Photoinduced electron transfer and cytochrome content in obligate aerobic phototrophic bacteria from genera Erythromicrobium,Sandaracinobacter, Erythromonas,Roseococcus and Erythrobacter

The photosynthetic apparatus and the electron carriers of seven species of five different genera of obligate aerobic phototrophic bacteria have been characterized by biochemical and biophysical techniques. A tetrahemic reaction center (RC) bound cytochrome (cyt) was found in Erythromonas (Em.) ursincola, Sandaracinobacter (S.) sibiricus and Roseococcus (R.) thiosulfatophilus, but not in Erythromicrobium (E.) ezovicum, Erythromicrobium ramosum, Erythromicrobium hydrolyticum and Erythrobacter (Eb.) litoralis. In none of the studied species, photochemical activity was observed under anaerobic conditions. Under aerobic conditions, the photoinduced cyclic electron transfer involves a soluble c-type cyt for the seven species. The cyt content of soluble and membrane fractions is highly dependent upon the species. The Erythromicrobium species (E. ezovicum, E. ramosum and E. hydrolyticum) contains a major soluble cyt while the other species possess several soluble cyts, up to four in the case of Eb. litoralis. These cyts have been characterized in terms of midpoint potential and apparent molecular mass. The presence of cyt bc₁ complexes has been clearly detected in Eb. litoralis, E. hydrolyticum, E. ezovicum and E. ramosum. These last three species also contain a high midpoint potential (350 mV) membrane-bound cyt c of unknown function.     

Membrane-bound cytochromes in Chloroflexus aurantiacus studied by EPR.

The heme components of chlorosome-depleted membranes of the green-gliding bacterium Chloroflexus aurantiacus were studied by EPR spectroscopy. The four major species, which are present in approximately equimolar quantities, are characterized by the following gz values, redox midpoint potentials and orientations of heme planes with respect to the plane of the membrane: gz = 3.40, Em = +280 mV, 30 degrees; gz = 3.33, Em = 0 mV, 45 degrees; gz = 3.03, Em = +95 mV, 40-50 degrees and gz = 2.95, Em = +150 mV, 90 degrees. These four hemes were attributed to cytochrome c554, the membrane-bound immediate electron donor to the photosynthetic reaction centre in Chloroflexus. All hemes except that with the highest potential were able to undergo photooxidation at 4 K. The photooxidation of the lowest potential heme was stable, whereas that of the +95 mV and the +150 mV hemes reversed on increasing the temperature to 100 K in darkness, due to charge recombination. The ability to photooxidize these hemes at 4 K was lost upon aging of samples. The results demonstrate that a reaction-centre-associated tetraheme cytochrome subunit, analogous to that of purple bacteria, is also present in C. aurantiacus.     

The photosynthetic apparatus and photoinduced electron transfer in the aerobic phototrophic bacteria <Emphasis Type="Italic">Roseicyclus mahoneyensis</Emphasis> and <Emphasis Type="Italic">Porphyrobacter meromictius</Emphasis>

Photosynthetic electron transfer has been examined in whole cells, isolated membranes and in partially purified reaction centers (RCs) of Roseicyclus mahoneyensis, strain ML6 and Porphyrobacter meromictius, strain ML31, two species of obligate aerobic anoxygenic phototrophic bacteria. Photochemical activity in strain ML31 was observed aerobically, but the photosynthetic apparatus was not functional under anaerobic conditions. In strain ML6 low levels of photochemistry were measured anaerobically, possibly due to incomplete reduction of the primary electron acceptor (Q_A) prior to light excitation, however, electron transfer occurred optimally under low oxygen conditions. Photoinduced electron transfer involves a soluble cytochrome c in both strains, and an additional reaction center (RC)-bound cytochrome c in ML6. The redox properties of the primary electron donor (P) and Q_A of ML31 are similar to those previously determined for other aerobic phototrophs, with midpoint redox potentials of +463 mV and −25 mV, respectively. Strain ML6 showed a very narrow range of ambient redox potentials appropriate for photosynthesis, with midpoint redox potentials of +415 mV for P and +94 mV for Q_A. Cytoplasm soluble and photosynthetic complex bound cytochromes were characterized in terms of apparent molecular mass. Fluorescence excitation spectra revealed that abundant carotenoids not intimately associated with the RC are not involved in photosynthetic energy conservation.     

