Engineering hydrogen bonding at tyrosine-201 in the orange carotenoid protein using halogenated analogues.

The Orange Carotenoid Protein (OCP) is a unique water-soluble photoactive protein that plays a critical role in regulating the balance between light harvesting and photoprotective responses in cyanobacteria. The challenge in understanding OCP´s photoactivation mechanism stems from the heterogeneity of the initial configurations of its embedded ketocarotenoid, which in the dark-adapted state can form up to two hydrogen bonds to critical amino acids in the protein's C-terminal domain, and the extremely low quantum yield of primary photoproduct formation. While a series of experiments involving point mutations within these contacts helped us to identify these challenges, they did not resolve them.

Specificity of Photochemical Energy Conversion in Photosystem I from the Green Microalga .

Primary excitation energy transfer and charge separation in photosystem I (PSI) from the extremophile desert green alga grown in low light were studied using broadband femtosecond pump-probe spectroscopy in the spectral range from 400 to 850 nm and in the time range from 50 fs to 500 ps. Photochemical reactions were induced by the excitation into the blue and red edges of the chlorophyll Qy absorption band and compared with similar processes in PSI from the cyanobacterium  sp. PCC 6803.

Raman Vibrational Signatures of Excited States of Echinenone in the Orange Carotenoid Protein (OCP) and Implications for its Photoactivation Mechanism.

In this study, the vibrational characteristics of optically excited echinenone in various solvents and the Orange Carotenoid Protein (OCP) in red and orange states are systematically investigated through steady-state and time-resolved spectroscopy techniques. Time-resolved experiments, employing both Transient Absorption (TA) and Femtosecond Stimulated Raman Spectroscopy (FSRS), reveal different states in the OCP photoactivation process. The time-resolved studies indicate vibrational signatures of exited states positioned above the S state during the initial 140 fs of carotenoid evolution in OCP, an absence of a vibrational signature for the relaxed S state of echinenone in OCP, and more robust signatures of a highly excited ground state (GS) in OCP.

Femtosecond optical studies of the primary charge separation reactions in far-red photosystem II from Synechococcus sp. PCC 7335.

Primary processes of light energy conversion by Photosystem II (PSII) were studied using femtosecond broadband pump-probe absorption difference spectroscopy. Transient absorption changes of core complexes isolated from the cyanobacterium Synechococcus sp. PCC 7335 grown under far-red light (FRL-PSII) were compared with the canonical Chl a containing spinach PSII core complexes upon excitation into the red edge of the Q band.

Femtosecond Dynamics of Excited States of Chlorophyll Tetramer in Water-Soluble Chlorophyll-Binding Protein BoWSCP.

The paper reports on the absorption dynamics of chlorophyll a in a symmetric tetrameric complex of the water-soluble chlorophyll-binding protein BoWSCP. It was measured by a broadband femtosecond laser pump-probe spectroscopy within the range from 400 to 750 nm and with a time resolution of 20 fs-200 ps. When BoWSCP was excited in the region of the Soret band at a wavelength of 430 nm, nonradiative intramolecular conversion S→S was observed with a characteristic time of 83 ± 9 fs.

Anti-stokes fluorescence of phycobilisome and its complex with the orange carotenoid protein.

Phycobilisomes (PBSs) are giant water-soluble light-harvesting complexes of cyanobacteria and red algae, consisting of hundreds of phycobiliproteins precisely organized to deliver the energy of absorbed light to chlorophyll chromophores of the photosynthetic electron-transport chain. Quenching the excess of excitation energy is necessary for the photoprotection of photosynthetic apparatus. In cyanobacteria, quenching of PBS excitation is provided by the Orange Carotenoid Protein (OCP), which is activated under high light conditions.

Role of pheophytin a in the primary charge separation of photosystem I from Acaryochloris marina: Femtosecond optical studies of excitation energy and electron transfer reactions.

Photosystem I (PSI) of the cyanobacterium Acaryochloris marina is capable of performing an efficient photoelectrochemical conversion of far-red light due to its unique suite of cofactors. Chlorophyll d (Chl-d) has been long known as the major antenna pigment in the PSI from A. marina, while the exact cofactor composition of the reaction centre (RC) was established only recently by cryo-electron microscopy.

Stages of OCP-FRP Interactions in the Regulation of Photoprotection in Cyanobacteria, Part 1: Time-Resolved Spectroscopy.

Most cyanobacteria utilize a water-soluble Orange Carotenoid Protein (OCP) to protect their light-harvesting complexes from photodamage. The Fluorescence Recovery Protein (FRP) is used to restore photosynthetic activity by inactivating OCP via dynamic OCP-FRP interactions, a multistage process that remains underexplored. In this work, applying time-resolved spectroscopy, we demonstrate that the interaction of FRP with the photoactivated OCP begins early in the photocycle.

Comparative Absorption Dynamics of the Singlet Excited States of Chlorophylls a and d.

