Design and engineering of photosynthetic light-harvesting and electron transfer using length, time, and energy scales

Decades of research on the physical processes and chemical reaction-pathways in photosynthetic enzymes have resulted in an extensive database of kinetic information. Recently, this database has been augmented by a variety of high and medium resolution crystal structures of key photosynthetic enzymes that now include the two photosystems (PSI and PSII) of oxygenic photosynthetic organisms. Here, we examine the currently available structural and functional information from an engineer's point of view with the long-term goal of reproducing the key features of natural photosystems in de novo designed and custom-built molecular solar energy conversion devices. We find that the basic physics of the transfer processes, namely, the time constraints imposed by the rates of incoming photon flux and the various decay processes allow for a large degree of tolerance in the engineering parameters. Moreover, we find that the requirements to guarantee energy and electron transfer rates that yield high efficiency in natural photosystems are largely met by control of distance between chromophores and redox cofactors. Thus, for projected de novo designed constructions, the control of spatial organization of cofactor molecules within a dense array is initially given priority. Nevertheless, constructions accommodating dense arrays of different cofactors, some well within 1 nm from each other, still presents a significant challenge for protein design.     

Natural photosystems from an engineer’s perspective: length,time, and energy scales of charge and energy transfer

The vast structural and functional information database of photosynthetic enzymes includes, in addition to detailed kinetic records from decades of research on physical processes and chemical reaction-pathways, a variety of high and medium resolution crystal structures of key photosynthetic enzymes. Here, it is examined from an engineer’s point of view with the long-term goal of reproducing the key features of natural photosystems in novel biological and non-biological solar-energy conversion systems. This survey reveals that the basic physics of the transfer processes, namely, the time constraints imposed by the rates of incoming photon flux and the various decay processes allow for a large degree of tolerance in the engineering parameters. Furthermore, the requirements to guarantee energy and electron transfer rates that yield high efficiency in natural photosystems are largely met by control of distance between chromophores and redox cofactors. This underlines a critical challenge for projected de novo designed constructions, that is, the control of spatial organization of cofactor molecules within dense array of different cofactors, some well within 1 nm from each other.     

Exploring amino-acid radical chemistry: protein engineering and de novo design

Amino-acid radical enzymes are often highly complex structures containing multiple protein subunits and cofactors. These properties have in many cases hampered the detailed characterization of their amino-acid redox cofactors. To address this problem, a range of approaches has recently been developed in which a common strategy is to reduce the complexity of the radical-containing system. This work will be reviewed and it includes the light-induced generation of aromatic radicals in small-molecule and peptide systems. Natural redox proteins, including the blue copper protein azurin and a bacterial photosynthetic reaction center, have been engineered to introduce amino-acid radical chemistry. The redesign strategies to achieve this remarkable change in the properties of these proteins will be described. An additional approach to gain insights into the properties of amino-acid radicals is to synthesize de novo designed model proteins in which the redox chemistry of these species can be studied. Here we describe the design, synthesis and characteristics of monomeric three-helix bundle and four-helix bundle proteins designed to study the redox chemistry of tryptophan and tyrosine. This work demonstrates that de novo protein design combined with structural, electrochemical and quantum chemical analyses can provide detailed information on how the protein matrix tunes the thermodynamic properties of tryptophan.     

Exploring amino-acid radical chemistry: protein engineering and de novo design

Amino-acid radical enzymes are often highly complex structures containing multiple protein subunits and cofactors. These properties have in many cases hampered the detailed characterization of their amino-acid redox cofactors. To address this problem, a range of approaches has recently been developed in which a common strategy is to reduce the complexity of the radical-containing system. This work will be reviewed and it includes the light-induced generation of aromatic radicals in small-molecule and peptide systems. Natural redox proteins, including the blue copper protein azurin and a bacterial photosynthetic reaction center, have been engineered to introduce amino-acid radical chemistry. The redesign strategies to achieve this remarkable change in the properties of these proteins will be described. An additional approach to gain insights into the properties of amino-acid radicals is to synthesize de novo designed model proteins in which the redox chemistry of these species can be studied. Here we describe the design, synthesis and characteristics of monomeric three-helix bundle and four-helix bundle proteins designed to study the redox chemistry of tryptophan and tyrosine. This work demonstrates that de novo protein design combined with structural, electrochemical and quantum chemical analyses can provide detailed information on how the protein matrix tunes the thermodynamic properties of tryptophan.     