Kinetics and energetics of electron transfer in reaction centers of the photosynthetic bacterium Roseiflexus castenholzii

The kinetics and thermodynamics of the photochemical reactions of the purified reaction center (RC)-cytochrome (Cyt) complex from the chlorosome-lacking, filamentous anoxygenic phototroph, Roseiflexus castenholzii are presented. The RC consists of L- and M-polypeptides containing three bacteriochlorophyll (BChl), three bacteriopheophytin (BPh) and two quinones (Q(A) and Q(B)), and the Cyt is a tetraheme subunit. Two of the BChls form a dimer P that is the primary electron donor. At 285K, the lifetimes of the excited singlet state, P*, and the charge-separated state P(+)H(A)(-) (where H(A) is the photoactive BPh) were found to be 3.2±0.3 ps and 200±20 ps, respectively. Overall charge separation P*→→ P(+)Q(A)(-) occurred with ≥90% yield at 285K. At 77K, the P* lifetime was somewhat shorter and the P(+)H(A)(-) lifetime was essentially unchanged. Poteniometric titrations gave a P(865)/P(865)(+) midpoint potential of +390mV vs. SHE. For the tetraheme Cyt two distinct midpoint potentials of +85 and +265mV were measured, likely reflecting a pair of low-potential hemes and a pair of high-potential hemes, respectively. The time course of electron transfer from reduced Cyt to P(+) suggests an arrangement where the highest potential heme is not located immediately adjacent to P. Comparisons of these and other properties of isolated Roseiflexus castenholzii RCs to those from its close relative Chloroflexus aurantiacus and to RCs from the purple bacteria are made.     

Photo-induced cyclic electron transfer involving cytochrome bc1 complex and reaction center in the obligate aerobic phototroph Roseobacter denitrificans.

Flash-induced redox changes of b-type and c-type cytochromes have been studied in chromatophores from the aerobic photosynthetic bacterium Roseobacter denitrificans under redox-controlled conditions. The flash-oxidized primary donor P+ of the reaction center (RC) is rapidly re-reduced by heme H1 (Em,7 = 290 mV), heme H2 (Em,7 = 240 mV) or low-potential hemes L1/L2 (Em,7 = 90 mV) of the RC-bound tetraheme, depending on their redox state before photoexcitation. By titrating the extent of flash-induced low-potential heme oxidation, a midpoint potential equal to -50 mV has been determined for the primary quinone acceptor QA. Only the photo-oxidized heme H2 is re-reduced in tens of milliseconds, in a reaction sensitive to inhibitors of the bc1 complex, leading to the concomitant oxidation of a cytochrome c spectrally distinct from the RC-bound hemes. This reaction involves cytochrome c551 in a diffusional process. Participation of the bc1 complex in a cyclic electron transfer chain has been demonstrated by detection of flash-induced reduction of cytochrome b561, stimulated by antimycin and inhibited by myxothiazol. Cytochrome b561, reduced upon flash excitation, is re-oxidized slowly even in the absence of antimycin. The rate of reduction of cytochrome b561 in the presence of antimycin increases upon lowering the ambient redox potential, most likely reflecting the progressive prereduction of the ubiquinone pool. Chromatophores contain approximately 20 ubiquinone-10 molecules per RC. At the optimal redox poise, approximately 0.3 cytochrome b molecules per RC are reduced following flash excitation. Cytochrome b reduction titrates out at Eh < 100 mV, when low-potential heme(s) rapidly re-reduce P+ preventing cyclic electron transfer. Results can be rationalized in the framework of a Q-cycle-type model.     

