Bernhard Dick

Switching from adduct formation to electron transfer in a light–oxygen–voltage domain containing the reactive cysteine

Co-reporter:Kathrin Magerl;Ivan Stambolic

Physical Chemistry Chemical Physics 2017 vol. 19(Issue 17) pp:10808-10819

Publication Date(Web):2017/05/03

DOI:10.1039/C6CP08370F

LOV (light-, oxygen- or voltage-sensitive) domains act as photosensory units of many prokaryotic and eukaryotic proteins. Upon blue light excitation they undergo a photocycle via the excited triplet state of their flavin chromophore yielding the flavin–cysteinyl adduct. Adduct formation is highly conserved among all LOV domains and constitutes the primary step of LOV domain signaling. But recently, it has been shown that signal propagation can also be triggered by flavin photoreduction to the neutral semiquinone offering new prospects for protein engineering. This, however, requires mutation of the photo-active Cys. Here, we report on LOV1 mutants of C. reinhardtii phototropin in which adduct formation is suppressed although the photo-active Cys is present. Introduction of a Tyr into the LOV core induces a proton coupled electron transfer towards the flavin chromophore. Flavin radical species are formed via either the excited flavin singlet or triplet state depending on the geometry of donor and acceptor. This photoreductive pathway resembles the photoreaction observed in other blue light photoreceptors, e.g. blue-light sensors using flavin adenine dinucleotide (BLUF) domains or cryptochromes. The ability to tune the photoreactivity of the flavin chromophore inside the LOV core has implications for the mechanism of adduct formation in the wild type and may be of use for protein engineering.

A search for radical intermediates in the photocycle of LOV domains

Co-reporter:Roger Jan Kutta, Kathrin Magerl, Uwe Kensy and Bernhard Dick

Photochemical & Photobiological Sciences 2015 vol. 14(Issue 2) pp:288-299

Publication Date(Web):17 Oct 2014

DOI:10.1039/C4PP00155A

LOV domains are the light sensitive parts of phototropins and many other light-activated enzymes that regulate the response to blue light in plants and algae as well as some fungi and bacteria. Unlike all other biological photoreceptors known so far, the photocycle of LOV domains involves the excited triplet state of the chromophore. This chromophore is flavin mononucleotide (FMN) which forms a covalent adduct with a cysteine residue in the signaling state. Since the formation of this adduct from the triplet state involves breaking and forming of two bonds as well as a change from the triplet to the singlet spin state, various intermediates have been proposed, e.g. a protonated triplet state 3FMNH+, the radical anion 2FMN˙−, or the neutral semiquinone radical 2FMNH˙. We performed an extensive search for these intermediates by two-dimensional transient absorption (2D-TA) with a streak camera. However, no transient with a rate constant between the decay of fluorescence and the decay of the triplet state could be detected. Analysis of the decay associated difference spectra results in quantum yields for the formation of the adduct from the triplet of ΦA(LOV1) ≈ 0.75 and ΦA(LOV2) ≈ 0.80. This is lower than the values ΦA(LOV1) ≈ 0.95 and ΦA(LOV2) ≈ 0.99 calculated from the rate constants, giving indirect evidence of an intermediate that reacts either to form the adduct or to decay back to the ground state. Since there is no measurable delay between the decay of the triplet and the formation of the adduct, we conclude that this intermediate reacts much faster than it is formed. The LOV1-C57S mutant shows a weak and slowly decaying (τ > 100 μs) transient whose decay associated spectrum has bands at 375 and 500 nm, with a shoulder at 400 nm. This transient is insensitive to the pH change in the range 6.5–10.0 but increases on addition of β-mercaptoethanol as the reducing agent. We assign this intermediate to the radical anion which is protected from protonation by the protein. We propose that the adduct is formed via the same intermediate by combination of the radical ion pair.

