A1 Binding Site Research Articles

Time-resolved FTIR difference spectroscopy for the study of photosystem I with high potential naphthoquinones incorporated into the A1 binding site 2: Identification of neutral state quinone bands.

Time-resolved step-scan FTIR difference spectroscopy at 77K has been used to study photosystem I (PSI) from Synechocystis sp. PCC 6803 with four high-potential, 1,4-naphthoquinones (NQs) incorporated into the A1 binding site. The incorporated quinones are 2-chloro-NQ (2ClNQ), 2-bromo-NQ (2BrNQ), 2,3-dichloro-NQ (Cl2NQ), and 2,3-dibromo-NQ (Br2NQ). For completeness 2-methyl-NQ (2MNQ) was also incorporated and studied. Previously, PSI with the same quinones incorporated were studied in the, so-called, anion spectral region between 1550 and 1400cm-1 (Agarwala et al. in Biochim Biophys Acta 1864(1):148918, 2023). Here we focus on spectra in the previously unexplored 1400-1200cm-1 spectral region. In this region several bands are identified and assigned to the neutral state of the incorporated quinones. This is important as identification of neutral state quinone bands in the regular 1700-1600cm-1 region has proven difficult in the past. For neutral PhQ in PSI a broad, intense band appears at ~ 1300cm-1. For the symmetric di-substituted NQs (Cl2NQ/Br2NQ) a single intense neutral state band is found at ~ 1280/1269cm-1, respectively. For both mono-substituted NQs, 2ClNQ and 2BrNQ, however, two neutral state bands are observed at ~ 1280 and ~ 1250cm-1, respectively. These observations from time-resolved spectra agree well with conclusions drawn from absorption spectra of the NQs in THF, which are also presented here. Density functional theory based vibrational frequency calculations were undertaken allowing an identification of the normal modes associated with the neutral state quinone bands.

A splicing silencer in SMN2 intron 6 is critical in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a fatal neuromuscular disease caused by homozygous deletions or mutations of the SMN1 gene. SMN2 is a paralogous gene of SMN1 and a modifying gene of SMA. A better understanding of how SMN2 exon 7 splicing is regulated helps discover new therapeutic targets for SMA therapy. Based on an antisense walk method to map exonic and intronic splicing silencers (ESSs and ISSs) in SMN2 exon 7 and the proximal regions of its flanking introns, we identified one ISS (ISS6-KH) at upstream of the branch point site in intron 6. By using mutagenesis-coupled RT-PCR with SMN1/2 minigenes, immunochromatography, overexpression and siRNA-knockdown, we found this ISS consists of a bipartite hnRNP A1 binding cis-element and a poly-U sequence located between the proximal hnRNP A1 binding site (UAGCUA) and the branch site. Both HuR and hnRNP C1 proteins promote exon 7 skipping through the poly-U stretch. Mutations or deletions of these motifs lead to efficient SMN2 exon 7 inclusion comparable to SMN1 gene. Furthermore, we identified an optimal antisense oligonucleotide that binds the intron six ISS and causes striking exon 7 inclusion in the SMN2 gene in patient fibroblasts and SMA mouse model. Our findings demonstrate that this novel ISS plays an important role in SMN2 exon 7 skipping and highlight a new therapeutic target for SMA therapy.

Time-resolved FTIR difference spectroscopy for the study of photosystem I with high potential naphthoquinones incorporated into the A1 binding site

Time–resolved step-scan Fourier transform infrared difference spectroscopy has been used to study cyanobacterial photosystem I photosynthetic reaction centers from Synechocystis sp. PCC 6803 (S6803) with four high-potential, 1,4-naphthoquinones incorporated into the A1 binding site. The high-potential naphthoquinones are 2-chloro-, 2-bromo-, 2,3-dichloro- and 2,3-dibromo-1,4-naphthoquinone. “Foreign minus native” double difference spectra (DDS) were constructed by subtracting difference spectra for native photosystem I (with phylloquinone in the A1 binding site) from corresponding spectra obtained using photosystem I with the different quinones incorporated. To help assess and assign bands in the difference and double difference spectra, density functional theory based vibrational frequency calculations for the different quinones in solvent, or in the presence of a single asymmetric H– bond to either a water molecule or a peptide backbone NH group, were undertaken. Calculated and experimental spectra agree best for the peptide backbone asymmetrically H– bonded system. By comparing multiple sets of double difference spectra, several new bands for the native quinone (phylloquinone) are identified. By comparing calculated and experimental spectra we conclude that the mono-substituted halogenated NQs can occupy the binding site in either of two different orientations, with the chlorine or bromine atom being either ortho or meta to the H– bonded CO group.