Structural and functional characterization of the unusual triheme cytochrome bound to the reaction center of Rhodovulum sulfidophilum

The cytochrome bound to the photosynthetic reaction center of Rhodovulum sulfidophilum presents two unusual characteristics with respect to the well characterized tetraheme cytochromes. This cytochrome contains only three hemes because it lacks the peptide motif CXXCH, which binds the most distal fourth heme. In addition, we show that the sixth axial ligand of the third heme is a cysteine (Cys-148) instead of the usual methionine ligand. This ligand exchange results in a very low midpoint potential (-160 +/- 10 mV). The influence of the unusual cysteine ligand on the midpoint potential of this distal heme was further investigated by site-directed mutagenesis. The midpoint potential of this heme is upshifted to +310 mV when cysteine 148 is replaced by methionine, in agreement with the typical redox properties of a His/Met coordinated heme. Because of the large increase in the midpoint potential of the distal heme in the mutant, both the native and modified high potential hemes are photooxidized at a redox poise where only the former is photooxidizable in the wild type. The relative orientation of the three hemes, determined by EPR measurements, is shown different from tetraheme cytochromes. The evolutionary basis of the concomitant loss of the fourth heme and the down-conversion of the third heme is discussed in light of phylogenetic relationships of the Rhodovulum species triheme cytochromes to other reaction center-associated tetraheme cytochromes.     

Redox Properties of Thermus thermophilus ba3: Different Electron-Proton Coupling in Oxygen Reductases?

A comprehensive study of the thermodynamic redox behavior of the hemes of the ba₃ enzyme from Thermus thermophilus, a B-type heme-copper oxygen reductase, is presented. This enzyme, in contrast to those having a single type of heme, allows the B- and A-type hemes to be monitored separately by visible spectroscopy and the reduction potential of each heme to be determined unequivocally. The relative order of the midpoint reduction potentials of each center changed in the pH range from 6 to 8.4, and both hemes present a significant redox-Bohr effect. For instance, at pH 7, the midpoint reduction potentials of the hemes B and A₃ are 213 mV and 285 mV, respectively, whereas at pH 8.4, the order is reversed: 246 mV for heme B and 199 mV for heme A₃. The existence of redox anticooperativity was established by introducing a redox interaction parameter in a model of pairwise interacting redox centers.     

Direct electron transfer of redox proteins at the bare glassy carbon electrode

A simple system is presented for the microscale, direct voltammetry of redox proteins, typically 25 micrograms, in the absence of mediators and/or modifiers. The sample consists of a droplet of anaerobic solution laid onto an oversized disc of nitric-acid-pretreated glassy carbon as the working electrode. Very reproducible, Nernstian responses are obtained with horse heart cytochrome c. The midpoint potential Em (pH 7.0) is dependent on the ionic strength, ranging from $293 mV in 1 mM potassium phosphate to $266 mV in 0.1 M phosphate. At fixed buffer and cytochrome concentrations the magnitude of the voltammetric response is found to be independent of pH over six pH units around neutrality. It is suggested that the response of the present system is not complicated by pH-dependent properties of the electrode surface around physiological pH and, therefore, that the use of this system is practical in biochemically oriented studies. Direct, quasi-reversible responses have also been obtained at pH 7.0 (5 mM phosphate) from Desulfovibrio vulgaris. Hildenborough strain, tetraheme cytochrome c3 (pI = 10.0 at 4 C; 3 X Em = -0.32 mV, Em = -0.26 V), and cytochrome c553 (pI = 9.3; Em = +60 mV), and from Megasphaera elsdenii rubredoxin (pI congruent to 3; Em = -353 mV). The latter protein absorbs onto the glassy carbon surface, thus forming a system with possible applications in the electrochemical study of ferredoxin-linked enzymes.     

Fast oxidation of the primary electron acceptor under anaerobic conditions requires the organization of the photosynthetic chain of Rhodobacter sphaeroides in supercomplexes

The kinetics of reoxidation of the primary acceptor Q_a has been followed by measuring the changes in the fluorescence yield induced by a series of saturating flashes in intact cells of Rhodobacter sphaeroides in anaerobic conditions. At 0 °C, about half of Q_a⁻ is reoxidized in about 200 ms while reoxidation of the remaining fraction is completed in several seconds to minutes. The fast phase is associated with the transfer of ubiquinone formed at site Q_o of the cytochrome bc₁ complex while the slowest phase is associated with the diffusion of ubiquinone present in the membrane prior to the flash excitation. The biphasic kinetics of Q_a⁻ oxidation is interpreted assuming that the electron chain is organized in supercomplexes that associate two RCs and one cyt bc₁ complex, which allows a fast transfer of quinone formed at the level of cyt bc₁ complex to the RCs. In agreement with this model, the fast phase of Q_a⁻ reoxidation is inhibited by myxothiazol, a specific inhibitor of cyt bc₁. The PufX-deleted mutant displays only the slowest phase of Q_a⁻ oxidation; it is interpreted by the lack of supramolecular organization of the photosynthetic chain that leads to a larger average distance between cyt bc₁ and RCs.     