Electronic spectroscopy of lumiflavin in superfluid helium nanodroplets

Co-reporter:Alexander Vdovin, Alkwin Slenczka, Bernhard Dick

Chemical Physics 2013 Volume 422() pp:195-203

Publication Date(Web):30 August 2013

DOI:10.1016/j.chemphys.2012.11.001

Abstract

We present the fluorescence excitation and dispersed emission spectra of lumiflavin doped into superfluid He nanodroplets. Both spectra show well resolved vibrational structure. The electronic origin transition at 21511 cm⁻¹ is the strongest line in both spectra. Quantum chemical calculations with DFT and CASSCF methods support the assignment of S₁ to a ππ^∗ $π π^{*}$ excited state. We obtain vibrational frequencies in the ground and lowest excited singlet state that can serve to test the validity of quantum chemical calculations. Multidimensional Franck–Condon factors are in good agreement with the intensities within the vibrational structure for S₀ and S₁. The strongest progression forming mode has a frequency of 164 cm⁻¹ in both states and is assigned to an in-plane bending mode of the whole flavin chromophore with a large amplitude on the two methyl groups at ring I.

Photodissociation dynamics of tert-butylnitrite following excitation to the S1 and S2 states. A study by velocity-map ion-imaging and 3D-REMPI spectroscopy

Co-reporter:Andreas M. Wenge, Andreas Schmaunz, Uwe Kensy and Bernhard Dick

Physical Chemistry Chemical Physics 2012 vol. 14(Issue 19) pp:7076-7089

Publication Date(Web):20 Mar 2012

DOI:10.1039/C2CP40349H

Excitation of tert-butylnitrite into the first and second UV absorption bands leads to efficient dissociation into the fragment radicals NO and tert-butoxy in their electronic ground states 2Π and 2E, respectively. Velocity distributions and angular anisotropies for the NO fragment in several hundred rotational and vibrational quantum states were obtained by velocity-map imaging and the recently developed 3D-REMPI method. Excitation into the well resolved vibronic progression bands (k = 0, 1, 2) of the NO stretch mode in the S1 ← S0 transition produces NO fragments mostly in the vibrational state with v = k, with smaller fractions in v = k − 1 and v = k − 2. It is concluded that dissociation occurs on the purely repulsive PES of S1 without barrier. All velocity distributions from photolysis via the S1(nπ*) state are monomodal and show high negative anisotropy (β ≈ −1). The rotational distributions peak near j = 30.5 irrespective of the vibronic state S1(k) excited and the vibrational state v of the NO fragment. On average 46% of the excess energy is converted to kinetic energy, 23% and 31% remain as internal energy in the NO fragment and the t-BuO radical, respectively. Photolysis via excitation into the S2 ← S0 transition at 227 nm yields NO fragments with about equal populations in v = 0 and v = 1. The rotational distributions have a single maximum near j = 59.5. The velocity distributions are monomodal with positive anisotropy β ≈ 0.8. The average fractions of the excess energy distributed into translation, internal energy of NO, and internal energy of t-BuO are 39%, 23%, and 38%, respectively. In all cases ∼8500 cm−1 of energy remain in the internal degrees of freedom of the t-BuO fragment. This is mostly assigned to rotational energy. An ab initio calculation of the dynamic reaction path shows that not only the NO fragment but also the t-BuO fragment gain large angular momentum during dissociation on the purely repulsive potential energy surface of S2.

Circular Dichroism of a Finite Number of Identical Chromophores in a Helical Arrangement

Co-reporter: Dr. Bernhard Dick

ChemPhysChem 2011 Volume 12( Issue 8) pp:1578-1587

Publication Date(Web):

DOI:10.1002/cphc.201000949

Abstract

Compact expressions to calculate the transition energies, absorption line strengths, and rotational line strengths of circular dichroism for the excitonic states in a helical arrangement of N identical chromophores are presented. The absorption spectrum A(ν) and the CD spectrum C(ν) are given in terms of the same function G(ν,α) as , . The function G(ν,α) depends only on the helical angle α and the number N of interacting chromophores. An analytical expression can be given when only next-neighbor interactions are considered. All other structural parameters of the system (e.g. orientation of transition dipoles and the translation vector of the helix) enter only into the prefactors a_j and s_j.