Evaluation of the Specific Roles of Anions in Electron Transport and Energy Transfer Reactions in Photosynthesis

Ionic environment is important in regulating photosynthetic reactions. The roles of cations, Mn²⁺, Mg²⁺, Ca²⁺, Na⁺, and K⁺ as cofactors in electron transport, energy transfer, phosphorylation, and carbon assimilation are better known than the roles of anions, except for chloride and bicarbonate. Only a limited information exists on the roles and effects of nitri formate, sulphate, and phosphate. In this review, we evaluate and highlight the roles of some specific anions on electron transport as well as on excitation energy transfer processes in photosynthesis. Anions exert significant effects on thyla membrane conformation and membrane fluidity, possibly by redistributing the thylakoid membrane surface charges. The anion/cation induced phase transitions in the hydrophilic domains of the thylakoid membranes are probably responsible for the various structural and co-related functional changes under stress. Anions are also important in regulation of energy distribution between the two photosystems. Anions do not only divert more energy from photosystem (PS) 2 to PS1, but can also reverse the effect of cations on energy distribution in a valence-dependent manner. Anions affect also the structure of the photosynthetic apparatus and excitation energy distribution between the two photosystems.     

Effect of water stress on the photosynthetic apparatus of plants and the protective role of cytokinins: a review

Characteristics of photosynthetic apparatus (the pool of pigments and proteins; the activity of photosystems; the intensities of in vivo photoassimilation of carbon dioxide and in vitro activity of enzymes of carbon metabolism; leaf structure; chloroplast structure), undergoing changes under the conditions of water deficiency, have been reviewed. The protective role of cytokinins is due to their regulatory effects on the renewal of disrupted cellular structures, the condition of the stomata, and de novo synthesis and activation of proteins that are required for increasing plant resistance to water stress.     

Energy transfer in a model of the photosynthetic unit of green plants

A model array is set up to represent a photosynthetic unit of 344 chlorophyll molecules of seven different spectral varieties and in definite orientations. The array is provided with two traps, representing the reaction centers of photosystems I and II. The number of jumps required to obtain a high probability of trapping is lower than on a similar array of undifferentiated chlorophylls by a factor of 15. Most of the molecules fall into two groups which transfer their energy predominantly into one or the other trap, and which may be regarded as functional photosystems I and II. The rate of transfer between these two functional photosystems can be controlled by redirecting the orientation of only six of the molecules, which occupy a key position in the array. The effect on trapping rates of reorientation of these molecules is especially pronounced when one of the traps is closed. This constitutes a model for the control of energy distribution between the two photosystems, as indicated in recent years through fluorescence studies.     

Mechanisms for photosystems I and II

Using structural information from recently published crystal structures of photosystems I and II, the processes of excitation energy transfer and electron transfer in oxygenic photosynthesis have been studied in great detail by experimental and theoretical methods. Although both systems share numerous common structural and functional features, efficiency and regulation are differently weighted in the individual processes that are involved in the transformation of light energy into chemical energy in the two complexes.     

