Functional redundancy between flavodiiron proteins and NDH-1 in Synechocystis sp. PCC 6803.

In oxygenic photosynthetic organisms, excluding angiosperms, flavodiiron proteins (FDPs) catalyze light-dependent reduction of O2 to H2 O. This alleviates electron pressure on the photosynthetic apparatus and protects it from photodamage. In Synechocystis sp. PCC 6803, four FDP isoforms function as hetero-oligomers of Flv1 and Flv3 and/or Flv2 and Flv4. An alternative electron transport pathway mediated by the NAD(P)H dehydrogenase-like complex (NDH-1) also contributes to redox hemostasis and the photoprotection of photosynthesis. Four NDH-1 types have been characterized in cyanobacteria: NDH-11 and NDH-12 , which function in respiration; and NDH-13 and NDH-14 , which function in CO2 uptake. All four types are involved in cyclic electron transport. Along with single FDP mutants (∆flv1 and Δflv3) and the double NDH-1 mutants (∆d1d2, which is deficient in NDH-11,2 and ∆d3d4, which is deficient in NDH-13,4 ), we studied triple mutants lacking one of Flv1 or Flv3, and NDH-11,2 or NDH-13,4 . We show that the presence of either Flv1/3 or NDH-11,2 , but not NDH-13,4 , is indispensable for survival during changes in growth conditions from high CO2 /moderate light to low CO2 /high light. Our results show functional redundancy between FDPs and NDH-11,2 under the studied conditions. We suggest that ferredoxin probably functions as a primary electron donor to both Flv1/3 and NDH-11,2 , allowing their functions to be dynamically coordinated for efficient oxidation of photosystem I and for photoprotection under variable CO2 and light availability.

Closing the Gap for Electronic Short-Circuiting: Photosystem†I Mixed Monolayers Enable Improved Anisotropic Electron Flow in Biophotovoltaic Devices.

Well-defined assemblies of photosynthetic protein complexes are required for an optimal performance of semi-artificial energy conversion devices, capable of providing unidirectional electron flow when light-harvesting proteins are interfaced with electrode surfaces. We present mixed photosystem I (PSI) monolayers constituted of native cyanobacterial PSI trimers in combination with isolated PSI monomers from the same organism. The resulting compact arrangement ensures a high density of photoactive protein complexes per unit area, providing the basis to effectively minimize short-circuiting processes that typically limit the performance of PSI-based bioelectrodes. The PSI film is further interfaced with redox polymers for optimal electron transfer, enabling highly efficient light-induced photocurrent generation. Coupling of the photocathode with a [NiFeSe]-hydrogenase confirms the possibility to realize light-induced H2 evolution.

Extended Operational Lifetime of a Photosystem-Based Bioelectrode.

The development of bioelectrochemical assemblies for sustainable energy transformation constitutes an increasingly important field of research. Significant progress has been made in the development of semiartificial devices for conversion of light into electrical energy by integration of photosynthetic biomolecules on electrodes. However, sufficient long-term stability of such biophotoelectrodes has been compromised by reactive species generated under aerobic operation. Therefore, meeting the requirements of practical applications still remains unsolved. We present the operation of a photosystem I-based photocathode using an electron acceptor that enables photocurrent generation under anaerobic conditions as the basis for a biodevice with substantially improved stability. A continuous operation lifetime considerably superior to previous reports and at higher light intensities is paving the way toward the potential application of semiartificial energy conversion devices.

In memory of Achim Trebst (1929-2017): a pioneer of photosynthesis research.

The life and work of Achim Trebst (1929-2017) was dedicated to photosynthesis, involving a wide span of seminal contributions which cumulated in more than five decades of active research: Major topics include the separation of light and dark phases in photosynthesis, the elucidation of photosynthesis by the use of inhibitors, the identification of the three-dimensional structure of photosystem II and its degradation, and an explanation of singlet oxygen formation. For this tribute, which has been initiated by Govindjee, twenty-two personal tributes by former coworkers, scientific friends, and his family have been compiled and combined with an introduction tracing the different stages of Achim Trebst's scientific life.

Global Protein Oxidation Profiling Suggests Efficient Mitochondrial Proteome Homeostasis During Aging.