High-Resolution Electronic Spectroscopy of the BODIPY Chromophore in Supersonic Beam and Superfluid Helium Droplets

Co-reporter:Dr. Anja Stromeck-Faderl;Dr. Dominik Pentlehner;Dr. Uwe Kensy ; Dr. Bernhard Dick

ChemPhysChem 2011 Volume 12( Issue 10) pp:1969-1980

Publication Date(Web):

DOI:10.1002/cphc.201001076

Abstract

We present the fluorescence excitation and dispersed emission spectra of the parent compound of the boron dipyrromethene (BODIPY) dye class measured in a supersonic beam and isolated in superfluid helium nanodroplets. The gas-phase spectrum of the isolated molecules displays many low-frequency transitions that are assigned to a symmetry-breaking mode with a strongly nonharmonic potential, presumably the out-of-plane wagging mode of the BF₂ group. The data are in good agreement with transition energies and Franck–Condon factors calculated for a double minimum potential in the upper electronic state. The corresponding transitions do not appear in the helium droplet. This is explained with the quasi-rigid first layer of helium atoms attached to the dopant molecule by van der Waals forces. The spectral characteristics are those of a cyanine dye rather than that of an aromatic chromophore.

Photoreaction of mutated LOV photoreceptor domains from Chlamydomonas reinhardtii with aliphatic mercaptans: implications for the mechanism of wild type LOV

Co-reporter:Karin Lanzl, Madlene v. Sanden-Flohe, Roger-Jan Kutta and Bernhard Dick

Physical Chemistry Chemical Physics 2010 vol. 12(Issue 25) pp:6594-6604

Publication Date(Web):06 May 2010

DOI:10.1039/B922408D

Irradiation of the LOV1 domain from the blue-light photoreceptor phototropin of the green alga Chlamydomonas reinhardtii leads to the formation of a covalent adduct of the sulfur atom of cysteine 57 to the carbon C4a in the chromophore FMN. This reaction is not possible in the mutant LOV1-C57G in which this cysteine is replaced by glycine. Irradiation of LOV1-C57G in the absence of oxygen but in the presence of aliphatic mercaptans or thioethers leads to the formation of a species with an absorption maximum at 615 nm, which is identified as the neutral radical FMNH˙. When oxygen is admitted, the reaction is completely reversible. Irradiation of LOV1-C57G in the presence of methylmercaptan CH3SH under oxygen-free conditions yields, in addition to FMNH˙, a third species with a single absorption maximum at 379 nm. This species is stable against oxygen and is also formed when the irradiation is performed in the presence of oxygen. This species is assigned to the adduct between CH3SH and FMN. In aqueous solution the photoreaction of CH3SH with FMN leads to the fully reduced hydroquinone form FMNH2 or its anion FMNH−. Adduct formation apparently requires the protein cage. After formation, the adduct is stable for hours inside the protein, but decomposes immediately upon denaturation. The implications of these observations for the mechanism of adduct formation in wild type LOV domains are discussed.

The photodissociation dynamics of N-nitrosopyrrolidine from the first and second excited singlet states studied by velocity map imaging

