage recombination to photo-Fries products. Thus, singlet excited states (S(1)) of the aromatic organosilanes (2, 3) prefer photo-Fries rearrangement products, while triplet excited states (1, 4) favor free radicals. Both reactions proceed via a common primary photodissociation step (C-Si bond homolysis) and differentiate obviously in the multiplicity of the resulting geminate radical pairs; singlet radical pairs give preferably photo-Fries products following an in-cage recombination, while triplet radical pairs escape the solvent cage (MeCN). The results demonstrate the crucial role which is played by the chromophore which prescribes in a sense, (i) the multiplicity of the intervening excited state and consequently that of the resulting geminate radical pair, and (ii) the dominant reaction path to be followed: the benzophenone- and anilino-chromophore present in silanes 1 and 4, respectively, impose effective intersystem crossing transitions (k(isc)= 10(11) s(-1) and 6 x 10(8) s(-1), respectively) leading to triplet states and finally to free radical products, while the phenyl chromophore in 2 and 3, possessing ineffective isc (k(isc)= 6 x 10(6) s(-1)) leads to photo-Fries product formation via the energetic high lying S(1) state [approximately 443 kJ mol(-1)(106 kcal mol(-1))].