Sulfhydryl modifying reagents inhibit Q A − oxidation in reaction centers from Rhodobacter sphaeroides and Capsulatus,but not Rhodopseudomonas viridis

The effects of various sulfhydryl-modifying reagents on reaction centers (RCs) from purple photosynthetic bacteria have been examined, with particular emphasis on the activity of the acceptor quinones, Q_A and Q_B, comprising the two electron gate. Mercurial reagents, especially p-chloromercuribenzenesulfonate (pCMBS), were effective in inhibiting Q_B function in RCs from Rhodobacter sphaeroides and Rb. capsulatus, but not in Rhodopseudomonas viridis. The inhibition was fully reversible by dialysis against dithiothreitol (DTT). The effect on Q_B function was not an apparent one mediated by an alteration in the redox potential of Q_A. N-ethylmaleimide (NEM) had no effect on any of the quinone functions, even at very high concentrations. Comparison of the X-ray structures of the RCs from Rb. sphaeroides and Rp. viridis and the known amino acid sequences for all three bacterial RCs suggest that a cysteine residue at position 108 in the L subunit of the Rhodobacter species is the most likely candidate for the site of action of the mercurial reagents. This was strongly supported by the absence of any effect of pCMBS on a site specific mutation of Rb. sphaeroides (L108CS) with residue L108 changed from cysteine to serine. These results imply a long distance (>20 Å) effect on the functioning of Q_B, perhaps involving a relatively gross structural alteration.     

Heme attachment motif mobility tunes cytochrome c redox potential

Hydrogen exchange (HX) rates and midpoint potentials (Em) of variants of cytochrome c from Pseudomonas aeruginosa (Pa cyt c551) and Hydrogenobacter thermophilus (Ht cyt c552) have been characterized in an effort to develop an understanding of the impact of properties of the Cys-X-X-Cys-His pentapeptide c-heme attachment (CXXCH) motif on heme redox potential. Despite structural conservation of the CXXCH motif, Ht cyt c552 exhibits a low level of protection from HX for amide protons within this motif relative to Pa cyt c551. Site-directed mutants have been prepared to determine the structural basis for and functional implications of these variations on HX behavior. The double mutant Ht-M13V/K22M displays suppressed HX within the CXXCH motif as well as a decreased Em (by 81 mV), whereas the corresponding double mutant of Pa cyt c551 (V13M/M22K) exhibits enhanced HX within the CXXCH pentapeptide and a modest increase in Em (by 30 mV). The changes in Em correlate with changes in axial His chemical shifts in the ferric proteins reflecting the extent of histidinate character. Thus, the mobility of the CXXCH pentapeptide is found to impact the His-Fe(III) interaction and therefore the heme redox potential.     

On the depletion and reconstitution of both QA and metal in reaction centers of the photosynthetic bacterium Rb. sphaeroides R-26