Experimental and calculated infrared spectra of disubstituted naphthoquinones

In recent years there has been interest in incorporating substituted 1,4-naphthoquinones (NQs) into the A1 binding site in photosystem I (PSI) photosynthetic protein complexes. This interest in part stems from the considerably altered bioenergetics of electron transfer that occur in PSI with such substitutions. Time resolved FTIR studies of PSI complexes with disubstituted NQs incorporated have and currently are being undertaken, and with this in mind it is worth considering FTIR absorption spectra of these disubstituted NQs in solution. Here we present FTIR absorbance spectra for 2-bromo-3-methyl-1,4-naphthoquinone (BrMeNQ), 2-chloromethyl-3-methyl-1,4-naphthoquinone (CMMeNQ) and 2-ethylthio-3-methyl-1,4-naphthoquinone (ETMeNQ) in tetrahydrofuran (THF). The FTIR spectra of these di-substituted naphthoquinones (NQs) were compared to FTIR spectra of 2-methyl-3-phytyl-1,4-naphthoquinone [phylloquinone (PhQ)], 2,3-dimethyl-1,4-naphthoquinone (DMNQ), and 2-methyl-1,4-naphthoquinone (2MNQ). To aid in the assignment of bands in the experimental spectra, density functional theory (DFT) based vibrational frequency calculations for all the substituted NQs in solution were undertaken. The calculated and experimental spectra agree well. By calculating normal mode potential energy distributions, unambiguous quantitative band assignments were made. The calculated and experimental spectra together make predictions about what may be observable in time resolved FTIR difference spectra obtained using PSI with the different NQs incorporated. Time resolved FTIR difference spectra are presented that support these predictions.

Assessment of the orientation and conformation of pigments in protein binding sites from infrared difference spectra

Time resolved FTIR difference spectroscopy (DS) has been used to study photosystem I (PSI) with the disubstituted 1,4-naphthoquinones acequinocyl (AcQ) and lapachol (Lpc) incorporated into the A1 binding site. AcQ is a 2-acetoxy-3-dodecyl-1,4-naphthoquinone, Lpc is a 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone. To assess whether the experimental spectra are specific to different orientations of the quinone and their substitutions ONIOM-type QM/MM vibrational frequency calculations were undertaken for various orientations of the pigments and side-chain conformations in the A1 binding site.Comparison of calculated and experimental spectra for the reduced species (semiquinone anion) suggests that the orientation for the naphthoquinone ring in the binding site and specific side-chain conformations can be identified based on the spectra. In native PSI phylloquinone (PhQ) in the A1 binding site binds with its phytyl chain ortho to the hydrogen bonded carbonyl group. This is not found to be the case for the hydrocarbon tail of AcQ, which is meta to the H-bonded carbonyl group. In contrast, Lpc in PSI binds with its hydrocarbon tail also ortho to the H-bonded carbonyl group. Furthermore, comparison of calculated and experimental spectra indicates which conformations the acetoxy group of AcQ and the hydroxy group of Lpc adopt in the A1 binding site.

Reversible inhibition and reactivation of electron transfer in photosystem I.

