Robert Bittl

Cryptochrome Blue Light Photoreceptors Are Activated through Interconversion of Flavin Redox States

Cryptochromes are blue light-sensing photoreceptors found in plants, animals, and humans. They are known to play key roles in the regulation of the circadian clock and in development. However, despite striking structural similarities to photolyase DNA repair enzymes, cryptochromes do not repair double-stranded DNA, and their mechanism of action is unknown. Recently, a blue light-dependent intramolecular electron transfer to the excited state flavin was characterized and proposed as the primary mechanism of light activation. The resulting formation of a stable neutral flavin semiquinone intermediate enables the photoreceptor to absorb green/yellow light (500–630 nm) in addition to blue light in vitro. Here, we demonstrate that Arabidopsis cryptochrome activation by blue light can be inhibited by green light in vivo consistent with a change of the cofactor redox state. We further characterize light-dependent changes in the cryptochrome1 (cry1) protein in living cells, which match photoreduction of the purified cry1 in vitro. These experiments were performed using fluorescence absorption/emission and EPR on whole cells and thereby represent one of the few examples of the active state of a known photoreceptor being monitored in vivo. These results indicate that cry1 activation via blue light initiates formation of a flavosemiquinone signaling state that can be converted by green light to an inactive form. In summary, cryptochrome activation via flavin photoreduction is a reversible mechanism novel to blue light photoreceptors. This photocycle may have adaptive significance for sensing the quality of the light environment in multiple organisms.

The Signaling State of Arabidopsis Cryptochrome 2 Contains Flavin Semiquinone

Cryptochrome (Cry) photoreceptors share high sequence and structural similarity with DNA repair enzyme DNA-photolyase and carry the same flavin cofactor. Accordingly, DNA-photolyase was considered a model system for the light activation process of cryptochromes. In line with this view were recent spectroscopic studies on cryptochromes of the CryDASH subfamily that showed photoreduction of the flavin adenine dinucleotide (FAD) cofactor to its fully reduced form. However, CryDASH members were recently shown to have photolyase activity for cyclobutane pyrimidine dimers in single-stranded DNA, which is absent for other members of the cryptochrome/photolyase family. Thus, CryDASH may have functions different from cryptochromes. The photocycle of other members of the cryptochrome family, such as Arabidopsis Cry1 and Cry2, which lack DNA repair activity but control photomorphogenesis and flowering time, remained elusive. Here we have shown that Arabidopsis Cry2 undergoes a photocycle in which semireduced flavin (FADH·) accumulates upon blue light irradiation. Green light irradiation of Cry2 causes a change in the equilibrium of flavin oxidation states and attenuates Cry2-controlled responses such as flowering. These results demonstrate that the active form of Cry2 contains FADH· (whereas catalytically active photolyase requires fully reduced flavin (FADH-)) and suggest that cryptochromes could represent photoreceptors using flavin redox states for signaling differently from DNA-photolyase for photorepair.

Magnetic-field effect on the photoactivation reaction of Escherichia coli DNA photolyase

One of the two principal hypotheses put forward to explain the primary magnetoreception event underlying the magnetic compass sense of migratory birds is based on a magnetically sensitive chemical reaction. It has been proposed that a spin-correlated radical pair is produced photochemically in a cryptochrome and that the rates and yields of the subsequent chemical reactions depend on the orientation of the protein in the Earths magnetic field. The suitability of cryptochrome for this purpose has been argued, in part, by analogy with DNA photolyase, although no effects of applied magnetic fields have yet been reported for any member of the cryptochrome/photolyase family. Here, we demonstrate a magnetic-field effect on the photochemical yield of a flavin–tryptophan radical pair in Escherichia coli photolyase. This result provides a proof of principle that photolyases, and most likely by extension also cryptochromes, have the fundamental properties needed to form the basis of a magnetic compass.

Time-resolved W-band (95 GHz) EPR spectroscopy of Zn-substituted reaction centers of Rhodobacter sphaeroides R-26

Abstract Transient EPR spectra of protonated and deuterated Zn-substituted reaction centres of Rhodobacter sphaeroides R-26, measured at a microwave frequency of 95 GHz and an external magnetic field of 3.5 T, are presented. The high-field/high-frequency spin-polarized spectrum of the correlated coupled radical pair P 865 +. Q A −. after laser flash excitation is a very sensitive monitor of the relative orientation of the two g -matrices and the dipolar tensor with respect to each other. Therefore, detailed structural information of the RC concerning the relative orientation of the primary donor P 865 +. and the acceptor Q A −. with respect to the axis connecting the two molecules in the RC can be derived. Together with the information obtained by high-field cw-EPR on single crystals, the enhanced resolution of the polarized high-field spectra allows an unambiguous assignment of the g -matrix of the donor P 865 +. to the molecular axis system. The experimental results are compared with earlier X-band (9 GHz) and K-band (24 GHz) EPR experiments.