The free radical theory of aging is based on the idea that reactive oxygen species (ROS) may lead to the accumulation of age-related protein oxidation. Because themajority of cellular ROS is generated at the respiratory electron transport chain, this study focuses on the mitochondrial proteome of the aging model Podospora anserina as target for ROS-induced damage. To ensure the detection of even low abundant modified peptides, separation by long gradient nLC-ESI-MS/MS and an appropriate statistical workflow for iTRAQ quantification was developed. Artificial protein oxidation was minimized by establishing gel-free sample preparation in the presence of reducing and iron-chelating agents. This first large scale, oxidative modification-centric study for P. anserina allowed the comprehensive quantification of 22 different oxidative amino acid modifications, and notably the quantitative comparison of oxidized and nonoxidized protein species. In total 2341 proteins were quantified. For 746 both protein species (unmodified and oxidatively modified) were detected and the modification sites determined. The data revealed that methionine residues are preferably oxidized. Further prominent identified modifications in decreasing order of occurrence were carbonylation as well as formation of N-formylkynurenine and pyrrolidinone. Interestingly, for the majority of proteins a positive correlation of changes in protein amount and oxidative damage were noticed, and a general decrease in protein amounts at late age. However, it was discovered that few proteins changed in oxidative damage in accordance with former reports. Our data suggest that P. anserina is efficiently capable to counteract ROS-induced protein damage during aging as long as protein de novo synthesis is functioning, ultimately leading to an overall constant relationship between damaged and undamaged protein species. These findings contradict a massive increase in protein oxidation during aging and rather suggest a protein damage homeostasis mechanism even at late age.

The cyanobacterial cytochrome b6f subunit PetP adopts an SH3 fold in solution.

PetP is a peripheral subunit of the cytochrome b(6)f complex (b(6)f) present in both, cyanobacteria and red algae. It is bound to the cytoplasmic surface of this membrane protein complex where it greatly affects the efficiency of the linear photosynthetic electron flow although it is not directly involved in the electron transfer reactions. Despite the crystal structures of the b(6)f core complex, structural information for the transient regulatory b(6)f subunits is still missing. Here we present the first structure of PetP at atomic resolution as determined by solution NMR. The protein adopts an SH3 fold, which is a common protein motif in eukaryotes but comparatively rare in prokaryotes. The structure of PetP enabled the identification of the potential interaction site for b(6)f binding by conservation mapping. The interaction surface is mainly formed by two large loop regions and one short 310 helix which also exhibit an increased flexibility as indicated by heteronuclear steady-state {(1)H}-(15)N NOE and random coil index parameters. The properties of this potential b(6)f binding site greatly differ from the canonical peptide binding site which is highly conserved in eukaryotic SH3 domains. Interestingly, three other proteins of the photosynthetic electron transport chain share this SH3 fold with PetP: NdhS of the photosynthetic NADH dehydrogenase-like complex (NDH-1), PsaE of the photosystem 1 and subunit α of the ferredoxin-thioredoxin reductase have, similar to PetP, a great impact on the photosynthetic electron transport. Finally, a model is presented to illustrate how SH3 domains modulate the photosynthetic electron transport processes in cyanobacteria.

Structural and functional characterisation of the cyanobacterial PetC3 Rieske protein family.

The cyanobacterium Synechocystis PCC 6803 possesses three Rieske isoforms: PetC1, PetC2 and PetC3. While PetC1 and PetC2 have been identified as alternative subunits of the cytochrome b6f complex (b6f), PetC3 was localized exclusively within the plasma membrane. The spatial separation of PetC3 from the photosynthetic and respiratory protein complexes raises doubt in its involvement in bioenergetic electron transfer. Here we report a detailed structural and functional characterization of the cyanobacterial PetC3 protein family indicating that PetC3 is not a component of the b6f and the photosynthetic electron transport as implied by gene annotation. Instead PetC3 has a distinct function in cell envelope homeostasis. Especially proteomic analysis shows that deletion of petC3 in Synechocystis PCC 6803 primarily affects cell envelope proteins including many nutrient transport systems. Therefore, the observed downregulation in the photosynthetic electron transport - mainly caused by photosystem 2 inactivation - might constitute a stress adaptation. Comprehensive in silico sequence analyses revealed that PetC3 proteins are periplasmic lipoproteins tethered to the plasma membrane with a subclass consisting of soluble periplasmic proteins, i.e. their N-terminal domain is inconsistent with their integration into the b6f. For the first time, the structure of PetC3 was determined by X-ray crystallography at an atomic resolution revealing significant high similarities to non-b6f Rieske subunits in contrast to PetC1. These results suggest that PetC3 affects processes in the periplasmic compartment that only indirectly influence photosynthetic electron transport. For this reason, we suggest to rename "Photosynthetic electron transport Chain 3" (PetC3) proteins as "periplasmic Rieske proteins" (Prp).

