The abundant pigment-protein membrane complex photosystem-I (PS-I) is at the heart of the Earth’s energy cycle. It is the central molecule in the “Z-scheme” of photosynthesis, converting sunlight into the chemical energy of life. Commandeering this intricately organized photosynthetic nanocircuitry and re-wiring it to produce electricity carries the promise of inexpensive and environmentally friendly solar power. We here report that dry PS-I stabilized by surfactant peptides functioned as both the light-harvester and charge separator in solar cells self-assembled on nanostructured semiconductors. Contrary to previous attempts at biophotovoltaics requiring elaborate surface chemistries, thin film deposition, and illumination concentrated into narrow wavelength ranges the devices described here are straightforward and inexpensive to fabricate and perform well under standard sunlight yielding open circuit photovoltage of 0.5 V, fill factor of 71%, electrical power density of 81 µW/cm2 and photocurrent density of 362 µA/cm2, over four orders of magnitude higher than any photosystem-based biophotovoltaic to date.
Hydrogenases catalyze the interconversion of protons and hydrogen according to the reversible reaction: 2H(+) + 2e(-) ⇆ H(2) while using only the earth-abundant metals nickel and/or iron for catalysis. Due to their high activity for proton reduction and the technological significance of the H(+)/H(2) half reaction, it is important to characterize the catalytic activity of [FeFe]-hydrogenases using both biochemical and electrochemical techniques. Following a detailed electrochemical and photoelectrochemical study of an [FeFe]-hydrogenase from Clostridium acetobutylicum (CaHydA), we now report electrochemical and single-molecule imaging studies carried out on a catalytically active hydrogenase preparation. The enzyme CaHydA, a homologue (70% identity) of the [FeFe]-hydrogenase from Clostridium pasteurianum , CpI, was adsorbed to a negatively charged, self-assembled monolayer (SAM) for investigation by electrochemical scanning tunneling microscopy (EC-STM) techniques and macroscopic electrochemical measurements. The EC-STM imaging revealed uniform surface coverage with sufficient stability to undergo repeated scanning with a STM tip as well as other electrochemical investigations. Cyclic voltammetry yielded a characteristic cathodic hydrogen production signal when the potential was scanned sufficiently negative. The direct observation of the single enzyme distribution on the Au-SAM surface coupled with macroscopic electrochemical measurements obtained from the same electrode allowed the evaluation of a turnover frequency (TOF) as a function of potential for single [FeFe]-hydrogenase molecules.
There is considerable interest in making use of solar energy through photosynthesis to create alternative forms of fuel. Here, we show that photosystem I from a thermophilic bacterium and cytochrome-c(6) can, in combination with a platinum catalyst, generate a stable supply of hydrogen in vitro upon illumination. The self-organized platinization of the photosystem I nanoparticles allows electron transport from sodium ascorbate to photosystem I via cytochrome-c(6) and finally to the platinum catalyst, where hydrogen gas is formed. Our system produces hydrogen at temperatures up to 55 degrees C and is temporally stable for >85 days with no decrease in hydrogen yield when tested intermittently. The maximum yield is approximately 5.5 micromol H(2) h(-1) mg(-1) chlorophyll and is estimated to be approximately 25-fold greater than current biomass-to-fuel strategies. Future work will further improve this yield by increasing the kinetics of electron transfer, extending the spectral response and replacing the platinum catalyst with a renewable hydrogenase.
We used a class of designed peptide detergents to stabilize photosystem I (PS-I) upon extended drying under N2 on a gold-coated-Ni-NTA glass surface. PS-I is a chlorophyll-containing membrane protein complex that is the primary reducer of ferredoxin and the electron acceptor of plastocyanin. We isolated the complex from the thylakoids of spinach chloroplasts using a chemical detergent. The chlorophyll molecules associated with the PS-I complex provide an intrinsic steady-state emission spectrum between 650 and 800 nm at −196.15 °C that reflects the organization of the pigment-protein interactions. In the absence of detergents, a large blue shift of the fluorescence maxima from approximately 735 nm to approximately 685 nm indicates a disruption in light-harvesting subunit organization, thus revealing chlorophyll−protein interactions. The commonly used membrane protein-stabilizing detergents, N-dodecyl-β-D-maltoside and N-octyl-β-D-glucoside, only partially stabilized the approximately 735-nm complex with approximately 685-nm spectroscopic shift. However, prior to drying, addition of the peptide detergent acetyl- AAAAAAK at increasing concentration significantly stabilized the PS-I complex. Moreover, in the presence of acetyl- AAAAAAK, the PS-I complex is stable in a dried form at room temperature for at least 3 wk. Another peptide detergent, acetyl-VVVVVVD, also stabilized the complex but to a lesser extent. These observations suggest that the peptide detergents may effectively stabilize membrane proteins in the solid-state. These designed peptide detergents may facilitate the study of diverse types of membrane proteins.