Co-reporter:Andreas Maximilian Wenge, Uwe Kensy and Bernhard Dick

Physical Chemistry Chemical Physics 2010 vol. 12(Issue 18) pp:4644-4655

Publication Date(Web):28 Jan 2010

DOI:10.1039/B920547K

The photodissociation reaction of N-nitrosopyrrolidine isolated and cooled in a supersonic jet has been studied following excitation to the S1 and S2 electronic states. The nascent NO (2Π½,, v, j) radicals were ionized by state-selective (1 + 1)-REMPI via the A2Σ+ state. The angularly resolved velocity distribution of these ions was measured with the velocity-map imaging (VMI) technique. Photodissociation from S1 produces NO in the vibrational ground state and the pyrrolidine radical in the electronic ground state 12B. About 73% of the excess energy is converted into kinetic energy of the fragments. The velocity distribution shows a strong negative anisotropy (β = −0.9) in accordance with the nπ*-character of the S0 → S1 transition. An upper limit for the N–NO dissociation energy of (14640 ± 340) cm−1 is determined. We conclude that photodissociation from S1 occurs very fast on a completely repulsive potential energy surface. Excitation into the S2 ππ*-state leads to a bimodal velocity distribution. Two dissociation channels can be distinguished which show both positive anisotropy (β = 1.3 and 1.6) but differ considerably in the total kinetic energy and the rotational energy of the NO fragment. We assign one channel to the direct dissociation on the S2 potential energy surface, leading to pyrrolidine radicals in the excited electronic state 12A. The second channel leads to pyrrolidine in the electronic ground state 12B, presumably after crossing to the S1 state via a conical intersection.

Photolysis of tert-Butylthionitrite via Excitation to the S1 and S2 States Studied by 3d-REMPI Spectroscopy†

Co-reporter:Andreas Schmaunz, Uwe Kensy, Alkwin Slenczka, and Bernhard Dick

The Journal of Physical Chemistry A 2010 Volume 114(Issue 36) pp:9948-9962

Publication Date(Web):July 15, 2010

DOI:10.1021/jp104399b

S-Nitrosothiols serve as carriers and donors of NO in several important biological signaling systems. In these compounds the S−NO bond is rather labile and NO can be released thermally or photochemically. This paper reports on the photolytical decomposition of tert-butylthionitrite (t-BuSNO) in the visible and near-UV spectral regions. Between 500 and 605 nm several vibronic levels of the S1 (nπ*) state were excited, including the electronic origin. At 360 nm t-BuSNO is excited near the maximum of the first UV band assigned to the S2 (ππ*) state. The velocity distributions of several hundred rovibrational states of the NO fragments were recorded with the recently developed 3d-REMPI method. A global fit to these data yields populations of the rovibrational states in both spin−orbit components of the 2Π electronic state of NO as well as their velocity distributions and angular anisotropies β. These data also carry the distribution functions for internal and kinetic energy of the counterfragment, the t-BuS radical. The range found for the anisotropy parameter confirms the nπ* character of the visible absorption band (−1.0 < β < −0.8), and the ππ* character of the UV band (β = 1.2). Mode-specific dissociation has been observed for excitation into several vibronic bands of the S0 → S1 transition. Some produce NO exclusively in the ν = 0 vibrational ground state, whereas some others produce NO almost entirely in the ν = 1 vibrationally excited state. It is concluded that photodissociation is faster than relaxation of the NO stretch vibration of t-BuSNO in S1 and that it proceeds on purely repulse potential energy surfaces in both electronic states.

Gradient Projection Method for Constraint Optimization and Relaxed Energy Paths on Conical Intersection Spaces and Potential Energy Surfaces

Co-reporter:Bernhard Dick

Journal of Chemical Theory and Computation 2009 Volume 5(Issue 1) pp:116-125

Publication Date(Web):December 8, 2008

DOI:10.1021/ct8003029

A gradient projection algorithm is presented that permits the application of several constraints during geometry optimization on electronic potential energy surfaces (PES) or conical intersection (CI) seams. The algorithm generalizes the idea recently published in this journal (Sicilia et al. J. Chem. Theory Comput. 2008, 4, 257) for the optimization of conical intersection geometries. Singular value decomposition is used to transform all constraints, including those related to maintaining the CI, to a new set of constraints with orthogonal gradients. The constraints need not be satisfied at the initial geometry but will be upon convergence. A procedure is presented that determines relaxed energy paths (REP) connecting two reference structures on a potential energy surface, or the conical intersection space, without the need to assign an internal coordinate as the reaction coordinate. Examples are presented of optimizations of minimum energy structures and REPs in the CI space and REPs on a single electronic PES.