In photosystem I (PSI) complexes at room temperature electron transfer from A1- to FX is an order of magnitude faster on the B-branch compared to the A-branch. One factor that might contribute to this branch asymmetry in time constants is TrpB673 (Thermosynechococcus elongatus numbering), which is located between A1B and FX. The corresponding residue on the A-branch, between A1A and FX, is GlyA693. Here, microsecond time-resolved step-scan FTIR difference spectroscopy at 77K has been used to study isolated PSI complexes from wild type and TrpB673Phe mutant (WB673F mutant) cells from Synechocystis sp. PCC 6803. WB673F mutant cells require glucose for growth and are light sensitive. Photoaccumulated FTIR difference spectra indicate changes in amide I and II protein vibrations upon mutation of TrpB673 to Phe, indicating the protein environment near FX is altered upon mutation. In the WB673F mutant PSI samples, but not in WT PSI samples, the phylloquinone molecule that occupies the A1 binding site is likely doubly protonated following long periods of repetitive flash illumination at room temperature. PSI with (doubly) protonated quinone in the A1 binding site are not functional in electron transfer. However, electron transfer functionality can be restored by incubating the light-treated mutant PSI samples in the presence of added phylloquinone.

Time-resolved FTIR difference spectroscopy for the study of quinones in the A1 binding site in photosystem I: Identification of neutral state quinone bands

Infrared absorption bands associated with the neutral state of quinones in the A1 binding site in photosystem I (PSI) have been difficult to identify in the past. This problem is addressed here, where time-resolved step-scan FTIR difference spectroscopy at 77 K has been used to study PSI with six different quinones incorporated into the A1 binding site. (P700+A1− – P700A1) and (A1− – A1) FTIR difference spectra (DS) were obtained for PSI with the different quinones incorporated, and several double-difference spectra (DDS) were constructed from the DS. From analysis of the DS and DDS, in combination with density functional theory based vibrational frequency calculations of the quinones, the neutral state bands of the incorporated quinones are identified and assigned. For neutral PhQ in the A1 binding site, infrared absorption bands were identified near 1665 and 1635 cm−1, that are due to the C1O and C4O stretching vibrations of the incorporated PhQ, respectively. These assignments indicate a 30 cm−1 separation between the C1O and C4O modes, considerably less than the ~80 cm−1 found for similar modes of PhQ−. The C4O mode downshifts due to hydrogen bonding, so the suggestion is that hydrogen bonding is weaker for the neutral state compared to the anion state, indicating radical-induced proton dynamics associated with the quinone in the A1 binding site in PSI.

Calculated vibrational properties of semiquinones in the A1 binding site in photosystem I

Time-resolved (P700+A1− – P700A1) FTIR difference spectra have been obtained using photosystem I (PSI) particles with several different quinones incorporated into the A1 protein binding site. Difference spectra were obtained for PSI with unlabeled and 18O labeled phylloquinone (2-methyl-3-phytyl-1,4-naphthoquinone) and 2-methyl-1,4-naphthaquinone (2MNQ) incorporated, and for PSI with unlabeled 2,3-dimethyl-1,4-naphthoquinone (DMNQ) incorporated. (18O – 16O), (2MNQ – PhQ) and (DMNQ – PhQ) FTIR double difference spectra were constructed from the difference spectra. These double difference spectra allow one to more easily distinguish protein and pigment bands in convoluted difference spectra. To further aid in the interpretation of the difference spectra, particularly the spectra associated with the semiquinones, we have used two-layer ONIOM methods to calculate corresponding difference and double difference spectra. In all cases, the experimental and calculated double difference spectra are in excellent agreement. In previous two and three-layer ONIOM calculations it was not possible to adequately simulate multiple difference and double difference spectra. So, the computational approach outlined here is an improvement over previous calculations. It is shown that the calculated spectra can vary depending on the details of the molecular model that is used. Specifically, a molecular model that includes several water molecules that are near the incorporated semiquinones is required.

Kinetics and Energetics of Phylloquinone Reduction in Photosystem I: Insight From Modeling of the Site Directed Mutants

Two phylloquinone molecules (A1), one being predominantly coordinated by PsaA subunit residues (A1A) the other by those of PsaB (A1B), act as intermediates in the two parallel electron transfer chains of Photosystem I. The oxidation kinetics of the two phyllosemiquinones by the iron-sulfur cluster FX differ by approximately one order of magnitude, with being oxidized in about 200 ns and in about 20 ns. These differences are generally explained in terms of asymmetries in the driving force for FX reduction on the two electron transfer chains. Site directed mutations of conserved amino acids composing the A1 binding site have been engineered on both reaction center subunits, and proved to affect selectively the oxidation lifetime of either , for PsaA mutants, or , for PsaB mutants. The mutation effects are here critically reviewed, also by novel modeling simulations employing the tunneling formalism to estimate the electron transfer rates. Three main classes of mutation effects are in particular addressed: (i) those leading to an acceleration, (ii) those leading to a moderated slowing (~5-folds), and (iii) those leading to a severe slowing (>20-folds) of the kinetics. The effect of specific amino acid perturbations contributing to the poising of the phylloquinones redox potential and, in turn, to PSI functionality, is discussed.

