BLUF Domain Function Does Not Require a Metastable Radical Intermediate State

BLUF (blue light using flavin) domain proteins are an important family of blue light-sensing proteins which control a wide variety of functions in cells. The primary light-activated step in the BLUF domain is not yet established. A number of experimental and theoretical studies points to a role for photoinduced electron transfer (PET) between a highly conserved tyrosine and the flavin chromophore to form a radical intermediate state. Here we investigate the role of PET in three different BLUF proteins, using ultrafast broadband transient infrared spectroscopy. We characterize and identify infrared active marker modes for excited and ground state species and use them to record photochemical dynamics in the proteins. We also generate mutants which unambiguously show PET and, through isotope labeling of the protein and the chromophore, are able to assign modes characteristic of both flavin and protein radical states. We find that these radical intermediates are not observed in two of the three BLUF domains studied, casting doubt on the importance of the formation of a population of radical intermediates in the BLUF photocycle. Further, unnatural amino acid mutagenesis is used to replace the conserved tyrosine with fluorotyrosines, thus modifying the driving force for the proposed electron transfer reaction; the rate changes observed are also not consistent with a PET mechanism. Thus, while intermediates of PET reactions can be observed in BLUF proteins they are not correlated with photoactivity, suggesting that radical intermediates are not central to their operation. Alternative nonradical pathways including a keto–enol tautomerization induced by electronic excitation of the flavin ring are considered

Hetero-cycloreversions Mediated by Photoinduced Electron Transfer

[EN] Discovered more than eight decades ago, the Diels-Alder (DA) cycloaddition (CA) remains one of the most versatile tools in synthetic organic chemistry. Hetero-DA processes are powerful methods for the synthesis of densely functionalized six-membered heterocycles, ubiquitous substructures found in natural products and bioactive compounds. These reactions frequently employ azadienes and oxadienes, but only a few groups have reported DA processes with thiadienes. The electron transfer (ET) version of the DA reaction, though less investigated, has emerged as a subject of increasing interest. In the last two decades, researchers have paid closer attention to radical ionic hetero-cycloreversions, mainly in connection with their possible involvement in the repair of pyrimidine(6-4)pyrimidone photolesions in DNA by photolyases. In biological systems, these reactions likely occur through a reductive photosensitization mechanism. In addition, photooxidation can lead to cycloreversion (CR) reactions, and researchers can exploit this strategy for DNA repair therapies. In this Account, we discuss electron-transfer (ET) mediated hetero-CR reactions. We focus on the oxidative and reductive ET splitting of oxetanes, azetidines, and thietanes. Photoinduced electron transfer facilitates the splitting of a variety of four-membered heterocycles. In this context, researchers have commonly examined oxetanes, both experimentally and theoretically. Although a few studies have reported the cycloreversion of azetidines and thietanes carried out under electron transfer conditions, the number of examples remains limited. In general, the cleavage of the ionized four-membered rings appears to occur via a nonconcerted two-step mechanism. The trapping of the intermediate 1,4-radical ions and transient absorption spectroscopy data support this hypothesis, and it explains the observed loss of stereochemistry in the products. In the initial step, either C-C or C-X bond breaking may occur, and the preferred route depends on the substitution pattern of the ring, the type of heteroatom, and various experimental conditions. To better accommodate spin and charge, C-X cleavage happens more frequently, especially in the radical anionic version of the reaction. The addition or withdrawal of a single electron provides a new complementary synthetic strategy to activate hetero-cycloreversions. Despite its potential, this strategy remains largely unexplored. However, it offers a useful method to achieve C=X/olefin metathesis or, upon ring expansion, to construct six-membered heterocyclic rings.Financial support from the Spanish Government (Grants CTQ2010-14882, SEV2012-0267, and JCI-2010-06204) and the Generalitat Valenciana (Prometeo II/2013/005) is gratefully acknowledged.Pérez Ruiz, R.; Jiménez Molero, MC.; Miranda Alonso, MÁ. (2014). Hetero-cycloreversions Mediated by Photoinduced Electron Transfer. Accounts of Chemical Research. 47(4):1359-1368. https://doi.org/10.1021/ar4003224S1359136847

