Paul Mathis

Intraprotein radical transfer during photoactivation of DNA photolyase

Amino-acid radicals play key roles in many enzymatic reactions. Catalysis often involves transfer of a radical character within the protein, as in class I ribonucleotide reductase where radical transfer occurs over 35 Å, from a tyrosyl radical to a cysteine. It is currently debated whether this kind of long-range transfer occurs by electron transfer, followed by proton release to create a neutral radical, or by H-atom transfer, that is, simultaneous transfer of electrons and protons. The latter mechanism avoids the energetic cost of charge formation in the low dielectric protein, but it is less robust to structural changes than is electron transfer. Available experimental data do not clearly discriminate between these proposals. We have studied the mechanism of photoactivation (light-induced reduction of the flavin adenine dinucleotide cofactor) of Escherichia coli DNA photolyase using time-resolved absorption spectroscopy. Here we show that the excited flavin adenine dinucleotide radical abstracts an electron from a nearby tryptophan in 30 ps. After subsequent electron transfer along a chain of three tryptophans, the most remote tryptophan (as a cation radical) releases a proton to the solvent in about 300 ns, showing that electron transfer occurs before proton dissociation. A similar process may take place in photolyase-like blue-light receptors.

Primary radical pair in the Photosystem II reaction centre

Abstract Primary Photosystem II (PS II) reactions have been studied by flash absorption spectroscopy with nanosecond resolution. In isolated reaction centers which are devoid of the 47- and 43-kDa chlorophyll-binding polypeptides, an absorption change (ΔA) is induced immediately by a flash. The difference spectrum (450–560 nm, 820 nm) indicates the formation of a radical pair (P-680+, Pheo−) decaying with t 1 2 = 40 ns at 120 K. At 140 ns after the flash the ΔA can be attributed to the triplet state of P-680 (3P-680) and of a carotenoid (3Car, λmax = 545 nm). 3Car is formed with a yield of ≈3% and it rises with t 1 2 ≈ 12 ns . At 276 K, the radical pair decays, with t 1 2 ≈ 32 ns . One radical pair is formed per 20 chlorophylls. The data are best interpreted if 3P-680 is formed as a product of radical pair recombination with a yield of 23% at 276 K (80% at 10 K) and if 3Car is formed only in a minority of pigment complexes. The ΔA at 820 nm disappear under conditions (addition of dithionite and methyl viologen, plus continuous illumination) designed to reduce pheophytin. The signals reappear after turning off the continuous light. More intact PS II particles (core complex) were also studied. At 820 nm, the data show that P-680+ is formed under oxidizing conditions and is re-reduced in the microsecond time range. Under reducing conditions the primary biradical decays with t 1 2 = 25 ns . 3P-680 is formed, with properties analogous to those in isolated reaction centers. 3Car is also formed (λmax = 535 nm). The results show that the PS II reaction center has strong functional analogies with the reaction center of purple bacteria. The behavior of the carotenoid is a remarkable exception. The primary biradical decays much more slowly than expected on the basis of fluorescence measurements.

Kinetics of reduction of the oxidized primary electron donor of Photosystem II in spinach chloroplasts and in Chlorella cells in the microsecond and nanosecond time ranges following flash excitation

Absorption changes (deltaA) at 820 nm, following laser flash excitation of spinach chloroplasts and Chlorella cells, were studied in order to obtain information on the reduction time of the photooxidized primary donor of Photosystem II at physiological temperatures. In the microsecond time range the difference spectrum of deltaA between 750 and 900 nm represents a peak at 820 nm, attributable to a radical-cation of chlorophyll a. In untreated dark-adapted material the signal can be attributed solely to P+-700; it decays in a polyphasic manner with half-times of 17 microseconds, 210 microseconds and over 1 ms. The oxidized primary donor of Photosystem II (P+II) is not detected with a time resolution of 3 microseconds. After treatment with 3--10 mM hydroxylamine, which inhibits the donor side of Photosystem II, P+II is observed and decays biphasically (a major phase with t1/2=20--40 microseconds, and a minor phase with t1/2 congruent to 200 microseconds), probably by reduction by an accessory electron donor. In the nanosecond range, which was made accessible by a new fast-response flash photometer operating at 820 nm, it was found the P+II is reduced with a half-time of 25--45 ns in untreated dark-adapted chloroplasts. It is assumed that the normal secondary electron donor is responsible for this fast reduction.