Effect of Carbohydrate Overfeeding on Whole Body and Adipose Tissue Metabolism in Humans

Objective: To evaluate the effect of a 4‐day carbohydrate overfeeding on whole body net de novo lipogenesis and on markers of de novo lipogenesis in subcutaneous adipose tissue of healthy lean humans. Research Methods and Procedures: Nine healthy lean volunteers (five men and four women) were studied after 4 days of either isocaloric feeding or carbohydrate overfeeding. On each occasion, they underwent a metabolic study during which their energy expenditure and net substrate oxidation rates (indirect calorimetry), and the fractional activity of the pentose‐phosphate pathway in subcutaneous adipose tissue (subcutaneous microdialysis with 1, 6¹³C₂, 6, 6²H₂ glucose) were assessed before and after administration of glucose. Adipose tissue biopsies were obtained at the end of the experiments to monitor mRNAs of key lipogenic enzymes. Results: Carbohydrate overfeeding increased basal and postglucose energy expenditure and net carbohydrate oxidation. Whole body net de novo lipogenesis after glucose loading was markedly increased at the expense of glycogen synthesis. Carbohydrate overfeeding also increased mRNA levels for the key lipogenic enzymes sterol regulatory element‐binding protein‐1c, acetyl‐CoA carboxylase, and fatty acid synthase. The fractional activity of adipose tissue pentose‐phosphate pathway was 17% to 22% and was not altered by carbohydrate overfeeding. Discussion: Carbohydrate overfeeding markedly increased net de novo lipogenesis at the expense of glycogen synthesis. An increase in mRNAs coding for key lipogenic enzymes suggests that de novo lipogenesis occurred, at least in part, in adipose tissue. The pentose‐phosphate pathway is active in adipose tissue of healthy humans, consistent with an active role of this tissue in de novo lipogenesis.     

The effects of acetaldehyde on nitrogenase, hydrogenase and photosynthesis in the cyanobacterium Anabaena cylindrica.

Acetaldehyde was shown to be an irreversible inhibitor of nitrogenase, hydrogenase, CO2 fixation and growth in the cyanobacterium Anabaena cylindrica, but had no effect on photosynthetic electron flow as measured by Methyl Viologen-dependent O2 uptake. The concentration-dependence of the inhibition of nitrogenase and hydrogenase activities was determined, and it was shown that acetaldehyde inhibition poses problems for anaerobic experiments in which the activities of these enzymes are measured in the presence of the frequently used glucose/glucose oxidase/catalase/ethanol O2 trap. It is suggested that acetaldehyde may find use as an inhibitor in experiments designed to separate electron flow through the photosystems from consequent fixation of CO2 and N2.     

State transitions reveal the dynamics and flexibility of the photosynthetic apparatus.

The chloroplast-based photosynthetic apparatus of plants and algae associates various redox cofactors and pigments with approximately 70 polypeptides to form five major transmembrane protein complexes. Among these are two photosystems that have distinct light absorption properties but work in series to produce reducing equivalents aimed at the fixation of atmospheric carbon. A short term chromatic adaptation known as 'State transitions' was discovered thirty years ago that allows photosynthetic organisms to adapt to changes in light quality and intensity which would otherwise compromise the efficiency of photosynthetic energy conversion. A two-decade research effort has finally unraveled the major aspects of the molecular mechanism responsible for State transitions, and their physiological significance has been revisited. This review describes how a-still elusive-regulatory kinase senses the physiological state of the photosynthetic cell and triggers an extensive supramolecular reorganization of the photosynthetic membranes. The resulting picture of the photosynthetic apparatus is that of a highly flexible energy convertor that adapts to the ever-changing intracellular demand for ATP and/or reducing power.     

Structure-function investigations of bacterial photosynthetic reaction centers

During photosynthesis light energy is converted into energy of chemical bonds through a series of electron and proton transfer reactions. Over the first ultrafast steps of photosynthesis that take place in the reaction center (RC) the quantum efficiency of the light energy transduction is nearly 100%. Compared to the plant and cyanobacterial photosystems, bacterial RCs are well studied and have relatively simple structure. Therefore they represent a useful model system both for manipulating of the electron transfer parameters to study detailed mechanisms of its separate steps as well as to investigate the common principles of the photosynthetic RC structure, function, and evolution. This review is focused on the research papers devoted to chemical and genetic modifications of the RCs of purple bacteria in order to study principles and mechanisms of their functioning. Investigations of the last two decades show that the maximal rates of the electron transfer reactions in the RC depend on a number of parameters. Chemical structure of the cofactors, distances between them, their relative orientation, and interactions to each other are of great importance for this process. By means of genetic and spectral methods, it was demonstrated that RC protein is also an essential factor affecting the efficiency of the photochemical charge separation. Finally, some of conservative water molecules found in RC not only contribute to stability of the protein structure, but are directly involved in the functioning of the complex.     

