Photosystems I and II convert solar energy into the chemical energy that powers life. Chlorophyll a photochemistry, using red light (680 to 700 nm), is near universal and is considered to define the ...energy "red limit" of oxygenic photosynthesis. We present biophysical studies on the photosystems from a cyanobacterium grown in far-red light (750 nm). The few long-wavelength chlorophylls present are well resolved from each other and from the majority pigment, chlorophyll a. Charge separation in photosystem I and II uses chlorophyll f at 745 nm and chlorophyll f (or d) at 727 nm, respectively. Each photosystem has a few even longer-wavelength chlorophylls f that collect light and pass excitation energy uphill to the photochemically active pigments. These photosystems function beyond the red limit using far-red pigments in only a few key positions.
We provide a new and definitive spectral assignment for the absorption, emission, high-resolution fluorescence excitation, linear dichroism, and/or magnetic circular dichroism spectra of 32 ...chlorophyllides in various environments. This encompases all data used to justify previous assignments and provides a simple interpretation of unexplained complex decoherence phenomena associated with Qx → Qy relaxation. Whilst most chlorophylls conform to the Gouterman model and display two independent transitions Qx (S2) and Qy (S1), strong vibronic coupling inseparably mixes these states in chlorophyll-a. This spreads x-polarized absorption intensity over the entire Q-band system to influence all exciton-transport, relaxation and coherence properties of chlorophyll-based photosystems. The fraction of the total absorption intensity attributed to Qx ranges between 7% and 33%, depending on chlorophyllide and coordination, and is between 10% and 25% for chlorophyll-a. CAM-B3LYP density-functional-theory calculations of the band origins, relative intensities, vibrational Huang-Rhys factors, and vibronic coupling strengths fully support this new assignment.
The identification of low-energy chlorophyll pigments in photosystem II (PSII) is critical to our understanding of the kinetics and mechanism of this important enzyme. We report parallel circular ...dichroism (CD) and circularly polarized luminescence (CPL) measurements at liquid helium temperatures of the proximal antenna protein CP47. This assembly hosts the lowest-energy chlorophylls in PSII, responsible for the well-known “F695” fluorescence band of thylakoids and PSII core complexes. Our new spectra enable a clear identification of the lowest-energy exciton state of CP47. This state exhibits a small but measurable excitonic delocalization, as predicated by its CD and CPL. Using structure-based simulations incorporating the new spectra, we propose a revised set of site energies for the 16 chlorophylls of CP47. The significant difference from previous analyses is that the lowest-energy pigment is assigned as Chl 612 (alternately numbered Chl 11). The new assignment is readily reconciled with the large number of experimental observations in the literature, while the most common previous assignment for the lowest energy pigment, Chl 627(29), is shown to be inconsistent with CD and CPL results. Chl 612(11) is near the peripheral light-harvesting system in higher plants, in a lumen-exposed region of the thylakoid membrane. The low-energy pigment is also near a recently proposed binding site of the PsbS protein. This result consequently has significant implications for our understanding of the kinetics and regulation of energy transfer in PSII.
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•Assignment of the lowest-energy chlorophyll of PSII, responsible for the F695 fluorescence band, to Chl 612 of CP47 (alternatively numbered Chl 11)•Location of the main emitting pigment is highly relevant for energy transfer and regulation in PSII.•Unambiguous observation of two distinct emission bands of CP47 via their circular polarization
Far-red light (FRL) Photosystem II (PSII) isolated from Chroococcidiopsis thermalis is studied using parallel analyses of low-temperature absorption, circular dichroism (CD) and magnetic circular ...dichroism (MCD) spectroscopies in conjunction with fluorescence measurements. This extends earlier studies (Nurnberg et al 2018 Science 360 (2018) 1210–1213). We confirm that the chlorophyll absorbing at 726 nm is the primary electron donor. At 1.8 K efficient photochemistry occurs when exciting at 726 nm and shorter wavelengths; but not at wavelengths longer than 726 nm. The 726 nm absorption peak exhibits a 21 ± 4 cm−1 electrochromic shift due to formation of the semiquinone anion, QA−. Modelling indicates that no other FRL pigment is located among the 6 central reaction center chlorins: PD1, PD2 ChlD1, ChlD2, PheoD1 and PheoD2. Two of these chlorins, ChlD1 and PD2, are located at a distance and orientation relative to QA− so as to account for the observed electrochromic shift. Previously, ChlD1 was taken as the most likely candidate for the primary donor based on spectroscopy, sequence analysis and mechanistic arguments. Here, a more detailed comparison of the spectroscopic data with exciton modelling of the electrochromic pattern indicates that PD2 is at least as likely as ChlD1 to be responsible for the 726 nm absorption. The correspondence in sign and magnitude of the CD observed at 726 nm with that predicted from modelling favors PD2 as the primary donor. The pros and cons of PD2 vs ChlD1 as the location of the FRL-primary donor are discussed.
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•Primary donor confirmed at 726 nm.•Determination of far-red chl pigment Qy excitation positions, widths, CD and MCD amplitudes•Quantification of electrochromic shifts and QA− photoconversion yield•Electrochromic shift consistent with primary donor at either ChlD1 or PD2•The CD amplitude favors the primary donor at PD2.