Transient absorption dynamics of chlorophylls a and d dissolved in tetrahydrofuran was measured by the broadband femtosecond laser pump-probe spectroscopy in a spectral range from 400 to 870 nm. The absorption spectra of the excited S singlet states of chlorophylls a and d were recorded, and the dynamics of the of the Q band shift of the stimulated emission (Stokes shift of fluorescence) was determined in a time range from 60 fs to 4 ps. The kinetics of the intramolecular conversion Q→Q (electronic transition S→S) was measured; the characteristic relaxation time was 54 ± 3 and 45 ± 9 fs for chlorophylls a and d, respectively.

Current state of the primary charge separation mechanism in photosystem I of cyanobacteria.

This review analyzes new data on the mechanism of ultrafast reactions of primary charge separation in photosystem I (PS I) of cyanobacteria obtained in the last decade by methods of femtosecond absorption spectroscopy. Cyanobacterial PS I from many species harbours 96 chlorophyll (Chl ) molecules, including six specialized Chls denoted Chl/Chl (dimer P, or PP), Chl/Chl, and Chl/Chl arranged in two branches, which participate in electron transfer reactions. The current data indicate that the primary charge separation occurs in a symmetric exciplex, where the special pair P is electronically coupled to the symmetrically located monomers Chl and Chl, which can be considered together as a symmetric exciplex ChlPPChl with the mixed excited (ChlPPChl) and two charge-transfer states P Chl and P Chl .

Symmetry breaking in photosystem I: ultrafast optical studies of variants near the accessory chlorophylls in the A- and B-branches of electron transfer cofactors.

Femtosecond absorption spectroscopy of Photosystem I (PS I) complexes from the cyanobacterium Synechocystis sp. PCC 6803 was carried out on three pairs of complementary amino acid substitutions located near the second pair of chlorophyll molecules Chl and Chl (also termed A and A). The absorption dynamics at delays of 0.

Conserved residue PsaB-Trp673 is essential for high-efficiency electron transfer between the phylloquinones and the iron-sulfur clusters in Photosystem I.

Despite the high level of symmetry between the PsaA and PsaB polypeptides in Photosystem I, some amino acids pairs are strikingly different, such as PsaA-Gly693 and PsaB-Trp673, which are located near a cluster of 11 water molecules between the A and A quinones and the F iron-sulfur cluster. In this work, we changed PsaB-Trp673 to PsaB-Phe673 in Synechocystis sp. PCC 6803.

Role of hydrogen bond alternation and charge transfer states in photoactivation of the Orange Carotenoid Protein.

Here, we propose a possible photoactivation mechanism of a 35-kDa blue light-triggered photoreceptor, the Orange Carotenoid Protein (OCP), suggesting that the reaction involves the transient formation of a protonated ketocarotenoid (oxocarbenium cation) state. Taking advantage of engineering an OCP variant carrying the Y201W mutation, which shows superior spectroscopic and structural properties, it is shown that the presence of Trp201 augments the impact of one critical H-bond between the ketocarotenoid and the protein. This confers an unprecedented homogeneity of the dark-adapted OCP state and substantially increases the yield of the excited photoproduct S*, which is important for the productive photocycle to proceed.

Primary charge separation within the structurally symmetric tetrameric ChlPPChl chlorophyll exciplex in photosystem I.

In Photosystem I (PS I), the role of the accessory chlorophyll (Chl) molecules, Chl and Chl (also termed A and A), which are directly adjacent to the special pair P and fork into the A- and B-branches of electron carriers, is incompletely understood. In this work, the Chl and Chl transient absorption ΔA(λ) at a time delay of 100 fs was identified by ultrafast pump-probe spectroscopy in three pairs of PS I complexes from Synechocystis sp. PCC 6803 with residues PsaA-N600 or PsaB-N582 (which ligate Chl or Chl through a HO molecule) substituted by Met, His, and Leu.

Comparative Femtosecond Spectroscopy of Primary Photoreactions of Rhodopsin and Bacteriorhodopsin.

The primary stages of the rhodopsin (ESR) photocycle were investigated by femtosecond absorption laser spectroscopy in the spectral range of 400-900 nm with a time resolution of 25 fs. The dynamics of the ESR photoreaction were compared with the reactions of bacteriorhodopsin (bR) in purple membranes (bR) and in recombinant form (bR). The primary intermediates of the ESR photocycle were similar to intermediates , , and in bacteriorhodopsin photoconversion.

Control of electron transfer by protein dynamics in photosynthetic reaction centers.

Trehalose and glycerol are low molecular mass sugars/polyols that have found widespread use in the protection of native protein states, in both short- and long-term storage of biological materials, and as a means of understanding protein dynamics. These myriad uses are often attributed to their ability to form an amorphous glassy matrix. In glycerol, the glass is formed only at cryogenic temperatures, while in trehalose, the glass is formed at room temperature, but only upon dehydration of the sample.