Functional characterization of the small regulatory subunit PetP from the cytochrome b6f complex in Thermosynechococcus elongatus.

The cyanobacterial cytochrome b(6)f complex is central for the coordination of photosynthetic and respiratory electron transport and also for the balance between linear and cyclic electron transport. The development of a purification strategy for a highly active dimeric b(6)f complex from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 enabled characterization of the structural and functional role of the small subunit PetP in this complex. Moreover, the efficient transformability of this strain allowed the generation of a ΔpetP mutant. Analysis on the whole-cell level by growth curves, photosystem II light saturation curves, and P700(+) reduction kinetics indicate a strong decrease in the linear electron transport in the mutant strain versus the wild type, while the cyclic electron transport via photosystem I and cytochrome b(6)f is largely unaffected. This reduction in linear electron transport is accompanied by a strongly decreased stability and activity of the isolated ΔpetP complex in comparison with the dimeric wild-type complex, which binds two PetP subunits. The distinct behavior of linear and cyclic electron transport may suggest the presence of two distinguishable pools of cytochrome b(6)f complexes with different functions that might be correlated with supercomplex formation.

The proteome and lipidome of Synechocystis sp. PCC 6803 cells grown under light-activated heterotrophic conditions.

Cyanobacteria are photoautotrophic prokaryotes with a plant-like photosynthetic machinery. Because of their short generation times, the ease of their genetic manipulation, and the limited size of their genome and proteome, cyanobacteria are popular model organisms for photosynthetic research. Although the principal mechanisms of photosynthesis are well-known, much less is known about the biogenesis of the thylakoid membrane, hosting the components of the photosynthetic, and respiratory electron transport chain in cyanobacteria. Here we present a detailed proteome analysis of the important model and host organism Synechocystis sp. PCC 6803 under light-activated heterotrophic growth conditions. Because of the mechanistic importance and severe changes in thylakoid membrane morphology under light-activated heterotrophic growth conditions, a focus was put on the analysis of the membrane proteome, which was supported by a targeted lipidome analysis. In total, 1528 proteins (24.5% membrane integral) were identified in our analysis. For 641 of these proteins quantitative information was obtained by spectral counting. Prominent changes were observed for proteins associated with oxidative stress response and protein folding. Because of the heterotrophic growth conditions, also proteins involved in carbon metabolism and C/N-balance were severely affected. Although intracellular thylakoid membranes were significantly reduced, only minor changes were observed in their protein composition. The increased proportion of the membrane-stabilizing sulfoqinovosyl diacyl lipids found in the lipidome analysis, as well as the increased content of lipids with more saturated acyl chains, are clear indications for a coordinated synthesis of proteins and lipids, resulting in stabilization of intracellular thylakoid membranes under stress conditions.

Filling the Green Gap of a Megadalton Photosystem I Complex by Conjugation of Organic Dyes.

Photosynthesis is Nature's major process for converting solar into chemical energy. One of the key players in this process is the multiprotein complex photosystem I (PSI) that through absorption of incident photons enables electron transfer, which makes this protein attractive for applications in bioinspired photoactive hybrid materials. However, the efficiency of PSI is still limited by its poor absorption in the green part of the solar spectrum. Inspired by the existence of natural phycobilisome light-harvesting antennae, we have widened the absorption spectrum of PSI by covalent attachment of synthetic dyes to the protein backbone. Steady-state and time-resolved photoluminescence reveal that energy transfer occurs from these dyes to PSI. It is shown by oxygen-consumption measurements that subsequent charge generation is substantially enhanced under broad and narrow band excitation. Ultimately, surface photovoltage (SPV) experiments prove the enhanced activity of dye-modified PSI even in the solid state.

X-ray Structure and Nuclear Magnetic Resonance Analysis of the Interaction Sites of the Ga-Substituted Cyanobacterial Ferredoxin.

