Pavel Müller

Light-activated Cryptochrome Reacts with Molecular Oxygen to Form a Flavin–Superoxide Radical Pair Consistent with Magnetoreception

Cryptochromes are flavin-based photoreceptors occurring throughout the biological kingdom, which regulate growth and development in plants and are involved in the entrainment of circadian rhythms of both plants and animals. A number of recent theoretical works suggest that cryptochromes might also be the receptors responsible for the sensing of the magnetic field of the earth (e.g. in insects, migratory birds, or migratory fish). Cryptochromes undergo forward light-induced reactions involving electron transfer to excited state flavin to generate radical intermediates, which correlate with biological activity. Here, we give evidence of a mechanism for the reverse reaction, namely dark reoxidation of protein-bound flavin in Arabidopsis thaliana cryptochrome (AtCRY1) by molecular oxygen that involves formation of a spin-correlated FADH•–superoxide radical pair. Formation of analogous radical pairs in animal cryptochromes might enable them to function as magnetoreceptors.

Electron Transfer in Peptides with Cysteine and Methionine as Relay Amino Acids

Caught on the hop: In multistep electron transfer (ET) reactions through peptides, aliphatic amino acids can also act as relay stations. With cysteine, the reaction occurs as a proton-coupled electron transfer (PCET) with water used as a mediator for the proton transfer (see picture).

Electron Transfer in Peptides: The Influence of Charged Amino Acids

The ammonium group in the peptide shown leads to a tenfold increase of the electron‐transfer rate compared to that in a fully protected system. This is explained by Coulombs law and the Marcus theory.

Electron relay race in peptides

A peptide assay was developed that allows the measurement of electron-transfer (ET) efficiencies in peptides. It turns out that two-step ET processes are faster than single-step reactions. This requires relay amino acids with appropriate redox potentials. Not only aromatic but also sulfur-containing aliphatic amino acids can act as stepping stones for the charge. With tryptophan, histidine, and cysteine the reaction is a more complex proton-coupled ET.

The electrodeless discharge lamp: a prospective tool for photochemistry Part 4. Temperature- and envelope material-dependent emission characteristics

This work extends our previous research on an original photochemical reactor—the electrodeless discharge lamp (EDL) inside a reaction mixture that generates ultraviolet radiation in the microwave (MW) field. This arrangement was found to be a straightforward solution for homogeneous as well as heterogeneous photochemical experiments that need to be carried out at higher temperatures. Here, we report the emission characteristics (250–600 nm) of EDL as a function of temperature, MW output power of the reactor, EDL envelope material, and properties of solvents used in photochemical reactions. Relative intensities of the individual emission peaks were found to be largely dependent on temperature (in the region of 35–174 °C): the short-wavelength bands (particularly the 254 nm peak) were suppressed with increasing temperature. Solvents absorbing MW significantly reduced the EDL emission intensity. It is concluded that the right choice of EDL envelope material and reaction conditions is essential for an efficient course of a photochemical process in this experimental arrangement.

Kinetics of cyclobutane thymine dimer splitting by DNA photolyase directly monitored in the UV

CPD photolyase uses light to repair cyclobutane pyrimidine dimers (CPDs) formed between adjacent pyrimidines in UV-irradiated DNA. The enzyme harbors an FAD cofactor in fully reduced state (FADH-). The CPD repair mechanism involves electron transfer from photoexcited FADH- to the CPD, splitting of its intradimer bonds, and electron return to restore catalytically active FADH-. The two electron transfer processes occur on time scales of 10-10 and 10-9 s, respectively. Until now, CPD splitting itself has only been poorly characterized by experiments. Using a previously unreported transient absorption setup, we succeeded in monitoring cyclobutane thymine dimer repair in the main UV absorption band of intact thymine at 266 nm. Flavin transitions that overlay DNA-based absorption changes at 266 nm were monitored independently in the visible and subtracted to obtain the true repair kinetics. Restoration of intact thymine showed a short lag and a biexponential rise with time constants of 0.2 and 1.5 ns. We assign these two time constants to splitting of the intradimer bonds (creating one intact thymine and one thymine anion radical T∘-) and electron return from T∘- to the FAD cofactor with recovery of the second thymine, respectively. Previous model studies and computer simulations yielded various CPD splitting times between < 1 ps and < 100 ns. Our experimental results should serve as a benchmark for future efforts to model enzymatic photorepair. The technique and methods developed here may be applied to monitor other photoreactions involving DNA.

Fluorescein analogues as photoremovable protecting groups absorbing at ∼520 nm.

A new photoremovable protecting group, (6-hydroxy-3-oxo-3H-xanthen-9-yl)methyl (1), with a molar absorption coefficient ε of ∼4 × 10(4) m(-1) cm(-1) at ∼520 nm for the release of carboxylates or phosphates is reported. Three derivatives of 1 (diethyl phosphate, acetate, and bromide) were isolated as complexes with DDQ and shown to release the ligands with quantum yields ≤2.4% in aqueous solution.

ATP Binding Turns Plant Cryptochrome Into an Efficient Natural Photoswitch

Cryptochromes are flavoproteins that drive diverse developmental light-responses in plants and participate in the circadian clock in animals. Plant cryptochromes have found application as photoswitches in optogenetics. We have studied effects of pH and ATP on the functionally relevant photoreduction of the oxidized FAD cofactor to the semi-reduced FADH· radical in isolated Arabidopsis cryptochrome 1 by transient absorption spectroscopy on nanosecond to millisecond timescales. In the absence of ATP, the yield of light-induced radicals strongly decreased with increasing pH from 6.5 to 8.5. With ATP present, these yields were significantly higher and virtually pH-independent up to pH 9. Analysis of our data in light of the crystallographic structure suggests that ATP-binding shifts the pKa of aspartic acid D396, the putative proton donor to FAD·−, from ~7.4 to >9, and favours a reaction pathway yielding long-lived aspartate D396−. Its negative charge could trigger conformational changes necessary for signal transduction.

Binding of concanavalin A to the outer membrane of potato tuber mitochondria detected by flow cytometry

Purified and intact mitochondria isolated from potato tuber show a specific concanavalin A (Con A) binding as deduced from fluorescence labelling studies in the presence of the specific sugar α‐D‐methylmannose. This specific binding also occurs in mitoplasts, indicating the presence of Con A‐binding sites on the inner mitochondrial membrane. In contrast, the binding of wheat germ agglutinin appears non‐specific. Flow cytometry with purified mitochondria enables assessment of the binding by individual mitochondria. Thus, three mitochondrial subpopulations were identified by their reactivity towards fluorescent Con A.

Discovery and functional analysis of a 4th electron-transferring tryptophan conserved exclusively in animal cryptochromes and (6-4) photolyases.

A 4th electron transferring tryptophan in animal cryptochromes and (6-4) photolyases is discovered and functionally analyzed by transient absorption. It yields a much longer-lived flavin-tryptophan radical pair than the mere tryptophan triad in related flavoproteins, questioning the putative role of the primary light reaction of cryptochrome in animal magnetoreception.