Electron acceptors associated with P-700 in Triton solubilized Photosystem I particles from spinach chloroplasts

Flash-induced absorption changes of Triton-solubilized Photosystem I particles from spinach were studied under reducing and/or illumination conditions that serve to alter the state of bound electron acceptors. By monitoring the decay of P-700 following each of a train of flashes, we found that P-430 or components resembling it can hold 2 equivalents of electrons transferred upon successive illuminations. This requires the presence of a good electron donor, reduced phenazine methosulfate or neutral red, otherwise the back reaction of P-700+ with P-430 occurs in about 30 ms. If the two P-430 sites, designated Centers A and B, are first reduced by preilluminating flashes or chemically by dithionite under anaerobic conditions, then subsequent laser flashes generate a 250 microseconds back reaction of P-700+, which we associate with a more primary electron acceptor A2. In turn, when A2 is reduced by background (continuous) illumination in presence of neutral red and under strongly reducing conditions, laser flashes then produce a much faster (3 microseconds) back reaction at wavelengths characteristic of P-700. We associate this with another more primary electron acceptor, A1, which functions very close to P-700. The organization of these components probably corresponds to the sequence P-700-A1-A2-P-430[AB]. The relation of the optical components to acceptor species detected by EPR, by electron-spin polarization or in terms of peptide components of Photosystem I is discussed. Preliminary experiments with broken chloroplasts suggest that an analogous situation occurs there, as well.

The effect of pH on the reduction kinetics of P-680 in tris-treated chloroplasts

The primary donor of Photosystem II (PS II), P-680, was photo-oxidized by a short flash and its rate of reduction was measured at different pH values by following the recovery of the absorption change at 820 nm in chloroplasts pretreated with a high concentration of Tris. The re-duction is biphasic with a fast phase (dominant after the first flash) attributed to the donation by a donor, D1, and a slow phase (usually dominant after the second flash) attributed to a back-reaction with the primary acceptor. It is found that pH has a strong influence on the donation from D1 (PI = 2 MICROSECONDS AT PH 9, 44 microseconds at pH 4), but no influence on the back reaction (pi approximately 200 microseconds). pH also influences the stability of the charge separation since the contribution of donation from D1 at the second flash increases at lower pH, getting close to 100% at pH 4.

Flash‐induced absorption changes in photosystem I at low temperature: evidence that the electron acceptor A1 is vitamin K1

Low temperature flash absorption spectroscopy has been applied to elucidate the chemical nature of the secondary electron acceptor A1 of photosystem I (PS‐I). The flash‐induced absorption changes measured in digitonin‐fractionated spinach PS‐I particles at 10 K between 240 and 525 nm are shown to comprise a major decay phase with t ~ 150 μs which has been attributed to the recombination reaction P‐700+·A1 → P‐700·A1 [(1984) Biochim. Biophys. Acta 767, 404‐414]. We present the absorption difference spectrum of this reaction and demonstrate that it contains contributions in the ultraviolet due to A1, which are characteristic of vitamin K1 (phylloquinone).

Quantum yield and rate of formation of the carotenoid triplet state in photosynthetic structures

The formation of the triplet state of carotenoids (detected by an absorption peak at 515 nm) and the photo-oxidation of the primary donor of Photosystem II, P-680 (detected by an absorption increase at 820 nm) have been measured by flash absorption spectroscopy in chloroplasts in which the oxygen evolution was inhibited by treatment with Tris. The amount of each transient form has been followed versus excitation flash intensity (at 590 or 694 nm). At low excitation energy the quantum yield of triplet formation (with the Photosystem II reaction center in the state Q-) is about 30% that of P-680 photo-oxidation. The yield of carotenoid triplet formation is higher in the state Q- than in the state Q, in nearly the same proportion as chlorophyll alpha fluorescence. It is concluded that, for excited chlorophyll alpha, the relative rates of intersystem crossing to the triplet state and of fluorescence emission are the same in vivo as in organic solvent. At high flash intensity the signal of P-680+ completely saturates, whereas that of carotenoid triplet continues to increase. The rate of triplet-triplet energy transfer from chlorophyll alpha to carotenoids has been derived from the rise time of the absorption change at 515 nm, in chloroplasts and in several light-harvesting pigment-protein complexes. In all cases the rate is very high, around 8 . 10(7) s-1 at 294 K. It is about 2--3 times slower at 5 K. The transitory formation of chlorophyll triplet has been verified in two pigment-protein complexes, at 5 K.

