Klaus Brettel

The Cryptochromes: Blue Light Photoreceptors in Plants and Animals

Cryptochromes are flavoprotein photoreceptors first identified in Arabidopsis thaliana, where they play key roles in growth and development. Subsequently identified in prokaryotes, archaea, and many eukaryotes, cryptochromes function in the animal circadian clock and are proposed as magnetoreceptors in migratory birds. Cryptochromes are closely structurally related to photolyases, evolutionarily ancient flavoproteins that catalyze light-dependent DNA repair. Here, we review the structural, photochemical, and molecular properties of cry-DASH, plant, and animal cryptochromes in relation to biological signaling mechanisms and uncover common features that may contribute to better understanding the function of cryptochromes in diverse systems including in man.

Electron transfer and arrangement of the redox cofactors in photosystem I

2. Summary of some basic features of photosystem I . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324 2.1. Composition and architecture of the photosystem I complex . . . . . . . . . . . . . . . . . . . 324 2.2. Antenna system and excitation energy transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 2.3. The electron transport chain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 2.4. Relationship with other ‘type I’ reaction centres . . . . . . . . . . . . . . . . . . . . . . . . . . 326

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.

Electron transfer in photosystem I

This mini-review focuses on recent experimental results and questions, which came up since the last more comprehensive reviews on the subject. We include a brief discussion of the different techniques used for time-resolved studies of electron transfer in photosystem I (PS I) and relate the kinetic results to new structural data of the PS I reaction centre.

Nanosecond reduction kinetics of photooxidized chlorophyll-aII (P-680) in single flashes as a probe for the electron pathway, H+-release and charge accumulation in the O2-evolving complex☆

(1) The re-reduction kinetics of chlorophyll a+II (P-680+) after the first, second, third etc. flash given to dark-adapted subchloroplasts have been monitored at 824 nm in the nanosecond range. After the first flash and, again, after the fifth flash, the re-reduction of chlorophyll a+II (Chl a+II) in the nanosecond range is nearly monophasic with t12 ≈ 23 ns. After the second and third flash, the re-reduction is significantly slower and biphasic; it can be well-adapted with t12 ≈ 50 ns and ≈260 ns. After the 4th flash, the re-reduction kinetics of Chl a+II are intermediate between the first/fifth and second/third flash. A similar dependence on flash number was obtained with a sample of oxygen-evolving Photosystem II particles from Synechococcus sp. (2) Considering the populations of the S-states of the O2-evolving complex before each flash, the following correlation of S-states to Chl a+II reduction kinetics and electron transfer times, respectively, is obtained: in state S0 as well as in state S1 Chl a+II is reduced with t12 ≈ 23 ns, whereas in state S2 as well as state S3 a biphasic reduction with t12 ≈ 50 ns and ≈260 ns (ratio of the amplitudes ≈1:1) occurs. (3) The observed multiphasic Chl a+II reduction under repetitive excitation is quantitatively explained by a superposition of the individual electron transfer times. (4) We suggest that the retardation of electron transfer to Chl a+II in states S2 and S3 as compared to S0 and S1 is caused by Coulomb attraction by one positive charge located in the O2-evolving complex. A positively charged O2-evolving complex in states S2 and S3 can be explained if the electron release pattern (1,1,1,1) is accompanied by a proton release pattern (1,0,1,2) for the transitions (S0 → S1, S1 → S2, S2 → S3, S3 → S0). (5) A kinetic model based on linear electron transfer from the O2-evolving complex (S) to Chl a+II via two carriers, D1 and D2, makes a quantitative description of the experimental results possible. (6) According to the kinetic model, the retardation of electron transfer to Chl a+II in states S2 and S3 is reflected by an increase in the change of standard free energy, ΔG0, of the reaction Chl a+IID1D2SChl aIID1+D2S from ΔG0 ≈ − 90 meV in states S0 and S1 to ΔG0 ≈ − 20 meV in states S2 and S3. (7) This increase by ≈ 70 meV can be quantitatively explained by the Coulomb potential of the positive charge in the O2-evolving complex, estimated by using the point charge approximation.

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).