Four possible ways to prepare Q_A-depleted, Fe-depleted and Q_A-reconstituted RCs were studied: (1) first depleting the Fe, then depleting Q_A and finally reconstituting Q_A (D-Fe, D-Q, R-Q), (2) first depleting Q_A, then depleting the Fe and finally reconstituting Q_A (D-Q, D-Fe, R-Q), (3) first depleting Q_A, then reconstituting Q_A and finally depleting Fe (D-Q, R-Q, D-Fe), (4) first depleting Q_A, then depleting the Fe and reconstituting Q_A in the same step (D-Q, D-Fe-R-Q). Our results showed that: method (1) results in the irreversible loss of photochemical activity; method (2) and (3) result in low recovery of the photochemical activity and poor yield of Fe-depleted, Q_A-reconstituted RCs; method (4) gives surprisingly good results. This method allows for the first time to prepare the Q_A-depleted, Fe-depleted, Q_A-reconstituted RCs with high recovery of the photochemical activity and good yield. The sample has 98% of photochemical activity (yield of P⁺ Q_A ^-) compared with that of the native RCs and shows strong polarization of the EPR signal of Q_A ^- under continuous illumination at 5K. The decay halftime of I^- is slow (5 ns) compared with that of the native RCs, but it is the same as that measured for the RCs from which only iron is removed. These results indicate that the depletion of iron and the reconstitution of Q_A have been successful. Reconstitution of the Q_A-depleted, Fe-depleted and Q_A-reconstituted RCs with Zn²⁺ gives also the spin-polarized Q_A ^-, and yields the same decay of I^- (halftime 200 ps) as that of the native RCs.Abbreviations LDAO lauryldimethylamine N-oxide - EDTA ethylenediaminetetraacetic acid - BSA albumin bovine - TL buffer 10 mM Tris.HCl, 0.1% LDAO and 0.1 mM EDTA     

Strategic roles of axial histidines in structure formation and redox regulation of tetraheme cytochrome c3

Tetraheme cytochrome c 3 (cyt c 3) exhibits extremely low reduction potentials and unique properties. Since axial ligands should be the most important factors for this protein, every axial histidine of Desulfovibrio vulgaris Miyazaki F cyt c 3 was replaced with methionine, one by one. On mutation at the fifth ligand, the relevant heme could not be linked to the polypeptide, revealing the essential role of the fifth histidine in heme linking. The fifth histidine is the key residue in the structure formation and redox regulation of a c-type cytochrome. A crystal structure has been obtained for only H25M cyt c 3. The overall structure was not affected by the mutation except for the sixth methionine coordination at heme 3. NMR spectra revealed that each mutated methionine is coordinated to the sixth site of the relevant heme in the reduced state, while ligand conversion takes place at hemes 1 and 4 during oxidation at pH 7. The replacement of the sixth ligand with methionine caused an increase in the reduction potential of the mutated heme of 222-244 mV. The midpoint potential of a triheme H52M cyt c 3 is higher than that of the wild type by approximately 50 mV, suggesting a contribution of the tetraheme architecture to the lowering of the reduction potentials. The hydrogen bonding of Thr24 with an axial ligand induces a decrease in reduction potential of approximately 50 mV. In conclusion, the bis-histidine coordination is strategically essential for the structure formation and the extremely low reduction potential of cyt c 3.     

Modulation of the redox state of quinones by light in Rhodobacter sphaeroides under anaerobic conditions

Illumination of intact cells of Rhodobacter sphaeroides under anaerobic conditions has a dual effect on the redox state of the quinone pool. A large oxidation of the quinone pool is observed during the first seconds following the illumination. This oxidation is suppressed by the addition of an uncoupler in agreement with a light-induced reverse electron transfer at the level of the complex I, present both in the non-invaginated part of the membrane and in the chromatophores. At longer dark times, this illumination increases the reducing power of the cells leading to a significant reduction of the others reaction centers (RCs). From the observation that a significant proportion of RCs could be reduced by the preillumination without affecting the numbers of charge separation for the RCs, we conclude that there is no rapid thermodynamic equilibrium between the quinones present in the non-invaginated part of the membrane and those localized in the chromatophores. Under anaerobic conditions where the chromatophores quinone pool is fully reduced, we deduce, on the basis of flash-induced fluorescence kinetics, that the reduced RCs are exclusively reoxidized by the quinone generated at the Q _o site of the cyt bc ₁ complex. The supramolecular association between a dimeric RC-LHI complex and one cyt bc ₁ complex allows the confinement of a quinone between the RC-LHI directly associated to the cyt bc ₁ complex.     

The oxidation-reduction potentials of cytochrome o + c4 and cytochrome o purified from Azotobacter vinelandii.

Oxidation-reduction titrations of Azotobacter vinelandii cytochrome o + c4 and cytochrome o were performed with simultaneous potential and absorbance measurements under anaerobic conditions. Cytochrome c4 has a midpoint potential (Em, 7.4) of 260mV and purified cytochrome o has an Em, 7.4 of -18mV. Little change in the midpoint potential of cytochrome o was observed when titrated in the pH range 6.2--9.8.     