Neutral histidine and photoinduced electron transfer in DNA photolyases

The two major UV−induced DNA lesions, the cyclobutane pyrimidine dimers (CPD) and (6−4) pyrimidine−pyrimidone photoproducts, can be repaired by the light−activated enzymes CPD and (6−4) photolyases, respectively. It is a long−standing question how the two classes of photolyases with alike molecular structure are capable of reversing the two chemically different DNA photoproducts. In both photolyases the repair reaction is initiated by photoinduced electron transfer from the hydroquinone−anion part of the flavin adenine dinucleotide (FADH−) cofactor to the photoproduct. Here, the state−of−the−art XMCQDPT2−CASSCF approach was employed to compute the excitation spectra of the respective active site models. It is found that protonation of His365 in the presence of the hydroquinone−anion electron donor causes spontaneous, as opposed to photoinduced, coupled proton and electron transfer to the (6−4) photoproduct. The resulting neutralized biradical, containing the neutral semiquinone and the N3'−protonated (6−4) photoproduct neutral radical, corresponds to the lowest energy electronic ground−state minimum. The high electron affinity of the N3'−protonated (6−4) photoproduct underlines this finding. Thus, it is anticipated that the (6−4) photoproduct repair is assisted by His365 in its neutral form, which is in contrast to the repair mechanisms proposed in the literature. The repair via hydroxyl group transfer assisted by neutral His365 is considered. The repair involves the 5'base radical anion of the (6−4) photoproduct which in terms of electronic structure is similar to the CPD radical anion. A unified model of the CPD and (6−4) photoproduct repair is propose

Spiers Memorial Lecture. Introductory lecture: the impact of structure on photoinduced processes in nucleic acids and proteins

Light is an important environmental variable and most organisms have evolved means to sense, exploit or avoid it and to repair detrimental effects on their genome. In general, light absorption is the task of specific chromophores, however other biomolecules such as oligonucleotides also do so which can result in undesired outcomes such as mutations and cancer. Given the biological importance of light-induced processes and applications for imaging, optogenetics, photodynamic therapy or photovoltaics, there is a great interest in understanding the detailed molecular mechanisms of photoinduced processes in proteins and nucleic acids. The processes are typically characterized by time-resolved spectroscopic approaches or computation, inferring structural information on transient species from stable ground state structures. Recently, however, structure determination of excited states or other short-lived species has become possible with the advent of X-ray free-electron lasers. This review gives an overview of the impact of structure on the understanding of photoinduced processes in macromolecules, focusing on systems presented at this Faraday Discussion meeting

Glutamine rotamers in BLUF photoreceptors: a mechanistic reappraisal

The blue light using FAD (BLUF) photosensory protein domain is activated by a unique photoreaction that results in a hydrogen-bond rearrangement around the flavin chromophore. The chemical structure of the hydrogen bond switch is a long-standing debate: The two main hypotheses postulate rotation as opposed to tautomerization of a conserved glutamine residue. Attempts to resolve the debate were inconclusive so far, despite numerous experimental and computational studies. Here we propose physical criteria for the dark and light state structures as well as for the light-activation process to evaluate existing models of BLUF using quantum-chemical calculations. The glutamine rotamer assignment of the crystal structure with the pdb code 1YRX does not satisfy our criteria because after equilibrating the intermolecular forces the glutamine rotamer in 1YRX is incompatible with the experimental density. We identified the root of the mechanistic controversy in the incorrect glutamine rotamer assignment of 1YRX . Furthermore, we show that the glutamine side chain may rotate without light activation in the BLUF dark state. Finally, we demonstrate that the tautomerized glutamine is consistent with our criteria and observations of the BLUF light stat

Challenges in computing electron-transfer energies of DNA repair using hybrid QM/MM models

The influence of the molecular environment on chemical activity is an important factor in biomolecular mechanisms. We studied the effects of ionic groups, that is, a protonated histidine side chain and deprotonated phosphates of DNA, on electron transfer in light-induced DNA repair. On the basis of the X-ray crystal structure, we prepared a hybrid QM/MM model of the macromolecular complex formed between the (6–4) photolyase enzyme and the DNA substrate containing the thymine–thymine (6–4) photoproduct. At the optimized geometries, we computed with the CASSCF and CASPT2 methods the excited states of the electron donor and electron acceptor complex, consisting of the reduced flavin and the (6–4) photoproduct. The donor–acceptor complex interacts with its environment comprised of the protein, the double-stranded DNA substrate with its counterions, and the solvating water molecules, which we modeled using the AMBER94 force field. The excited states of our interest include two locally excited (LE) states of the flavin chromophore and intermolecular electron-transfer (ET) states. We identify only minor changes of the LE excitation energies by interactions with the environment, but in drastic contrast to that, we found significant changes of the ET excitation energies. In the presence of the positively charged His365H+, the ET excitation energies decrease, indicating facilitated electron transfer. In addition, the excitation energy of the second LE state, explaining the flavin’s absorption at 380 nm, undergoes a 0.2 eV downshift. In contrast to the active-site protonation, reduced screening of the DNA phosphates increases the ET excitation energies but not the LE excitation energies. Accordingly, the electron affinities of the (6–4) photoproduct are significantly reduced, which should hinder electron transfer from the excited flavin. We also show that dynamic electron correlation accounted by the second order perturbation theory CASPT2 does not alter the energy trends obtained with the CASSCF method. Including the histidine side chain in the QM part enhances the effect of the histidine protonation on the ET energies. We also note that protonated His365H+ can serve as an electron acceptor