Single-molecule study of redox control involved in establishing the spinach plastocyanin-cytochrome b6f electron transfer complex

Small diffusible redox proteins play a ubiquitous role in bioenergetic systems, facilitating electron transfer (ET) between membrane bound complexes. Sustaining high ET turnover rates requires that the association between extrinsic and membrane-bound partners is highly specific, yet also sufficiently weak to promote rapid post-ET separation. In oxygenic photosynthesis the small soluble electron carrier protein plastocyanin (Pc) shuttles electrons between the membrane integral cytochrome b6f (cytb6f) and photosystem I (PSI) complexes. Here we use peak-force quantitative nanomechanical mapping (PF-QNM) atomic force microscopy (AFM) to quantify the dynamic forces involved in transient interactions between cognate ET partners. An AFM probe functionalised with Pc molecules is brought into contact with cytb6f complexes, immobilised on a planar silicon surface. PF-QNM interrogates the unbinding force of the cytb6f-Pc interactions at the single molecule level with picoNewton force resolution and on a time scale comparable to the ET time in vivo (ca. 120 μs). Using this approach, we show that although the unbinding force remains unchanged the interaction frequency increases over five-fold when Pc and cytb6f are in opposite redox states, so complementary charges on the cytb6f and Pc cofactors likely contribute to the electrostatic forces that initiate formation of the ET complex. These results suggest that formation of the docking interface is under redox state control, which lowers the probability of unproductive encounters between Pc and cytb6f molecules in the same redox state, ensuring the efficiency and directionality of this central reaction in the ‘Z-scheme’ of photosynthetic ET

Photosynthesis in the near infrared: the γ subunit of Blastochloris viridis LH1 red-shifts absorption beyond 1000 nm

The reaction centre (RC) in purple phototrophic bacteria is encircled by the primary light-harvesting complex 1 (LH1) antenna, forming the RC–LH1 ‘core’ complex. The Qy absorption maximum of LH1 complexes ranges from ∼875–960 nm in bacteriochlorophyll (BChl) a-utilising organisms, to 1018 nm in the BChl b-containing complex from Blastochloris (Blc.) viridis. The red-shifted absorption of the Blc. viridis LH1 was predicted to be due in part to the presence of the γ subunit unique to Blastochloris spp., which binds to the exterior of the complex and is proposed to increase packing and excitonic coupling of the BChl pigments. The study by Namoon et al. provides experimental evidence for the red-shifting role of the γ subunit and an evolutionary rationale for its incorporation into LH1. The authors show that cells producing RC–LH1 lacking the γ subunit absorb maximally at 972 nm, 46 nm to the blue of the wild-type organism. Wavelengths in the 900–1000 nm region of the solar spectrum transmit poorly through water, thus γ shifts absorption of LH1 to a region where photons have lower energy but are more abundant. Complementation of the mutant with a divergent copy of LH1γ resulted in an intermediate red shift, revealing the possibility of tuning LH1 absorption using engineered variants of this subunit. These findings provide new insights into photosynthesis in the lowest energy phototrophs and how the absorption properties of light-harvesting complexes are modified by the recruitment of additional subunits

Directed assembly of defined oligomeric photosynthetic reaction centres through adaptation with programmable extra-membrane coiled-coil interfaces

A challenge associated with the utilisation of bioenergetic proteins in new, synthetic energy transducing systems is achieving efficient and predictable self-assembly of individual components, both natural and man-made, into a functioning macromolecular system. Despite progress with water-soluble proteins, the challenge of programming self-assembly of integral membrane proteins into non-native macromolecular architectures remains largely unexplored. In this work it is shown that the assembly of dimers, trimers or tetramers of the naturally monomeric purple bacterial reaction centre can be directed by augmentation with an α-helical peptide that self-associates into extra-membrane coiled-coil bundle. Despite this induced oligomerisation the assembled reaction centres displayed normal spectroscopic properties, implying preserved structural and functional integrity. Mixing of two reaction centres modified with mutually complementary α-helical peptides enabled the assembly of heterodimers in vitro, pointing to a generic strategy for assembling hetero-oligomeric complexes from diverse modified or synthetic components. Addition of two coiled-coil peptides per reaction centre monomer was also tolerated despite the challenge presented to the pigment-protein assembly machinery of introducing multiple self-associating sequences. These findings point to a generalised approach where oligomers or longer range assemblies of multiple light harvesting and/or redox proteins can be constructed in a manner that can be genetically-encoded, enabling the construction of new, designed bioenergetic systems in vivo or in vitro