Time-resolved step-scan FTIR difference spectroscopy for the study of photosystem I with different benzoquinones incorporated into the A1 binding site

Time-resolved step-scan FTIR difference spectroscopy has been used to study photosystem I (PSI) with plastoquinone-9 (PQ) and two other benzoquinones (2,6-dimethyl-1,4-benzoquinone and 2,3,5,6-tetrachloro-1,4-benzoquinone) incorporated into the A1 binding site. By subtracting a (P700+A1− − P700A1) FTIR difference spectrum for PSI with the native phylloquinone (PhQ) incorporated from corresponding spectra for PSI with different benzoquinones (BQs) incorporated, FTIR double difference spectra are produced, that display bands associated with vibrational modes of the quinones, without interference from features associated with protein vibrational modes.Molecular models for BQs involved in asymmetric hydrogen bonding were constructed and used in vibrational mode frequency calculations. The calculated data were used to aid in the interpretation and assignment of bands in the experimental spectra. We show that the calculations capture the general trends found in the experimental spectra.By comparing four different FTIR double difference spectra we are able to verify unambiguously bands associated with phyllosemiquinone in PSI at 1495 and 1415 cm−1. We also resolve a previously unrecognized band of phyllosemiquinone at 1476 cm−1 that calculations suggest is due in part to a C4−⃛O stretching mode.For PSI with PQ incorporated, calculations and experiment taken together indicate that the C1−⃛O and C4−⃛O vibrational modes of the semiquinone give rise to bands at 1487 and 1444 cm−1, respectively. This is very distinct compared to PSI with PhQ incorporated.From the calculated and experimental spectra, we show that it is possible to distinguish between two possible orientations of PQ in the A1 protein binding site.

Blocking of an intronic splicing silencer completely rescues IKBKAP exon 20 splicing in familial dysautonomia patient cells.

Familial dysautonomia (FD) is a severe genetic disorder causing sensory and autonomic dysfunction. It is predominantly caused by a c.2204+6T>C mutation in the IKBKAP gene. This mutation decreases the 5′ splice site strength of IKBKAP exon 20 leading to exon 20 skipping and decreased amounts of full-length IKAP protein. We identified a binding site for the splicing regulatory protein hnRNP A1 downstream of the IKBKAP exon 20 5′-splice site. We show that hnRNP A1 binds to this splicing regulatory element (SRE) and that two previously described inhibitory SREs inside IKBKAP exon 20 are also bound by hnRNP A1. Knockdown of hnRNP A1 in FD patient fibroblasts increases IKBKAP exon 20 inclusion demonstrating that hnRNP A1 is a negative regulator of IKBKAP exon 20 splicing. Furthermore, by mutating the SREs in an IKBKAP minigene we show that all three SREs cause hnRNP A1-mediated exon repression. We designed splice switching oligonucleotides (SSO) that blocks the intronic hnRNP A1 binding site, and demonstrate that this completely rescues splicing of IKBKAP exon 20 in FD patient fibroblasts and increases the amounts of IKAP protein. We propose that this may be developed into a potential new specific treatment of FD.

Photosystem I with benzoquinone analogues incorporated into the A1 binding site.