Photosynthetic electrogenic events in native membranes ofChloroflexus aurantiacus. Flash-induced charge displacements within the reaction center-cytochromec 554 complex

The thermophilic phototrophChloroflexus aurantiacus possesses a photosynthetic reaction center (RC) containing a pair of menaquinones as primary (Q_A) and secondary (Q_B) electron acceptors and a bacteriochlorophyll dimer (P) as a primary donor. A tetraheme cytochromec ₅₅₄ with two high(H)- and two low(L)-potential hemes operates as an immediate electron donor for P. The following equilibrium E_m,7 values were determined by ESR for the hemes in whole membrane preparations: 280 mV (H1), 150 mV (H2), 95 mV (L1) and 0 mV (L2) (Van Vliet et al. (1991) Eur. J. Biochem. 199: 317–323). Partial electrogenic reactions induced by a laser flash inChl. aurantiacus chromatophores adsorbed to a phospholipid-impregnated collodion film were studied electrometrically at pH 8.3. The photoelectric response included a fast phase of generation ( < 10 ns, phase A). It was ascribed to the charge separation between P⁺ and Q_A ^– as its amplitude decreased both at high and low E_h values (E_m,high=360±10 mV, estimated E_m,low\s-160 mV) in good agreement with E_m values for P/P⁺ and Q_A/Q_A ^– redox couples. A slower kinetic component appeared upon reduction of the cytochromec ₅₅₄ hemes (phase C). With H1 reduced before the flash the amplitude of phase C was equal to 15–20% of that of phase A and its rise time was 1.2–1.3 s: we attribute this phase to the electrogenic electron transfer from H1 to P⁺. Pre-reduction of H2 decreased the value to about 700–800 ns and increased the amplitude of phase C to 30–35% of that of phase A. Pre-reduction of L1 further accelerated phase C (up to of 500 ns) and induced a reverse electrogenic phase with of 12 s and amplitude equal to 10% of phase A. Upon pre-reduction of L2 the rise time of phase C was decreased to about 300 ns and its amplitude decreased by 30%. The acceleration in the onset of phase C is explained by the acceleration of the rate-limiting H1 P electrogenic reaction after reduction of the other hemes due to their electrostatic influence; a P-H1-(L1-L2)-H2 alignment of redox centers with an approximately rhombic arrangement of the cytochromec ₅₅₄ hemes is proposed. The observed reverse phase is ascribed to the post-flash charge redistribution between the hemes. Redox titration of the amplitude of phase C yielded the E_m,8.3 values of H1, H2 and L2 hemes: 340±10 mV for H1, 160±20 mV for H2 and –40±40 mV for L2.     

Direct and mediated electron transfer between intact succinate:quinone oxidoreductase from Bacillus subtilis and a surface modified gold electrode reveals redox state-dependent conformational changes

Succinate:quinone oxidoreductase (SQR) from Bacillus subtilis consists of two hydrophilic protein subunits comprising succinate dehydrogenase, and a di-heme membrane anchor protein harboring two putative quinone binding sites, Q_p and Q_d. In this work we have used spectroelectrochemistry to study the electronic communication between purified SQR and a surface modified gold capillary electrode. In the presence of two soluble quinone mediators the midpoint potentials of both hemes were revealed essentially as previously determined by conventional redox titration (heme b_H, E_m = + 65 mV, heme b_L, E_m = − 95 mV). In the absence of mediators the enzyme still communicated with the electrode, albeit with a reproducible hysteresis, resulting in the reduction of both hemes occurring approximately at the midpoint potential of heme b_L, and with a pronounced delay of reoxidation. When the specific inhibitor 2-n-heptyl-4 hydroxyquinoline N-oxide (HQNO), which binds to Q_d in B. subtilis SQR, was added together with the two quinone mediators, rapid reductive titration was still possible which can be envisioned as an electron transfer occurring via the HQNO insensitive Q_p site. In contrast, the subsequent oxidative titration was severely hampered in the presence of HQNO, in fact it completely resembled the unmediated reaction. If mediators communicate with Q_p or Q_d, either event is followed by very rapid electron redistribution within the enzyme. Taken together, this strongly suggests that the accessibility of Q_p depended on the redox state of the hemes. When both hemes were reduced, and Q_d was blocked by HQNO, quinone-mediated communication via the Q_p site was no longer possible, revealing a redox-dependent conformational change in the membrane anchor domain.     