Light Harvesting and State Transitions in Cyanobacteria

Cyanobacteria are oxygenic phototrophic prokaryotes and are considered to be the ancestors of chloroplasts. Their photosynthetic machinery is functionally equivalent in terms of primary photochemistry and photosynthetic electron transport. Fluorescence measurements and other techniques indicate that cyanobacteria, like plants, are capable of redirecting pathways of excitation energy transfer from light harvesting antennae to both photosystems. Cyanobacterial cells can reach two energetically different states, which are defined as “State 1” (obtained after preferential excitation of photosystem I) and “State 2” (preferential excitation of photosystem II). These states can be distinguished by static and time resolved fluorescence techniques. One of the most important conclusions reached so far is that the presence of both photosystems, as well as certain antenna components, are necessary for state transitions to occur. Spectroscopic evidence suggests that changes in the coupling state of the light harvesting antenna complexes (the phycobilisomes) to both photosystems occur during state transitions. The finding that the phycobilisome complexes are highly mobile on the surface of the thylakoid membrane (the mode of interaction with the thylakoid membrane is essentially unknown), has led to the proposal that they are in dynamic equilibrium with both photosystems and regulation of energy transfer is mediated by changes in affinity for either photosystem.     

Slow and Bimolecular Folding of a De Novo Designed Monomeric Protein DS119

De novo protein design offers a unique means to test and advance our understanding of how proteins fold. However, most current design methods are native structure eccentric and folding kinetics has rarely been considered in the design process. Here, we show that a de novo designed mini-protein DS119, which folds into a βαβ structure, exhibits unusually slow and concentration-dependent folding kinetics. For example, the folding time for 50 μM of DS119 was estimated to be ∼2 s. Stopped-flow fluorescence resonance energy transfer experiments further suggested that its folding was likely facilitated by a transient dimerization process. Taken together, these results highlight the need for consideration of the entire folding energy landscape in de novo protein design and provide evidence suggesting nonnative interactions can play a key role in protein folding.     

The assembly of protein subunits and cofactors in photosystem I

The recently determined crystal structures of photosystems I and II at 2.5 A and 3.8 A resolution, respectively, have improved the structural basis for understanding the processes of light trapping, exciton transfer and electron transfer occurring in the primary steps of oxygenic photosynthesis. Understanding the assembly of the 12 protein subunits and 128 cofactors in photosystem I allows us to study the possible functions of the individual players in this protein-cofactor complex.     

Nanozymes: created by learning from nature

Nanozymes, a type of nanomaterials with enzyme-like activity, have shown great potential to replace natural enzymes in many fields such as biochemical detection, environmental management and disease treatment. However, the catalytic efficiency and substrate specificity of nanozymes still need improvement. To further optimize the enzymatic properties of nanozymes, recent studies have introduced the structural characteristics of natural enzymes into the rational design of nanozymes, either by employing small molecules to mimic the cofactors of natural enzymes to boost nanozymes' catalytic potential, or by simulating the active center of natural enzymes to construct the nanostructure of nanozymes. This review introduces the commonly used bio-inspired strategies to create nanozymes, aiming at clarifying the current progress and bottlenecks. Advances and challenges focusing on the research of bio-inspired nanozymes are outlined to provide ideas for the de novo design of ideal nanozymes.     

Variety in excitation energy transfer processes from phycobilisomes to photosystems I and II

Photosynthesis Research - The light-harvesting antennas of oxygenic photosynthetic organisms capture light energy and transfer it to the reaction centers of their photosystems. The light-harvesting...     