Time-resolved FTIR difference spectroscopy has been used to study photosystem I (PSI) particles with three different benzoquinones [plastoquinone-9 (PQ), 2,6-dimethyl-1,4-benzoquinone (DMBQ), 2,3,5,6-tetrachloro-1,4-benzoquinone (Cl4BQ)] incorporated into the A1 binding site. If PSI samples are cooled in the dark to 77K, the incorporated benzoquinones are shown to be functional, allowing the production of time-resolved (P700+A1--P700A1) FTIR difference spectra. If samples are subjected to repetitive flash illumination at room temperature prior to cooling, however, the time-resolved FTIR difference spectra at 77K display contributions typical of the P700 triplet state (3P700), indicating a loss of functionality of the incorporated benzoquinones, that occurs because of double protonation of the incorporated benzoquinones. The benzoquinone protonation mechanism likely involves nearby water molecules but does not involve the terminal iron-sulfur clusters FA and FB. These results and conclusions resolve discrepancies between results from previous low-temperature FTIR and EPR studies on similar PSI samples with PQ incorporated.

Quinones in the A1 binding site in photosystem I studied using time-resolved FTIR difference spectroscopy

Time-resolved step-scan FTIR difference spectroscopy at low temperature (77 K) has been used to study photosystem I particles with phylloquinone (2-methyl-3-phytyl-1,4-naphthaquinone) and menadione (2-methyl-1,4-naphthaquinone) incorporated into the A1 binding site. By subtracting spectra for PSI with phylloquinone incorporated from spectra for PSI with menadione incorporated a (menadione - phylloquinone) double difference spectrum was constructed. In the double difference spectrum bands associated with protein vibrational modes effectively cancel, and the bands in the spectrum are primarily associated with the neutral and reduced states of the two quinones in the A1 binding site.To aid in the assignment of bands in the experimental double difference spectrum, a double difference spectrum was calculated using three-layer ONIOM methods. The calculated and experimental spectra agree well, allowing unambiguous band assignments to be made. The ONIOM calculations show that both quinones in the A1 binding site are similarly oriented, with only a single hydrogen bond between the C4=O quinone carbonyl group and the backbone NH group of a leucine residue. For the semi-quinone species, but not for the neutral species, this hydrogen bond appears to be very strong.Finally, we have for the first time been able to unmask and identify infrared difference bands associated with neutral naphthoquinone species occupying the A1 binding site in PSI.

Kinetic modeling of electron transfer reactions in photosystem I complexes of various structures with substituted quinone acceptors.

The reduction kinetics of the photo-oxidized primary electron donor P700 in photosystem I (PS I) complexes from cyanobacteria Synechocystis sp. PCC 6803 were analyzed within the kinetic model, which considers electron transfer (ET) reactions between P700, secondary quinone acceptor A1, iron-sulfur clusters and external electron donor and acceptors-methylviologen (MV), 2,3-dichloro-naphthoquinone (Cl2NQ) and oxygen. PS I complexes containing various quinones in the A1-binding site (phylloquinone PhQ, plastoquinone-9 PQ and Cl2NQ) as well as F X-core complexes, depleted of terminal iron-sulfur F A/F B clusters, were studied. The acceleration of charge recombination in F X-core complexes by PhQ/PQ substitution indicates that backward ET from the iron-sulfur clusters involves quinone in the A1-binding site. The kinetic parameters of ET reactions were obtained by global fitting of the P700+ reduction with the kinetic model. The free energy gap ΔG 0 between F X and F A/F B clusters was estimated as -130meV. The driving force of ET from A1 to F X was determined as -50 and -220meV for PhQ in the A and B cofactor branches, respectively. For PQ in A1A-site, this reaction was found to be endergonic (ΔG 0 = +75meV). The interaction of PS I with external acceptors was quantitatively described in terms of Michaelis-Menten kinetics. The second-order rate constants of ET from F A/F B, F X and Cl2NQ in the A1-site of PS I to external acceptors were estimated. The side production of superoxide radical in the A1-site by oxygen reduction via the Mehler reaction might comprise ≥0.3% of the total electron flow in PS I.

Serine/Arginine-Rich Splicing Factor 3 and Heterogeneous Nuclear Ribonucleoprotein A1 Regulate Alternative RNA Splicing and Gene Expression of Human Papillomavirus 18 through Two Functionally Distinguishable cis Elements.