The effectiveness of styrene-maleic acid (SMA) copolymers for solubilisation of integral membrane proteins from SMA-accessible and SMA-resistant membranes

Solubilisation of biological lipid bilayer membranes for analysis of their protein complement has traditionally been carried out using detergents, but there is increasing interest in the use of amphiphilic copolymers such as styrene maleic acid (SMA) for the solubilisation, purification and characterisation of integral membrane proteins in the form of protein/lipid nanodiscs. Here we survey the effectiveness of various commercially-available formulations of the SMA copolymer in solubilising Rhodobacter sphaeroides reaction centres (RCs) from photosynthetic membranes. We find that formulations of SMA with a 2:1 or 3:1 ratio of styrene to maleic acid are almost as effective as detergent in solubilising RCs, with the best solubilisation by short chain variants ( < 30 kDa weight average molecular weight). The effectiveness of 10 kDa 2:1 and 3:1 formulations of SMA to solubilise RCs gradually declined when genetically-encoded coiled-coil bundles were used to artificially tether normally monomeric RCs into dimeric, trimeric and tetrameric multimers. The ability of SMA to solubilise reaction centre-light harvesting 1 (RC-LH1) complexes from densely packed and highly ordered photosynthetic membranes was uniformly low, but could be increased through a variety of treatments to increase the lipid:protein ratio. However, proteins isolated from such membranes comprised clusters of complexes in small membrane patches rather than individual proteins. We conclude that short-chain 2:1 and 3:1 formulations of SMA are the most effective in solubilising integral membrane proteins, but that solubilisation efficiencies are strongly influenced by the size of the target protein and the density of packing of proteins in the membrane

The structure and assembly of reaction centre-light-harvesting 1 complexes in photosynthetic bacteria

Chlorophototrophic organisms have a charge-separating reaction centre (RC) complex that receives energy from a dedicated light-harvesting (LH) antenna. In the purple phototrophic bacteria, these two functions are embodied by the ‘core’ photosynthetic component, the RC-LH1 complex. RC-LH1 complexes sit within a membrane bilayer, with the central RC wholly or partly surrounded by a curved array of LH1 subunits that bind a series of bacteriochlorophyll (BChl) and carotenoid pigments. Decades of research have shown that the absorption of light initiates a cascade of energy, electron, and proton transfers that culminate in the formation of a quinol, which is subsequently oxidized by the cytochrome bc1 complex. However, a full understanding of all these processes, from femtosecond absorption of light to millisecond quinone diffusion, requires a level of molecular detail that was lacking until the remarkable recent upsurge in the availability of RC-LH1 structures. Here, we survey 13 recently determined RC-LH1 assemblies, and we compare the precise molecular arrangements of pigments and proteins that allow efficient light absorption and the transfer of energy, electrons and protons. We highlight shared structural features, as well as differences that span the bound pigments and cofactors, the structures of individual subunits, the overall architecture of the complexes, and the roles of additional subunits newly identified in just one or a few species. We discuss RC-LH1 structures in the context of prior biochemical and spectroscopic investigations, which together enhance our understanding of the molecular mechanisms of photosynthesis in the purple phototrophic bacteria. A particular emphasis is placed on how the remarkable and unexpected structural diversity in RC-LH1 complexes demonstrates different evolutionary solutions for maximising pigment density for optimised light harvesting, whilst balancing the requirement for efficient quinone diffusion between RC and cytochrome bc1 complexes through the encircling LH1 complex

Cryo-EM structure of the Rhodospirillum rubrum RC-LH1 complex at 2.5 Å.

The reaction centre light-harvesting 1 (RC-LH1) complex is the core functional component of bacterial photosynthesis. We determined the cryo-electron microscopy (cryo-EM) structure of the RC-LH1 complex from Rhodospirillum rubrum at 2.5 Å resolution, which reveals a unique monomeric bacteriochlorophyll with a phospholipid ligand in the gap between the RC and LH1 complexes. The LH1 complex comprises a circular array of 16 αβ-polypeptide subunits that completely surrounds the RC, with a preferential binding site for a quinone, designated QP, on the inner face of the encircling LH1 complex. Quinols, initially generated at the RC QB site, are proposed to transiently occupy the QP site prior to traversing the LH1 barrier and diffusing to the cytochrome bc1 complex. Thus, the QP site, which is analogous to other such sites in recent cryo-EM structures of RC-LH1 complexes, likely reflects a general mechanism for exporting quinols from the RC-LH1 complex