A single-crystal ENDOR and density functional theory study of the oxidized states of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F.

The catalytic center of the [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F in the oxidized states was investigated by electron paramagnetic resonance and electron–nuclear double resonance spectroscopy applied to single crystals of the enzyme. The experimental results were compared with density functional theory (DFT) calculations. For the Ni-B state, three hyperfine tensors could be determined. Two tensors have large isotropic hyperfine coupling constants and are assigned to the β-CH2 protons of the Cys-549 that provides one of the bridging sulfur ligands between Ni and Fe in the active center. From a comparison of the orientation of the third hyperfine tensor with the tensor obtained from DFT calculations an OH− bridging ligand has been identified in the Ni-B state. For the Ni-A state broader signals were observed. The signals of the third proton, as observed for the “ready” state Ni-B, were not observed at the same spectral position for Ni-A, confirming a structural difference involving the bridging ligand in the “unready” state of the enzyme.

Transient EPR of light-induced radical pairs in plant photosystem I: observation of quantum beats

Abstract Electron spin polarization of light-induced radical pairs in plant photosystem I is studied by transient EPR following pulsed laser excitation. The time evolution of the transverse magnetization is monitored at various static and microwave magnetic fields. Quantum beat oscillations are observed at early times after light excitation of fully deuterated whole algae Synechococcus lividus . Model calculations for the time profiles, based on the correlated radical pair mechanism, provide information on the spin—spin coupling and lifetime of the secondary radical pair.

Transient EPR of radical pairs in photosynthetic reaction centers: prediction of quantum beats

Abstract Transient nutation EPR of light-induced radical pairs in photosynthetic reaction centers is studied theoretically using the stochastic Liouville equation. The spin Hamiltonian employed considers Zeeman, exchange and dipolar interactions of the spin-correlated radical pair. Particular emphasis is given to the slow-motional regime, where anisotropic magnetic interactions dominate. The calculated dependence of the transverse magnetization on the static magnetic field agrees well with previous simulations of the transient spectra. Quantum-beat oscillations are predicted for the time evolution of the transverse magnetization. The beat frequency is determined by the spin-spin coupling and g-factor difference of the radical pair.

Direct detection of photoinduced charge transfer complexes in polymer fullerene blends

We report transient electron paramagnetic resonance (trEPR) measurements with sub-microsecond time resolution performed on a P3HT:PCBM blend at low temperature. The trEPR spectrum immediately following photoexcitation reveals signatures of spin-correlated polaron pairs. The pair partners (positive polarons in P3HT and negative polarons in PCBM) can be identified by their characteristic g-values. The fact that the polaron pair states exhibit strong non-Boltzmann population unambiguously shows that the constituents of each pair are geminate, i.e. originate from one exciton. We demonstrate that coupled polaron pairs are present even several microseconds after charge transfer and suggest that they embody the intermediate charge transfer complexes which form at the donor/acceptor interface and mediate the conversion from excitons into free charge carriers.

Radicals and Radical Pairs in Photosynthesis

Light-induced radicals and radical pairs occur naturally in the primary photochemical reaction of photosynthesis. They have been at the focus of intense study ever since the first observation of an electron paramagnetic resonance (EPR) signal in plants and photosynthetic bacteria ( 1 , 2). The EPR and electron nuclear double resonance (ENDOR) techniques, along with a variety of optical methods, have been the main source of information about the identity and electronic structure of radicals that occur in photosynthetic systems during the various electron transfer (ET) reactions taking place as a result of light uptake. The chemical nature of most of the species that serve as electron donors and acceptors along the primary ET paths have been iden-

Photosystem II single crystals studied by EPR spectroscopy at 94 GHz: the tyrosine radical Y(D)(*).

Electron paramagnetic resonance (EPR) spectroscopy at 94 GHz is used to study the dark-stable tyrosine radical Y\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{D}^{{\bullet}}}}\end{equation*}\end{document} in single crystals of photosystem II core complexes (cc) isolated from the thermophilic cyanobacterium Synechococcus elongatus. These complexes contain at least 17 subunits, including the water-oxidizing complex (WOC), and 32 chlorophyll a molecules/PS II; they are active in light-induced electron transfer and water oxidation. The crystals belong to the orthorhombic space group P212121, with four PS II dimers per unit cell. High-frequency EPR is used for enhancing the sensitivity of experiments performed on small single crystals as well as for increasing the spectral resolution of the g tensor components and of the different crystal sites. Magnitude and orientation of the g tensor of Y\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{D}^{{\bullet}}}}\end{equation*}\end{document} and related information on several proton hyperfine tensors are deduced from analysis of angular-dependent EPR spectra. The precise orientation of tyrosine Y\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{D}^{{\bullet}}}}\end{equation*}\end{document} in PS II is obtained as a first step in the EPR characterization of paramagnetic species in these single crystals.