Redox titration of electron acceptor Q and the plastoquinone pool in Photosystem II

The primary photochemical quencher Q and the secondary electron acceptor pool in Photosystem II have been titrated. We used particles of Scenedesmus mutant No. 8 that lack System I and allowed the system to equilibrate with external redox mediators in darkness prior to measurement of the fluorescence rise curve.The titration of Q, as indicated by the dark level of F_i, occurs in two discrete steps. The high-potential component (Q_h) has a midpoint potential of +68 mV (pH 7.2) and accounts for ～67% of Q. The pH sensitivity of the midpoint potential is ?60 mV, indicating the involvement of 1 $\text{H}{}^{\mathrm{+}}\text{e}$. The low-potential component (Q₁) accounts for the remaining 33% of Q and shows a midpoint potential near?300 mV (pH 7.2).The plastoquinone pool, assayed as the half-time of the fluorescence rise curve, titrates as a single component with a midpoint potential 30–40 mV more oxidizing than that of Q_h, i.e., at 106 mV (pH 7.2). The E_m shows a pH sensitivity of ?60 mV/pH unit, indicating the involvement of 1 $\text{H}{}^{\mathrm{+}}\text{e}$. The observation that all 12–14 electron equivalents in the pool titrate as a single component indicates that the heterogeneity otherwise observed in the secondary acceptor system is a kinetic rather than a thermodynamic property.Illumination causes peculiar, and as yet unclarified, changes of both Q and the secondary pool under anaerobic conditions that are reversed by oxygen.     

Carboxylate shifts steer interquinone electron transfer in photosynthesis

Understanding the mechanisms of electron transfer (ET) in photosynthetic reaction centers (RCs) may inspire novel catalysts for sunlight-driven fuel production. The electron exit pathway of type II RCs comprises two quinone molecules working in series and in between a non-heme iron atom with a carboxyl ligand (bicarbonate in photosystem II (PSII), glutamate in bacterial RCs). For decades, the functional role of the iron has remained enigmatic. We tracked the iron site using microsecond-resolution x-ray absorption spectroscopy after laser-flash excitation of PSII. After formation of the reduced primary quinone, Q_A⁻, the x-ray spectral changes revealed a transition (t_½ ≈ 150 μs) from a bidentate to a monodentate coordination of the bicarbonate at the Fe(II) (carboxylate shift), which reverted concomitantly with the slower ET to the secondary quinone Q_B. A redox change of the iron during the ET was excluded. Density-functional theory calculations corroborated the carboxylate shift both in PSII and bacterial RCs and disclosed underlying changes in electronic configuration. We propose that the iron-carboxyl complex facilitates the first interquinone ET by optimizing charge distribution and hydrogen bonding within the Q_AFeQ_B triad for high yield Q_B reduction. Formation of a specific priming intermediate by nuclear rearrangements, setting the stage for subsequent ET, may be a common motif in reactions of biological redox cofactors.     

Usefulness of thermoluminescence in herbicide research

Many herbicides of different chemical structure inhibit photosynthetic electron flow by interrupting the photosyn‐thetic electron flow by interrupting the photosynthetic electron transport chain between the primary acceptor (Q_A) and the secondary acceptor (Q_B) of photosystem 2 (PS2). Thermoluminescence (TL) originates from PS2, and the bands of the glow curve can be related to the charge recombination between positively charged donors and negatively charged acceptors. The glow curve of TL is strongly influenced by addition of PS2 herbicides. The herbicide treatment shifts the peak position and activation energy of the TL band related to Q_A, suggesting that herbicide binding affects the midpoint redox potential not only of Q _B but also that of Q_A. On the basis of the band shift the herbicides of various chemical structures can be classified into different “thermodynamical” groups which relfect the differences in the binding properties of these herbicides. As a new approach TL seems to be a useful technique in studying the mechanism and site of action of herbicides that inhibit electron transport of PS2.