Dynamic Polar Solvation Is Reported by Fluorescing 4-Aminophthalimide Faithfully Despite H-Bonding

Co-reporter:Mohsen Sajadi, Thorsten Obernhuber, Sergey A. Kovalenko, Manuel Mosquera, Bernhard Dick and Nikolaus P. Ernsting

The Journal of Physical Chemistry A 2009 Volume 113(Issue 1) pp:44-55

Publication Date(Web):December 15, 2008

DOI:10.1021/jp807605b

Solvation dynamics of 4-aminophthalimide (4AP) in methanol is measured by broadband upconversion of the fluorescence band. The peak emission frequency ν̃(t) is determined from 100 fs onward with 85 fs time resolution. Polar solvation based on simple continuum theory, including solute polarizability, describes the temporal shape of ν̃(t) quantitatively. Extrapolation ν̃(t→0) points to an initial emission frequency which agrees with the result from stationary spectroscopy in a nonpolar solvent. The extent (4300 cm−1) of the dynamic Stokes shift is largely due (50%) to H-bonding, however. The observations imply that H-bonds with 4AP adiabatically follow the dielectric relaxation of the methanol network. The stimulated emission band is also used to measure solvation dynamics. The evolving band is monitored by transient absorption spectroscopy of supercontinuum probe pulses. But the excited-state absorption spectrum, its relative amplitude, and its evolution are needed to extract ν̃(t) from such measurements. These key data are obtained by comparison with the upconversion results. Thus calibrated photometrically, 4AP transient absorption can be used to monitor solvation dynamics in any solvent. The excited-state absorption spectrum is assigned with the help of time-dependent density-functional calculations. Fluorescence excitation and double-resonance spectroscopy of isolated 4AP, cooled in a supersonic jet, is used to determine optically active modes. An intramolecular reorganization energy is inferred which is consistent with the value in 2-methylbutane (2025 cm−1). The crystal structure is also provided.

Locating conical intersections relevant to photochemical reactions

Co-reporter:Bernhard Dick, Yehuda Haas, Shmuel Zilberg

Chemical Physics 2008 Volume 347(1–3) pp:65-77

Publication Date(Web):23 May 2008

DOI:10.1016/j.chemphys.2007.10.022

Abstract

A new computerized method for locating conical intersections of interest in photochemistry is presented. The search is based on the Longuet-Higgins phase change theorem (Berry phase) which provides the subspace required for the initial search. The subspace is approximated as a plane containing three stable structures lying on a Longuet-Higgins loop. The search is conducted for a minimum of ΔE, the energy difference between two electronic states. It is started using up to three points within the circle defined by the three structures; symmetry, if relevant, is helpful but not essential. Since a two-dimensional subspace of the large 3N − 6 space is used, the search that uses either Cartesian or internal coordinates is efficient and yields a degeneracy after a few iterations. Given that not all degrees of freedom are included in the search, usually a high lying part of the conical intersection is initially located. The system is subsequently optimized along all coordinates keeping ΔE as close to zero as desired. The method is demonstrated for the symmetric H₃ system and also for the butadiene–cyclobutene–bicyclobutane system in which the three stable structures are not equivalent. The method is general and can be extended to any photochemical system.

LOV1 Protein From Chlamydomonas Reinhardtii is a Template For the Photoadduct Formation of FMN and Methylmercaptane.