Evidence that chlorophyll f functions solely as an antenna pigment in far-red-light photosystem I from Fischerella thermalis PCC 7521.

The Photosystem I (PSI) reaction center in cyanobacteria is comprised of ~96 chlorophyll (Chl) molecules, including six specialized Chl molecules denoted Chl1A/Chl1B (P), Chl2A/Chl2B, and Chl3A/Chl3B that are arranged in two branches and function in primary charge separation. It has recently been proposed that PSI from Chroococcidiopsis thermalis (Nürnberg et al. (2018) Science 360, 1210-1213) and Fischerella thermalis PCC 7521 (Hastings et al.

Generation of ion-radical chlorophyll states in the light-harvesting antenna and the reaction center of cyanobacterial photosystem I.

The energy and charge-transfer processes in photosystem I (PS I) complexes isolated from cyanobacteria Thermosynechococcus elongatus and Synechocystis sp. PCC 6803 were investigated by pump-to-probe femtosecond spectroscopy. The formation of charge-transfer (CT) states in excitonically coupled chlorophyll a complexes (exciplexes) was monitored by measuring the electrochromic shift of β-carotene in the spectral range 500-510 nm.

PSI-SMALP, a Detergent-free Cyanobacterial Photosystem I, Reveals Faster Femtosecond Photochemistry.

Cyanobacterial photosystem I (PSI) functions as a light-driven cyt c-ferredoxin/oxidoreductase located in the thylakoid membrane. In this work, the energy and charge transfer processes in PSI complexes isolated from Thermosynechococcus elongatus via conventional n-dodecyl-β-D-maltoside solubilization (DM-PSI) and a, to our knowledge, new detergent-free method using styrene-maleic acid copolymers (SMA-PSI) have been investigated by pump-to-probe femtosecond laser spectroscopy. In DM-PSI preparations excited at 740 nm, the excitation remained localized on the long-wavelength chlorophyll forms within 0.

G protein-coupled receptors of class A harness the energy of membrane potential to increase their sensitivity and selectivity.

The human genome contains about 700 genes of G protein-coupled receptors (GPCRs) of class A; these seven-helical membrane proteins are the targets of almost half of all known drugs. In the middle of the helix bundle, crystal structures reveal a highly conserved sodium-binding site, which is connected with the extracellular side by a water-filled tunnel. This binding site contains a sodium ion in those GPCRs that are crystallized in their inactive conformations but does not in those GPCRs that are trapped in agonist-bound active conformations.

Proton leakage across lipid bilayers: Oxygen atoms of phospholipid ester linkers align water molecules into transmembrane water wires.

Up to half of the cellular energy gets lost owing to membrane proton leakage. The permeability of lipid bilayers to protons is by several orders of magnitude higher than to other cations, which implies efficient proton-specific passages. The nature of these passages remains obscure.

Evolution of cation binding in the active sites of P-loop nucleoside triphosphatases in relation to the basic catalytic mechanism.

The ubiquitous P-loop fold nucleoside triphosphatases (NTPases) are typically activated by an arginine or lysine 'finger'. Some of the apparently ancestral NTPases are, instead, activated by potassium ions. To clarify the activation mechanism, we combined comparative structure analysis with molecular dynamics (MD) simulations of Mg-ATP and Mg-GTP complexes in water and in the presence of potassium, sodium, or ammonium ions.

Monitoring the electric field in CdSe quantum dots under ultrafast interfacial electron transfer via coherent phonon dynamics.

Coherent phonon dynamics in CdSe quantum dots (QD) under an ultrafast electron transfer (ET) reaction of the (1S-1S) exciton quenched by methyl viologen (MV) adsorbed onto the QD surface was studied by ultrafast pump-probe spectroscopy. Frequency and amplitude modulations (FM, AM) of the transient absorption ΔA(ω,t) in the pure CdSe and coupled CdSe/MV QDs were identified in the bleach band dynamics of the red-edge exciton. The fast Fourier transform (FFT) and continuous wavelet transform analysis of the FM and AM oscillations revealed peaks at 0.

Critical evaluation of electron transfer kinetics in P-F/F, P-F, and P-A Photosystem I core complexes in liquid and in trehalose glass.

This work aims to fully elucidate the effects of a trehalose glassy matrix on electron transfer reactions in cyanobacterial Photosystem I (PS I). Forward and backward electron transfer rates from A and A to F and charge recombination rates from A, A, A, F, and [F/F] to P were measured in P-F/F complexes, P-F cores, and P-A cores, both in liquid and in a trehalose glassy matrix at 11% humidity. By comparing CONTIN-resolved kinetic events over 6 orders of time in increasingly simplified versions of PS I at 480 nm, a wavelength that reports primarily A/A oxidation, and over 9 orders of time at 830 nm, a wavelength that reports P reduction and A oxidation, assignments could be made for nearly all of the resolved kinetic phases.