The nitrogen-vacancy (NV) center in diamond is a widely utilized system due to its useful quantum properties. Almost all research focuses on the negative charge state (NV
) and comparatively little ...is understood about the neutral charge state (NV
). This is surprising as the charge state often fluctuates between NV
and NV
during measurements. There are potentially under-utilized technical applications that could take advantage of NV
, either by improving the performance of NV
or utilizing NV
directly. However, the fine structure of NV
has not been observed. Here, we rectify this lack of knowledge by performing magnetic circular dichroism measurements that quantitatively determine the fine structure of NV
. The observed behavior is accurately described by spin-Hamiltonians in the ground and excited states with the ground state yielding a spin-orbit coupling of
= 2.24 ± 0.05 GHz and a orbital
-factor of 0.0186 ± 0.0005. The reasons why this fine structure has not been previously measured are discussed and strain-broadening is concluded to be the likely reason.
The IR absorptions of several first-shell carboxylate ligands of the water oxidizing complex (WOC) have been experimentally shown to be unaffected by oxidation state changes in the WOC during its ...catalytic cycle. Several model clusters that mimic the Mn4O5Ca core of the WOC in the S1 state, with electronic configurations that correspond to both the so-called “high” and “low” oxidation paradigms, were investigated. Deprotonation at W2, W1, or O3 sites was found to strongly reduce carboxylate ligand frequency shifts on oxidation of the metal cluster. The frequency shifts were smallest in neutrally charged clusters where the initial mean Mn oxidation state was +3, with W2 as an hydroxide and O5 a water. Deprotonation also reduced and balanced the oxidation energy of all clusters in successive oxidations.
Photosynthetic pigments are inherently intense optical absorbers and have strong polarisation characteristics. They can also luminesce strongly. These properties have led optical spectroscopies to ...be, quite naturally, key techniques in photosynthesis. However, there are typically many pigments in a photosynthetic assembly, which when combined with the very significant inhomogeneous and homogeneous linewidths characteristic of optical transitions, leads to spectral congestion. This in turn has made it difficult to provide a definitive and detailed electronic structure for many photosynthetic assemblies. An electronic structure is, however, necessary to provide a foundation for any complete description of fundamental processes in photosynthesis, particularly those in reaction centres. A wide range of selective and differential spectral techniques have been developed to help overcome the problems of spectral complexity and congestion. The techniques can serve to either reduce spectral linewidths and/or extract chromophore specific information from unresolved spectral features. Complementary spectral datasets, generated by a number of techniques, may then be combined in a ‘multi-dimensional’ theoretical analysis so as to constrain and define effective models of photosynthetic assemblies and their fundamental processes. A key example is the work of Renger and his group (Raszewski, Biophys J 88(2):986–998, 2005) on PS II reaction centre assemblies. This article looks to provide an overview of some of these techniques and indicate where their strengths and weaknesses may lie. It highlights some of our own contributions and indicates areas where progress may be possible.
The protonation state of the deazaflavin dependent nitroreductase (Ddn) enzyme bound cofactor F420 was investigated using UV-visible spectroscopy and computational simulations. The reduced cofactor ...F420H2 was determined to be present in its deprotonated state in the holoenzyme form. The mechanistic implications of these findings are discussed.
The illumination of oxygen-evolving PSII core complexes at very low temperatures in spectral regions not expected to excite P680 leads to charge separation in a majority of centers. The fraction of ...centers photoconverted as a function of the number of absorbed photons per PSII core is determined by quantification of electrochromic shifts on Pheo
D1. These shifts arise from the formation of metastable plastoquinone anion (Q
A
−) configurations. Spectra of concentrated samples identify absorption in the 700–730 nm range. This is well beyond absorption attributable to CP47. Spectra in the 690–730 nm region can be described by the ‘trap’ CP47 absorption at 689 nm, with dipole strength of ∼1 chlorophyll
a (chl
a)
, partially overlapping a broader feature near 705 nm with a dipole strength of ∼0.15 chl
a. This absorption strength in the 700–730 nm region falls by 40% in the photoconverted configuration. Quantum efficiencies of photoconversion following illumination in the 690–700 nm region are similar to those obtained with green illumination but fall significantly in the 700–730 nm range. Two possible assignments of the long-wavelength absorption are considered. Firstly, as a low intensity component of strongly exciton-coupled reaction center chlorin excitations and secondly as a nominally ‘dark’ charge-transfer excitation of the ‘special pair’ P
D1–P
D2. The opportunities offered by these observations towards the understanding of the nature of P680 and PSII fluorescence are discussed.
Routinely prepared PS II core samples are often contaminated by a significant (~1–5%) fraction of PS I, as well as related proteins. This contamination is of little importance in many experiments, ...but masks the optical behaviour of the deep red state in PS II, which absorbs in the same spectral range (700–730nm) as PS I (Hughes et al. 2006). When contamination levels are less than ~1%, it becomes difficult to quantify the PS I related components by gel-based, chromatographic, circular dichroism or EPR techniques. We have developed a fluorescence-based technique, taking advantage of the distinctively different low-temperature emission characteristics of PS II and PS I when excited near 700nm. The approach has the advantage of providing the relative concentration of the two photosystems in a single spectral measurement. A sensitivity limit of 0.01% PS I (or better) can be achieved. The procedure is applied to PS II core preparations from spinach and Thermosynechococcus vulcanus. Measurements made of T. vulcanus PS II preparations prepared by re-dissolving crystallised material indicate a low but measurable PS I related content. The analysis provides strong evidence for a previously unreported fluorescence of PS II cores peaking near 780nm. The excitation dependence of this emission as well as its appearance in both low PS I cyanobacterial and plant based PS II core preparations suggests its association with the deep red state of PS II.
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•Distinct 700–730nm-excitation fluorescence signatures of PS I and PS II identified•Selective fluorescence-enabled quantification of the PS I content of PS II preparations•An ~0.01% PS I content in PS II sensitivity was achieved.•Quantitative studies of the deep red state of PS II in cyanobacteria become enabled.•New reaction centre-based emission in both plant and cyanobacterial PS II at 780nm
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