Human papillomavirus 18 (HPV18) is the second most common oncogenic HPV type associated with cervical, anogenital, and oropharyngeal cancers. Like other oncogenic HPVs, HPV18 encodes two major (one early and one late) polycistronic pre-mRNAs that are regulated by alternative RNA splicing to produce a repertoire of viral transcripts for the expression of individual viral genes. However, RNA cis-regulatory elements and trans-acting factors contributing to HPV18 alternative RNA splicing remain unknown. In this study, an exonic splicing enhancer (ESE) in the nucleotide (nt) 3520 to 3550 region in the HPV18 genome was identified and characterized for promotion of HPV18 929^3434 splicing and E1^E4 production through interaction with SRSF3, a host oncogenic splicing factor differentially expressed in epithelial cells and keratinocytes. Introduction of point mutations in the SRSF3-binding site or knockdown of SRSF3 expression in cells reduces 929^3434 splicing and E1^E4 production but activates other, minor 929^3465 and 929^3506 splicing. Knockdown of SRSF3 expression also enhances the expression of E2 and L1 mRNAs. An exonic splicing silencer (ESS) in the HPV18 nt 612 to 639 region was identified as being inhibitory to the 233^416 splicing of HPV18 E6E7 pre-mRNAs via binding to hnRNP A1, a well-characterized, abundantly and ubiquitously expressed RNA-binding protein. Introduction of point mutations into the hnRNP A1-binding site or knockdown of hnRNP A1 expression promoted 233^416 splicing and reduced E6 expression. These data provide the first evidence that the alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host trans-acting splicing factors. Expression of HPV18 genes is regulated by alternative RNA splicing of viral polycistronic pre-mRNAs to produce a repertoire of viral early and late transcripts. RNA cis elements and trans-acting factors contributing to HPV18 alternative RNA splicing have been discovered in this study for the first time. The identified ESS at the E7 open reading frame (ORF) prevents HPV18 233^416 splicing in the E6 ORF through interaction with a host splicing factor, hnRNP A1, and regulates E6 and E7 expression of the early E6E7 polycistronic pre-mRNA. The identified ESE at the E1^E4 ORF promotes HPV18 929^3434 splicing of both viral early and late pre-mRNAs and E1^E4 production through interaction with SRSF3. This study provides important observations on how alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host splicing factors and offers potential therapeutic targets to overcome HPV-related cancer.

Global identification of hnRNP A1 binding sites for SSO-based splicing modulation.

BackgroundMany pathogenic genetic variants have been shown to disrupt mRNA splicing. Besides splice mutations in the well-conserved splice sites, mutations in splicing regulatory elements (SREs) may deregulate splicing and cause disease. A promising therapeutic approach is to compensate for this deregulation by blocking other SREs with splice-switching oligonucleotides (SSOs). However, the location and sequence of most SREs are not well known.ResultsHere, we used individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) to establish an in vivo binding map for the key splicing regulatory factor hnRNP A1 and to generate an hnRNP A1 consensus binding motif. We find that hnRNP A1 binding in proximal introns may be important for repressing exons. We show that inclusion of the alternative cassette exon 3 in SKA2 can be significantly increased by SSO-based treatment which blocks an iCLIP-identified hnRNP A1 binding site immediately downstream of the 5’ splice site. Because pseudoexons are well suited as models for constitutive exons which have been inactivated by pathogenic mutations in SREs, we used a pseudoexon in MTRR as a model and showed that an iCLIP-identified hnRNP A1 binding site downstream of the 5′ splice site can be blocked by SSOs to activate the exon.ConclusionsThe hnRNP A1 binding map can be used to identify potential targets for SSO-based therapy. Moreover, together with the hnRNP A1 consensus binding motif, the binding map may be used to predict whether disease-associated mutations and SNPs affect hnRNP A1 binding and eventually mRNA splicing.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0279-9) contains supplementary material, which is available to authorized users.

Time-resolved visible and infrared absorption spectroscopy data obtained using photosystem I particles with non-native quinones incorporated into the A1 binding site.