Co-reporter:Karin Lanzl;Gilbert Nöll

ChemBioChem 2008 Volume 9( Issue 6) pp:861-864

Publication Date(Web):

DOI:10.1002/cbic.200700737

Velocity-map ion-imaging of the NO fragment from the UV-photodissociation of nitrosobenzene

Co-reporter:Thorsten J. Obernhuber, Uwe Kensy and Bernhard Dick

Physical Chemistry Chemical Physics 2003 vol. 5(Issue 13) pp:2799-2806

Publication Date(Web):27 May 2003

DOI:10.1039/B302319B

The velocity and angular distribution of NO fragments produced by UV photodissociation of nitrosobenzene have been determined by velocity-map ion-imaging. Excitation of the S2-state by irradiation into the peak of the first UV absorption band at 290.5 nm leads to a completely isotropic velocity distribution with Gaussian shape. The average kinetic energy in both fragments correlates with the rotational energy of the NO fragment and increases from 6% of the excess energy for j=6.5 to 11% for j=29.5. A similar isotropic distribution albeit with larger average velocity is observed when the ionization laser at 226 nm is also used for photodissociation, corresponding to excitation into a higher electronic state Sn (n≥3) of nitrosobenzene. It is concluded that photodissociation occurs on a timescale much slower than rotation of the parent molecule, and after redistribution of the excess energy into the vibrational degrees of freedom.

Fluorescence excitation and UV–UV double-resonance spectroscopy of the S0→S1(Lb) transition of 1,6-methano[10]annulene cooled in a supersonic jet

Co-reporter:Reinhold Seiler, Uwe Kensy and Bernhard Dick

Physical Chemistry Chemical Physics 2001 vol. 3(Issue 24) pp:5373-5382

Publication Date(Web):21 Dec 2001

DOI:10.1039/B107365F

The first electronic singlet transition S0→S1 of the 10π-aromatic compound 1,6-methano[10]annulene (MA) cooled in a supersonic jet has been studied up to an excess energy of 4000 cm−1. The strongest line at 25154 cm−1 is assigned as the electronic origin. Analysis of the rotational envelope of this line proved that the transition dipole is parallel to the long axis of the molecule. Optical–optical double resonance was used to identify the lines which share the same ground state with the origin transition. These lines occur all at higher energies. A few weaker lines which are always present but do not lead to double resonances are tentatively attributed to a van-der-Waals dimer of MA. The rich vibrational structure is interpreted in terms of 13 fundamental vibrations of a1 symmetry and 11 of a2 symmetry, based on the analysis of the rotational contours. The fundamental vibrational frequencies of the excited state are in very good agreement with ab initio calculations. Based on these calculations 8 further lines which are not combination bands are tentatively assigned to double quantum transitions in b1 and b2 modes. These results strongly support the assignment of a delocalized structure without bond length alternation to the electronic ground state as well as to the first electronically excited singlet state.

1,6-Methano[10]annulen hat eine delokalisierte Struktur in S0 und S1: hochaufgelöste Spektroskopie im Überschall-Düsenstrahl

Co-reporter:Reinhold Seiler Dipl.-Chem. Dr.

Angewandte Chemie 2001 Volume 113(Issue 21) pp:

Publication Date(Web):31 OCT 2001

DOI:10.1002/1521-3757(20011105)113:21<4144::AID-ANGE4144>3.0.CO;2-Z

Für den Grundzustand des Hückel-Arens 1,6-Methano[10]annulen (MA) wurde in der Vergangenheit je nach der verwendeten theoretischen Methode eine lokalisierte oder eine delokalisierte Struktur postuliert (siehe Schema). Hochaufgelöste elektronische Spektren im Überschall-Düsenstrahl belegen eindeutig, dass isoliertes, ultrakaltes MA sowohl im elektronischen Grundzustand S₀ als auch im angeregten Zustand S₁ eine delokalisierte Struktur aufweist.

1,6-Methano[10]annulene has a Delocalized Structure in S0 and S1: High-Resolution Spectroscopy in a Supersonic Jet

Co-reporter:Reinhold Seiler Dipl.-Chem. Dr.