In chloroplasts, ferredoxin (Fd) is reduced by Photosystem I (PSI) and oxidized by Fd-NADP(+) reductase (FNR) that is involved in NADP(+) reduction. To understand the structural basis for the dynamics and efficiency of the electron transfer reaction via Fd, we complementary used X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. In the NMR analysis of the formed electron transfer complex with Fd, the paramagnetic effect of the [2Fe-2S] cluster of Fd prevented us from detecting the NMR signals around the cluster. To solve this problem, the paramagnetic iron-sulfur cluster was replaced with a diamagnetic metal cluster. We determined the crystal structure of the Ga-substituted Fd (GaFd) from Synechocystis sp. PCC6803 at 1.62 Šresolution and verified its functional complementation using affinity chromatography. NMR analysis of the interaction sites on GaFd with PSI (molecular mass of ∼1 MDa) and FNR from Thermosynechococcus elongatus was achieved with high-field NMR spectroscopy. With reference to the interaction sites with FNR of Anabaena sp. PCC 7119 from the published crystal data, the interaction sites of Fd with FNR and PSI in solution can be classified into two types: (1) the core hydrophobic residues in the proximity of the metal center and (2) the hydrophilic residues surrounding the core. The former sites are shared in the Fd:FNR and Fd:PSI complex, while the latter ones are target-specific and not conserved on the residual level.

A simple method for decomposition of peracetic acid in a microalgal cultivation system.

A cost-efficient process devoid of several washing steps was developed, which is related to direct cultivation following the decomposition of the sterilizer. Peracetic acid (PAA) is known to be an efficient antimicrobial agent due to its high oxidizing potential. Sterilization by 2 mM PAA demands at least 1 h incubation time for an effective disinfection. Direct degradation of PAA was demonstrated by utilizing components in conventional algal medium. Consequently, ferric ion and pH buffer (HEPES) showed a synergetic effect for the decomposition of PAA within 6 h. On the contrary, NaNO3, one of the main components in algal media, inhibits the decomposition of PAA. The improved growth of Chlorella vulgaris and Synechocystis PCC6803 was observed in the prepared BG11 by decomposition of PAA. This process involving sterilization and decomposition of PAA should help cost-efficient management of photobioreactors in a large scale for the production of value-added products and biofuels from microalgal biomass.

Light-mediated hydrogen generation in Photosystem I: attachment of a naphthoquinone-molecular wire-Pt nanoparticle to the A1A and A1B sites.

The molecular wire-appended naphthoquinone 1-[15-(3-methyl-1,4-naphthoquinone-2-yl)]pentadecyl disulfide [(NQ(CH2)15S)2] has been incorporated into the A1A and A1B sites of Photosystem I (PS I) in the menB variant of Synechocystis sp. PCC 6803. Transient electron paramagnetic resonance studies show that the naphthoquinone headgroup displaces plastoquinone-9 from the A1A (and likely A1B) sites to a large extent. When a Pt nanoparticle is attached to the molecular wire by reductive cleavage of the disulfide and reaction with the resulting thiol, the PS I-NQ(CH2)15S-Pt nanoconstruct evolves dihydrogen at a rate of 67.3 µmol of H2 (mg of Chl)(-1) h(-1) [3.4 e(-) (PS I)(-1) s(-1)] after illumination for 1 h at pH 6.4. No dihydrogen is detected if wild-type PS I, which does not incorporate the quinone, is used or if either (NQ(CH2)15S)2 or the Pt nanoparticle is absent. Time-resolved optical studies of the PS I-NQ(CH2)15S-Pt nanoconstruct show that the lifetimes of the forward electron transfer to and reverse electron transfer from the iron-sulfur clusters are the same as in native PS I. Thus, electrons are not shuttled directly from the quinone to the Pt nanoparticle during either forward or reverse electron transfer. It is found that the rate of dihydrogen evolution in the PS I-NQ(CH2)15S-Pt nanoconstruct depends strongly on the concentration the sacrificial electron donor cytochrome c6. These observations can be explained if the iron-sulfur clusters are involved in stabilizing the electron; the ~50 ms residence time of the electron on FA or FB is sufficiently long to allow cytochrome c6 to reduce P700(+), thereby eliminating the recombination channel. In the absence of P700(+), slow electron transfer through the molecular wire to the Pt catalyst can occur, and hence, H2 evolution is observed.

The plasma membrane of the cyanobacterium Gloeobacter violaceus contains segregated bioenergetic domains.