Reaction mechanisms of DNA photolyase

DNA photolyase uses visible light and a fully reduced flavin cofactor FADH(-) to repair major UV-induced lesions in DNA, the cyclobutane pyrimidine dimers (CPDs). Electron transfer from photoexcited FADH(-) to CPD, splitting of the two intradimer bonds, and back electron transfer to the transiently formed flavin radical FADH° occur in overall 1ns. Whereas the kinetics of FADH° was resolved, the DNA-based intermediates escaped unambiguous detection yet. Another light reaction, named photoactivation, reduces catalytically inactive FADH° to FADH(-) without implication of DNA. It involves electron hopping along a chain of three tryptophan residues in 30ps, as elucidated in detail by transient absorption spectroscopy. The same triple tryptophan chain is found in cryptochrome blue-light photoreceptors and may be involved in their primary photoreaction.

Electron transfer from A−1 to an iron‐sulfur center with t = 200 ns at room temperature in photosystem I Characterization by flash absorption spectroscopy

Forward electron transfer in intact photosystem I particles from Synechococcus sp. at room temperature has been studied by flash absorption spectroscopy with a time resolution of 5 ns. A kinetic phase with t = 200 ns was resolved and characterized by its absorption difference spectrum between 325 and 495 nm. This phase is attributed to electron transfer from the reduced redox center A1 to an iron‐sulfur center, probably Fx. The difference spectrum for the reduction of A1 is evaluated and the previously proposed identification of A1 with vitamin K1 is discussed.

Rapid formation of the stable tyrosyl radical in photosystem II.

Two symmetrically positioned redox active tyrosine residues are present in the photosystem II (PSII) reaction center. One of them, TyrZ, is oxidized in the ns–μs time scale by P680+ and reduced rapidly (μs to ms) by electrons from the Mn complex. The other one, TyrD, is stable in its oxidized form and seems to play no direct role in enzyme function. Here, we have studied electron donation from these tyrosines to the chlorophyll cation (P680+) in Mn-depleted PSII from plants and cyanobacteria. In particular, a mutant lacking TyrZ was used to investigate electron donation from TyrD. By using EPR and time-resolved absorption spectroscopy, we show that reduced TyrD is capable of donating an electron to P680+ with t1/2 ≈ 190 ns at pH 8.5 in approximately half of the centers. This rate is ≈105 times faster than was previously thought and similar to the TyrZ donation rate in Mn-depleted wild-type PSII (pH 8.5). Some earlier arguments put forward to rationalize the supposedly slow electron donation from TyrD (compared with that from TyrZ) can be reassessed. At pH 6.5, TyrZ (t1/2 = 2–10 μs) donates much faster to P680+ than does TyrD (t1/2 > 150 μs). These different rates may reflect the different fates of the proton released from the respective tyrosines upon oxidation. The rapid rate of electron donation from TyrD requires at least partial localization of P680+ on the chlorophyll (PD2) that is located on the D2 side of the reaction center.

Optical characterization of the immediate electron donor to chlorophyll a+II in O2-evolving photosystem II complexes Tyrosine as possible electron carrier between chlorophyll aII and the water-oxidizing manganese complex

The number and chemical nature of the electron carrier(s) between Chl a II and the water‐oxidizing enzyme, S, were analyzed through flash‐induced absorption changes in the UV with nanosecond time resolution. (i) At all wavelengths where the reaction of the donor with Chl a + II has been characterized, this donor is oxidized in the nanosecond time range in exact accordance with the reduction kinetics of Chl a + II. The donor is in turn re‐reduced with t > 10,μs, i.e. in the range where S is oxidized. From this time course it is concluded that there exists only one electron carrier between Chl a + II and S. (ii) The UV‐diference spectrum due to the electron transfer from the immediate donor to Chl a + II in the nanosecond time range in O2‐evolving PS II complexes is characterized by a maximum around 260 nm and smaller minimum around 310 nm. This spectrum is identical with that observed for the reaction of the donor with Chl a + II in the microsecond time range in Tris‐treated PS II. Therefore, the donors in both reactions must be of the same chemical nature. (iii) This result, together with the well‐established similarity of EPR signal IIf of the oxidized donor in Tris‐treated PS II to the EPR signal IIIs, recently assigned to Tyr‐160 of the D2 protein of PS II [(1988) Proc. Natl. Acad. Sci. USA 85, 427–430], provides strong evidence that the immediate donor to Chl a + II in water‐oxidizing PS II is also a tyrosine. (iv) It is shown that the UV‐difference spectra of the oxidation of the immediate donor in O2‐evolving as well as that of Tris‐treated PS II complexes are similar to the in vitro difference spectrum of the oxidation of tyrosine in water. This independent result supports the conclusion that the donor is a tyrosine.