Time-resolved visible and infrared absorption difference spectroscopy data at both 298 and 77 K were obtained using cyanobacterial menB− mutant photosystem I particles with several non-native quinones incorporated into the A1 binding site. Data was obtained for photosystem I particles with phylloquinone (2-methyl-3-phytyl-1,4-naphthoquinone), 2-bromo-1,4-naphthoquinone, 2-chloro-1,4-naphthoquinone, 2-methyl-1,4-naphthoquinone, 2,3-dibromo-1,4-naphthoquinone, 2,3-dichloro-1,4-naphthoquinone, and 9,10-anthraquinone incorporated. Transient absorption data were obtained at 487 and 703 nm in the visible spectral range, and 1950–1100 cm−1 in the infrared region. Time constants obtained from fitting the time-resolved infrared and visible data are in good agreement. The measured time constants are crucial for the development of appropriate kinetic models that can describe electron transfer processes in photosystem I, “Modeling Electron Transfer in Photosystem I” Makita and Hastings (2016) [1].

Modeling electron transfer in photosystem I

Nanosecond to millisecond time-resolved absorption spectroscopy has been used to study electron transfer processes in photosystem I particles from Synechocystis sp. PCC 6803 with eight different quinones incorporated into the A1 binding site, at both 298 and 77K. A detailed kinetic model was constructed and solved within the context of Marcus electron transfer theory, and it was found that all of the data could be well described only if the in situ midpoint potentials of the quinones fell in a tightly defined range. For photosystem I with phylloquinone incorporated into the A1 binding site all of the time-resolved optical data is best modeled when the in situ midpoint potential of phylloquinone on the A/B branch is −635/−690mV, respectively. With the midpoint potential of the FX iron sulfur cluster set at −680mV, this indicates that forward electron transfer from A1− to FX is slightly endergonic/exergonic on the A/B branch, respectively. Additionally, for forward electron transfer from A1− to FX, on both the A and B branches the reorganization energy is close to 0.7eV. Reorganization energies of 0.4 or 1.0eV are not possible.For the eight different quinones incorporated, the same kinetic model was used, allowing us to establish in situ redox potentials for all of the incorporated quinones on both branches. A linear correlation was found between the in situ and in vitro midpoint potentials of the quinones on both branches.

Directionality of electron transfer in cyanobacterial photosystem I at 298 and 77 K

Electron transfer processes in cyanobacterial photosystem I particles from Synechocystis sp. PCC 6803 with a high potential naphthoquinone (2,3-dichloro-1,4-naphthoquinone) incorporated into the A1 binding site have been studied at 298 and 77K using time-resolved visible and infrared difference spectroscopy. The high potential naphthoquinone inhibits electron transfer past A1, and biphasic P700+A1− radical pair recombination is observed. The two phases are assigned to P700+A1B− and P700+A1A− recombination. Analyses of the transient absorption changes indicate that the ratio of A- and B-branch electron transfer is 95:5 at 77 K and 77:23 at 298K.

Time-resolved visible and infrared difference spectroscopy for the study of photosystem I with different quinones incorporated into the A1 binding site

Room (298K) and low (77K) temperature time-resolved visible and infrared difference spectroscopy has been used to study photosystem I particles with phylloquinone (2-methyl-3-phytyl-1,4-naphthoquinone), menadione (2-methyl-1,4-naphthoquinone) and plastoquinone 9 (2,3-dimethyl-5-prenyl-l,4-benzoquinone), incorporated into the A1 binding site. Concentrated samples in short path-length (~5μm) sample cells are typically used in FTIR experiments. Measurements were undertaken using standard “dilute” samples at 298K, and concentrated (~5×) samples at both 298 and 77K. No concentration induced alterations in the flash-induced absorption changes were observed. Concentrated samples in short path-length cells form a transparent film at 77K, and could therefore be studied spectroscopically at 77K without addition of a cryoprotectant. At 298K, for photosystem I with plastoquinone 9/menadione/phylloquinone incorporated, P700+FA/B− radical pair recombination is characterized by a time constant of 3/14/80ms, and forward electron transfer from A1A− to Fx by a time constant of 211/3.1/0.309μs, respectively. At 77K, for concentrated photosystem I with menadione/phylloquinone incorporated, P700+A1− radical pair recombination is characterized by a time constant of 240/340μs, with this process occurring in 58/39% of the PSI particles, respectively. The origin of these differences is discussed. Marcus electron transfer theory in combination with kinetic modeling is used to simulate the observed electron transfer time constants at 298K. This simulation allows an estimate of the redox potential for the different quinones in the A1 binding site.