A photosynthetic antenna complex foregoes unity carotenoid-to-bacteriochlorophyll energy transfer efficiency to ensure photoprotection

Carotenoids play a number of important roles in photosynthesis, primarily providing light-harvesting and photoprotective energy dissipation functions within pigment–protein complexes. The carbon–carbon double bond (C=C) conjugation length of carotenoids (N), generally between 9 and 15, determines the carotenoid-to-(bacterio)chlorophyll [(B)Chl] energy transfer efficiency. Here we purified and spectroscopically characterized light-harvesting complex 2 (LH2) from Rhodobacter sphaeroides containing the N = 7 carotenoid zeta (ζ)-carotene, not previously incorporated within a natural antenna complex. Transient absorption and time-resolved fluorescence show that, relative to the lifetime of the S1 state of ζ-carotene in solvent, the lifetime decreases ∼250-fold when ζ-carotene is incorporated within LH2, due to transfer of excitation energy to the B800 and B850 BChls a. These measurements show that energy transfer proceeds with an efficiency of ∼100%, primarily via the S1 → Qx route because the S1 → S0 fluorescence emission of ζ-carotene overlaps almost perfectly with the Qx absorption band of the BChls. However, transient absorption measurements performed on microsecond timescales reveal that, unlike the native N ≥ 9 carotenoids normally utilized in light-harvesting complexes, ζ-carotene does not quench excited triplet states of BChl a, likely due to elevation of the ζ-carotene triplet energy state above that of BChl a. These findings provide insights into the coevolution of photosynthetic pigments and pigment–protein complexes. We propose that the N ≥ 9 carotenoids found in light-harvesting antenna complexes represent a vital compromise that retains an acceptable level of energy transfer from carotenoids to (B)Chls while allowing acquisition of a new, essential function, namely, photoprotective quenching of harmful (B)Chl triplets

Carotenoid-to-(bacterio)chlorophyll energy transfer in LH2 antenna complexes from Rba. sphaeroides reconstituted with non-native (bacterio)chlorophylls

Six variants of the LH2 antenna complex from Rba. sphaeroides, comprising the native B800-B850, B800-free LH2 (B850) and four LH2s with various (bacterio)chlorophylls reconstituted into the B800 site, have been investigated with static and time-resolved optical spectroscopies at room temperature and at 77 K. The study particularly focused on how reconstitution of a non-native (bacterio)chlorophylls affects excitation energy transfer between the naturally bound carotenoid spheroidene and artificially substituted pigments in the B800 site. Results demonstrate there is no apparent trend in the overall energy transfer rate from spheroidene to B850 bacteriochlorophyll a; however, a trend in energy transfer rate from the spheroidene S1 state to Qy of the B800 (bacterio)chlorophylls is noticeable. These outcomes were applied to test the validity of previously proposed energy values of the spheroidene S1 state, supporting a value in the vicinity of 13,400 cm−1 (746 nm)

A Mechanoresponsive Phase-Changing Electrolyte Enables Fabrication of High-Output Solid-State Photobioelectrochemical Devices from Pigment-Protein Multilayers

Exploitation of natural photovoltaic reaction center pigment proteins in biohybrid architectures for solar energy harvesting is attractive due to their global abundance, environmental compatibility, and near-unity quantum efficiencies. However, it is challenging to achieve high photocurrents in a device setup due to limitations imposed by low light absorbance by protein monolayers and/or slow long-range diffusion of liquid-phase charge carriers. In an attempt to enhance the photocurrent density achievable by pigment proteins, here, an alternative solid-state device architecture enabled by a mechanoresponsive gel electrolyte that can be applied under nondenaturing conditions is demonstrated. The phase-changing electrolyte gel provides a pervading biocompatible interface for charge conduction through highly absorbing protein multilayers that are fabricated in a simple fashion. Assembled devices exhibit enhanced current stability and a maximal photoresponse of ≈860 μA cm -2 , a fivefold improvement over the best previous comparable devices under standard illumination conditions. Photocurrent generation is enhanced by directional energy transfer through extended layers of light-harvesting complexes, mimicking the modular antenna/transducer architecture of natural photosystems, and by metastable radical pair formation when photovoltaic reaction centers are embedded throughout light-harvesting regions of the device