Photosynthetic Acclimation to Temperature in the Desert Shrub, Larrea divaricata: II. Light-harvesting Efficiency and Electron Transport

The response of photosynthetic electron transport and light-harvesting efficiency to high temperatures was studied in the desert shrub Larrea divaricata Cav. Plants were grown at day/night temperatures of 20/15, 32/25, or 45/33 C in rough approximation of natural seasonal temperature variations. The process of acclimation to high temperatures involves an enhancement of the stability of the interactions between the light-harvesting pigments and the photosystem reaction centers. As temperature is increased, the heat-induced dissociation of these complexes results in a decrease in the quantum yield of electron transport at limiting light intensity, followed by a loss of electron transport activity at rate-saturating light intensity. The decreased quantum yield can be attributed to a block of excitation energy transfer from chlorophyll b to chlorophyll a, and changes in the distribution of the excitation energy between photosystems II and I. The block of excitation energy transfer is characterized by a loss of the effectiveness of 480 nm light (absorbed primarily by chlorophyll b) to drive protochemical processes, as well as fluorescence emission by chlorophyll b.     

Light harvesting in photosystem I supercomplexes

In photosynthetic membranes of cyanobacteria, algae, and higher plants, photosystem I (PSI) mediates light-driven transmembrane electron transfer from plastocyanin or cytochrome c6 to the ferredoxin-NADP complex. The oxidoreductase function of PSI is sensitized by a reversible photooxidation of primary electron donor P700, which launches a multistep electron transfer via a series of redox cofactors of the reaction center (RC). The excitation energy for the functioning of the primary electron donor in the RC is delivered via the chlorophyll core antenna in the complex with peripheral light-harvesting antennas. Supermolecular complexes of the PSI acquire remarkably different structural forms of the peripheral light-harvesting antenna complexes, including distinct pigment types and organizational principles. The PSI core antenna, being the main functional unit of the supercomplexes, provides an increased functional connectivity in the chlorophyll antenna network due to dense pigment packing resulting in a fast spread of the excitation among the neighbors. Functional connectivity within the network as well as the spectral overlap of antenna pigments allows equilibration of the excitation energy in the depth of the whole membrane within picoseconds and loss-free delivery of the excitation to primary donor P700 within 20-40 ps. Low-light-adapted cyanobacteria under iron-deficiency conditions extend this capacity via assembly of efficiently energy coupled rings of CP43-like complexes around the PSI trimers. In green algae and higher plants, less efficient energy coupling in the eukaryotic PSI-LHCI supercomplexes is probably a result of the structural adaptation of the Chl a/b binding LHCI peripheral antenna that not only extends the absorption cross section of the PSI core but participates in regulation of excitation flows between the two photosystems as well as in photoprotection.     

Low potential manganese ions as efficient electron donors in native anoxygenic bacteria

Systematic control over molecular driving forces is essential for understanding the natural electron transfer processes as well as for improving the efficiency of the artificial mimics of energy converting enzymes. Oxygen producing photosynthesis uniquely employs manganese ions as rapid electron donors. Introducing this attribute to anoxygenic photosynthesis may identify evolutionary intermediates and provide insights to the energetics of biological water oxidation. This work presents effective environmental methods that substantially and simultaneously tune the redox potentials of manganese ions and the cofactors of a photosynthetic enzyme from native anoxygenic bacteria without the necessity of genetic modification or synthesis. A spontaneous coordination with bis-tris propane lowered the redox potential of the manganese (II) to manganese (III) transition to an unusually low value (~400?mV) at pH?9.4 and allowed its binding to the bacterial reaction center. Binding to a novel buried binding site elevated the redox potential of the primary electron donor, a dimer of bacteriochlorophylls, by up to 92?mV also at pH?9.4 and facilitated the electron transfer that is able to compete with the wasteful charge recombination. These events impaired the function of the natural electron donor and made BTP-coordinated manganese a viable model for an evolutionary alternative.