Photosystem I photochemistry at low temperature. Heterogeneity in pathways for electron transfer to the secondary acceptors and for recombination processes

Abstract Electron transport has been studied by flash absorption and EPR spectroscopies at 10–30 K in Photosystem I particles prepared with digitonin under different redox conditions. In the presence of ascorbate, an irreversible charge separation is progressively induced at 10 K between P-700 and iron-sulfur center A by successive laser flashes, up to a maximum which corresponds to about two-thirds of the reaction centers. In these centers, heterogeneity of the rate for center A reduction is also shown. In the other third of reaction centers, the charge separation is reversible and relaxes with a t 1 2 ≈ 120 μ s . When the iron-sulfur centers A and B are prereduced, the 120 μs relaxation becomes the dominant process (70–80% of the reaction centers), while a slow component ( t 1 2 = 50–400 ms ) reflecting the recombination between P-700+ and center X− occurs in a minority of reaction centers (10–15%). Flash absorption and EPR experiments show that the partner of P-700+ in the 120 μs recombination is neither X nor a chlorophyll but more probably the acceptor A−1 as defined by Bonnerjea and Evans (Bonnerjea, J. and Evans, M.C.W. (1982) FEBS Lett. 148, 313–316). The role of center X in low-temperature electron flow is also discussed.

The effect of magnesium and phosphorylation of light-harvesting chlorophyll ab-protein on the yield of P-700-photooxidation in pea chloroplasts

The yield of P-700 photooxidation has been studied in isolated chloroplast membranes by measuring the extent of the flash-induced absorption increase at 820 nm (ΔA820) in the microsecond time range. The extent of ΔA820 induced by non-saturating laser flashes was increased by the following treatments. (1) Suspension of chloroplast membranes in Mg2+ free medium (plus 15 mM K+) which leads to unstacking of grana (as detected by a decrease in chlorophyll fluorescence). (2) Reduction of Q, the primary acceptor of Photosystem II, in the presence of 20 μM 3-(3,4 dichlorophenyl)-1,1-dimethylurea by a saturating xenon flash, fired 300 ms before the laser flash. (3) Phosphorylation of light harvesting chlorophyll ab-protein complex, which occurs in the presence of ATP after activation of protein kinase in the dark with NADPH and ferredoxin. We conclude that the Mg2+ concentration, the redox state of Q and the protein-phosphorylation all can control the photochemical efficiency of P-700 photooxidation in isolated chloroplasts, and we discuss these results in relation to control of excitation energy distribution between the two photosystems. We also discuss the significance of these results in relation to the regulation of photosynthetic electron transport in vivo.

Flash-induced absorption changes in Photosystem I, Radical pair or triplet state formation?

Abstract Absorption changes induced in chlorophyll protein (CP 1) particles by short laser flashes have been analyzed in order to decide whether a state lasting for a few microseconds at 21°C or 800 μs at 10 K corresponds to the biradical P-700+ ... A−1 (A1 being a chlorophyll a) or to a triplet state produced in a submicrosecond recombination of the preceding state. At 21°C the spectrum of the flash-induced ΔA (720–870 nm) presents a flat-topped band from 740 to 820 nm, clearly different from that of P-700+. A saturation curve (ΔA vs. laser energy), obtained with a 2 or 10 ns laser pulse, indicates that ΔA saturates at a value 2- or 3-times smaller than that expected on the basis of the chemical oxidation of P-700. At 21°C the size of flash-induced ΔA is slightly decreased (5–15%) when the sample is subjected to a 400 G magnetic field. The kinetics of decay are not affected; they are not affected either by the oxygen concentration. At 10 K the spectrum of the flash-induced ΔA has been measured between 650 and 1700 nm. Between 650 and 720 nm, the spectrum presents only one major negative peak at 702 nm; it is quite different from that due to the chemical oxidation of P-700 (which has additional peaks at 688 and 677 nm). Between 720 and 870 nm, the spectrum is identical to that obtained at 21°C. Above 870 nm, the spectrum includes a broad band around 1250 nm, which is absent in P-700+. A saturation curve leads to a maximum ΔA greater than that at 21°C and which is also greater with a 1 μs dye laser flash than with a 10 ns ruby laser flash. An analysis of the spectral data indicates that these do not fit correctly with the hypothesis of a contribution of P-700+ and of a chlorophyll a anion radical. They fit more closely with the hypothesis of a triplet state of P-700, a hypothesis which is discussed in relation to other experimental data.