Angewandte Chemie International Edition 2001 Volume 40(Issue 21) pp:

Publication Date(Web):31 OCT 2001

DOI:10.1002/1521-3773(20011105)40:21<4020::AID-ANIE4020>3.0.CO;2-G

For the ground state of the Hückel aromatic ring 1,6-methano[10]annulene (MA) both a localized and a delocalized structure (see scheme) have been proposed in the past, depending on the theoretical methods used. A high-resolution electronic spectrum in a supersonic jet unambiguously shows that isolated, ultracold MA has a delocalized structure in its S₀ ground state as well as in its S₁ excited state.

Photodissociation dynamics of tert-butylnitrite following excitation to the S1 and S2 states. A study by velocity-map ion-imaging and 3D-REMPI spectroscopy

Co-reporter:Andreas M. Wenge, Andreas Schmaunz, Uwe Kensy and Bernhard Dick

Physical Chemistry Chemical Physics 2012 - vol. 14(Issue 19) pp:NaN7089-7089

Publication Date(Web):2012/03/20

DOI:10.1039/C2CP40349H

Excitation of tert-butylnitrite into the first and second UV absorption bands leads to efficient dissociation into the fragment radicals NO and tert-butoxy in their electronic ground states 2Π and 2E, respectively. Velocity distributions and angular anisotropies for the NO fragment in several hundred rotational and vibrational quantum states were obtained by velocity-map imaging and the recently developed 3D-REMPI method. Excitation into the well resolved vibronic progression bands (k = 0, 1, 2) of the NO stretch mode in the S1 ← S0 transition produces NO fragments mostly in the vibrational state with v = k, with smaller fractions in v = k − 1 and v = k − 2. It is concluded that dissociation occurs on the purely repulsive PES of S1 without barrier. All velocity distributions from photolysis via the S1(nπ*) state are monomodal and show high negative anisotropy (β ≈ −1). The rotational distributions peak near j = 30.5 irrespective of the vibronic state S1(k) excited and the vibrational state v of the NO fragment. On average 46% of the excess energy is converted to kinetic energy, 23% and 31% remain as internal energy in the NO fragment and the t-BuO radical, respectively. Photolysis via excitation into the S2 ← S0 transition at 227 nm yields NO fragments with about equal populations in v = 0 and v = 1. The rotational distributions have a single maximum near j = 59.5. The velocity distributions are monomodal with positive anisotropy β ≈ 0.8. The average fractions of the excess energy distributed into translation, internal energy of NO, and internal energy of t-BuO are 39%, 23%, and 38%, respectively. In all cases ∼8500 cm−1 of energy remain in the internal degrees of freedom of the t-BuO fragment. This is mostly assigned to rotational energy. An ab initio calculation of the dynamic reaction path shows that not only the NO fragment but also the t-BuO fragment gain large angular momentum during dissociation on the purely repulsive potential energy surface of S2.

The photodissociation dynamics of N-nitrosopyrrolidine from the first and second excited singlet states studied by velocity map imaging

Co-reporter:Andreas Maximilian Wenge, Uwe Kensy and Bernhard Dick

Physical Chemistry Chemical Physics 2010 - vol. 12(Issue 18) pp:NaN4655-4655

Publication Date(Web):2010/01/28

DOI:10.1039/B920547K

The photodissociation reaction of N-nitrosopyrrolidine isolated and cooled in a supersonic jet has been studied following excitation to the S1 and S2 electronic states. The nascent NO (2Π½,, v, j) radicals were ionized by state-selective (1 + 1)-REMPI via the A2Σ+ state. The angularly resolved velocity distribution of these ions was measured with the velocity-map imaging (VMI) technique. Photodissociation from S1 produces NO in the vibrational ground state and the pyrrolidine radical in the electronic ground state 12B. About 73% of the excess energy is converted into kinetic energy of the fragments. The velocity distribution shows a strong negative anisotropy (β = −0.9) in accordance with the nπ*-character of the S0 → S1 transition. An upper limit for the N–NO dissociation energy of (14640 ± 340) cm−1 is determined. We conclude that photodissociation from S1 occurs very fast on a completely repulsive potential energy surface. Excitation into the S2 ππ*-state leads to a bimodal velocity distribution. Two dissociation channels can be distinguished which show both positive anisotropy (β = 1.3 and 1.6) but differ considerably in the total kinetic energy and the rotational energy of the NO fragment. We assign one channel to the direct dissociation on the S2 potential energy surface, leading to pyrrolidine radicals in the excited electronic state 12A. The second channel leads to pyrrolidine in the electronic ground state 12B, presumably after crossing to the S1 state via a conical intersection.