Detailed structural analyses of membrane proteins as well as their uses in advanced nanobiotechnological applications require extended stabilization of the functional protein conformation. Here we report that a new class of designer surfactant like peptides can significantly increase the activity and stabilize the functional form of the multidomain protein complex Photosystem-I (PS-I) in solution better than other commonly used chemical detergents. We carried out a systematic analysis using a series of such peptides to identify the chemical and structural features that enhance the photochemical activity of PS-I. We observed that peptide surfactant amphiphilicity is necessary but not sufficient to stabilize PS-I in its functional form. A number of factors are essential for designing the optimal peptide including amino acid sequence, N-terminal acetylation and C-terminal amidation. Furthermore, we showed that the polarity and number of charges on the hydrophilic head are important as well as hydrophobicity and size of the amino acid side groups in the hydrophobic tail play an important role. The best performing peptides for the stabilization of functional PS-I are, in order of effectiveness, ac-I(6)K(2)-CONH(2), ac-A(6)K-CONH(2), ac-V(6)K(2)-CONH(2), and ac-V(6)R(2)-CONH(2). These simple and inexpensive peptide surfactants will likely make significant contributions to stabilize the functional form of diverse and currently elusive membrane proteins and their complexes with important applications.
Hydrogen is an attractive fuel with potential for production scalability, provided that inexpensive, efficient molecular catalysts utilizing base metals can be developed for hydrogen production. Here we show for the first time that cobalt myoglobin (CoMyo) catalyzes hydrogen production in mild aerobic conditions with turnover number of 520 over 8 hours. Compared to free Co-protoporphyrin IX, incorporation into the myoglobin scaffold results in a 4-fold increase in photoinduced hydrogen production activity. Engineered variants in which specific histidine resides in proximity of the active site were mutated to alanine result in modulation of the catalytic activity, with the H64A/H97A mutant displaying activity 2.5-fold higher than wild type. Our results demonstrate that protein scaffolds can augment and modulate the intrinsic catalytic activity of molecular hydrogen production catalysts.
A unique aspect of protein transport into plastids is the coordinate involvement of two GTPases in the translocon of the outer chloroplast membrane (Toc). There are two subfamilies in Arabidopsis, the small GTPases (Toc33 and Toc34) and the large acidic GTPases (Toc90, Toc120, Toc132, and Toc159). In chloroplasts, Toc34 and Toc159 are implicated in precursor binding, yet mechanistic details are poorly understood. How the GTPase cycle is modulated by precursor binding is complex and in need of careful dissection. To this end, we have developed novel in vitro assays to quantitate nucleotide binding and hydrolysis of the Toc GTPases. Here we present the first systematic kinetic characterization of four Toc GTPases (cytosolic domains of atToc33, atToc34, psToc34, and the GTPase domain of atToc159) to permit their direct comparison. We report the K M , V max , and E a values for GTP hydrolysis and the K d value for nucleotide binding for each protein. We demonstrate that GTP hydrolysis by psToc34 is stimulated by chloroplast transit peptides; however, this activity is not stimulated by homodimerization and is abolished by the R133A mutation. Furthermore, we show peptide stimulation of hydrolytic rates are not because of accelerated nucleotide exchange, indicating that transit peptides function as GTPase-activating proteins and not guanine nucleotide exchange factors in modulating the activity of psToc34. Finally, by using the psToc34 structure, we have developed molecular models for atToc33, atToc34, and atToc159G. By combining these models with the measured enzymatic properties of the Toc GTPases, we provide new insights of how the chloroplast protein import cycle may be regulated.Both mitochondria and chloroplasts arose through endosymbiotic events that followed phagocytotic internalization of either a free-living ␣-proteobacteria or cyanobacteria. During the course of evolution, in both cases, the vast majority of the endosymbiont genes was relocated to the host nucleus (1). Moreover, in higher plants, most of the proteome from both organelles is derived from nuclearly encoded proteins that are translated on free cytosolic ribosomes and then post-translationally translocated into the organelle via distinct protein complexes located in each of the two membranes that enclose these organelles (2, 3). These mitochondrial and chloroplast translocators are denoted as Tim/Tom and Tic/Toc, respectively (Translocator of the inner/outer membrane of mitochondria/chloroplast). The Toc 5 complex consists of three key proteins denoted by their apparent molecular masses, Toc34, Toc75, and Toc159, and most likely exist with a stoichiometry of 4:4:1, respectively (4). Proteins targeted from the cytosol to these mitochondrial and chloroplast translocators require additional "information" in the form of an N-terminal targeting sequence known as a presequence and transit peptide, respectively (1). This additional targeting sequence has been added during evolution, yielding a larger precursor protein, and is cleaved once the precursor...
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