Switching from adduct formation to electron transfer in a light–oxygen–voltage domain containing the reactive cysteine

Co-reporter:Kathrin Magerl, Ivan Stambolic and Bernhard Dick

Physical Chemistry Chemical Physics 2017 - vol. 19(Issue 17) pp:NaN10819-10819

Publication Date(Web):2017/02/20

DOI:10.1039/C6CP08370F

LOV (light-, oxygen- or voltage-sensitive) domains act as photosensory units of many prokaryotic and eukaryotic proteins. Upon blue light excitation they undergo a photocycle via the excited triplet state of their flavin chromophore yielding the flavin–cysteinyl adduct. Adduct formation is highly conserved among all LOV domains and constitutes the primary step of LOV domain signaling. But recently, it has been shown that signal propagation can also be triggered by flavin photoreduction to the neutral semiquinone offering new prospects for protein engineering. This, however, requires mutation of the photo-active Cys. Here, we report on LOV1 mutants of C. reinhardtii phototropin in which adduct formation is suppressed although the photo-active Cys is present. Introduction of a Tyr into the LOV core induces a proton coupled electron transfer towards the flavin chromophore. Flavin radical species are formed via either the excited flavin singlet or triplet state depending on the geometry of donor and acceptor. This photoreductive pathway resembles the photoreaction observed in other blue light photoreceptors, e.g. blue-light sensors using flavin adenine dinucleotide (BLUF) domains or cryptochromes. The ability to tune the photoreactivity of the flavin chromophore inside the LOV core has implications for the mechanism of adduct formation in the wild type and may be of use for protein engineering.

Photoreaction of mutated LOV photoreceptor domains from Chlamydomonas reinhardtii with aliphatic mercaptans: implications for the mechanism of wild type LOV

Co-reporter:Karin Lanzl, Madlene v. Sanden-Flohe, Roger-Jan Kutta and Bernhard Dick

Physical Chemistry Chemical Physics 2010 - vol. 12(Issue 25) pp:NaN6604-6604

Publication Date(Web):2010/05/06

DOI:10.1039/B922408D

Irradiation of the LOV1 domain from the blue-light photoreceptor phototropin of the green alga Chlamydomonas reinhardtii leads to the formation of a covalent adduct of the sulfur atom of cysteine 57 to the carbon C4a in the chromophore FMN. This reaction is not possible in the mutant LOV1-C57G in which this cysteine is replaced by glycine. Irradiation of LOV1-C57G in the absence of oxygen but in the presence of aliphatic mercaptans or thioethers leads to the formation of a species with an absorption maximum at 615 nm, which is identified as the neutral radical FMNH˙. When oxygen is admitted, the reaction is completely reversible. Irradiation of LOV1-C57G in the presence of methylmercaptan CH3SH under oxygen-free conditions yields, in addition to FMNH˙, a third species with a single absorption maximum at 379 nm. This species is stable against oxygen and is also formed when the irradiation is performed in the presence of oxygen. This species is assigned to the adduct between CH3SH and FMN. In aqueous solution the photoreaction of CH3SH with FMN leads to the fully reduced hydroquinone form FMNH2 or its anion FMNH−. Adduct formation apparently requires the protein cage. After formation, the adduct is stable for hours inside the protein, but decomposes immediately upon denaturation. The implications of these observations for the mechanism of adduct formation in wild type LOV domains are discussed.

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