The light reactions of oxygenic photosynthesis almost invariably take place in the thylakoid membranes, a highly specialized internal membrane system located in the stroma of chloroplasts and the cytoplasm of cyanobacteria. The only known exception is the primordial cyanobacterium Gloeobacter violaceus, which evolved before the appearance of thylakoids and harbors the photosynthetic complexes in the plasma membrane. Thus, studies on G. violaceus not only shed light on the evolutionary origin and the functional advantages of thylakoid membranes but also might include insights regarding thylakoid formation during chloroplast differentiation. Based on biochemical isolation and direct in vivo characterization, we report here structural and functional domains in the cytoplasmic membrane of a cyanobacterium. Although G. violaceus has no internal membranes, it does have localized domains with apparently specialized functions in its plasma membrane, in which both the photosynthetic and the respiratory complexes are concentrated. These bioenergetic domains can be visualized by confocal microscopy, and they can be isolated by a simple procedure. Proteomic analysis of these domains indicates their physiological function and suggests a protein sorting mechanism via interaction with membrane-intrinsic terpenoids. Based on these results, we propose specialized domains in the plasma membrane as evolutionary precursors of thylakoids.

Engineered electron-transfer chain in photosystem 1 based photocathodes outperforms electron-transfer rates in natural photosynthesis.

Photosystem 1 (PS1) triggers the most energetic light-induced charge-separation step in nature and the in vivo electron-transfer rates approach 50 e(-) s(-1) PS1(-1). Photoelectrochemical devices based on this building block have to date underperformed with respect to their semiconductor counterparts or to natural photosynthesis in terms of electron-transfer rates. We present a rational design of a redox hydrogel film to contact PS1 to an electrode for photocurrent generation. We exploit the pH-dependent properties of a poly(vinyl)imidazole Os(bispyridine)2Cl polymer to tune the redox hydrogel film for maximum electron-transfer rates under optimal conditions for PS1 activity. The PS1-containing redox hydrogel film displays electron-transfer rates of up to 335±14 e(-) s(-1) PS1(-1), which considerably exceeds the rates observed in natural photosynthesis or in other semiartificial systems. Under O2 supersaturation, photocurrents of 322±19 µA cm(-2) were achieved. The photocurrents are only limited by mass transport of the terminal electron acceptor (O2). This implies that even higher electron-transfer rates may be achieved with PS1-based systems in general.

Redox hydrogels with adjusted redox potential for improved efficiency in Z-scheme inspired biophotovoltaic cells.

The improvement of Z-scheme inspired biophotovoltaics is achieved by fine tuning the properties of redox hydrogels applied as immobilization and electron conducting matrices for the photosystem-protein complexes. The formal potentials of the redox hydrogels are adjusted to the respective redox sites in the photosystems for optimized electron transfer without substantial voltage loss. The anode is based on photosystem 2 (PS2) integrated in a phenothiazine modified redox hydrogel with a formal potential of -1 mV vs. SHE, which is 59 mV more positive than the QB acceptor site in PS2. The cathode is based on photosystem 1 (PS1) contacted via an Os-complex based redox hydrogel with a formal potential of 395 mV vs. SHE, i.e. 28 mV more negative than the primary P700 electron acceptor of PS1. The potential difference between the two redox hydrogels is 396 mV. An open circuit voltage (VOC) of 372.5 ± 2.1 mV could be achieved for the biophotovoltaic cell. The maximum power output is 1.91 ± 0.56 µW cm(-2) and the conversion efficiency (η) is 4.5 × 10(-5), representing a 125-fold improvement in comparison to the previously proposed device exploiting the photosynthetic Z-scheme for electrical energy production.

Deletion of psbJ leads to accumulation of Psb27-Psb28 photosystem II complexes in Thermosynechococcus elongatus.

The life cycle of Photosystem II (PSII) is embedded in a network of proteins that guides the complex through biogenesis, damage and repair. Some of these proteins, such as Psb27 and Psb28, are involved in cofactor assembly for which they are only transiently bound to the preassembled complex. In this work we isolated and analyzed PSII from a ΔpsbJ mutant of the thermophilic cyanobacterium Thermosynechococcus elongatus. From the four different PSII complexes that could be separated the most prominent one revealed a monomeric Psb27-Psb28 PSII complex with greatly diminished oxygen-evolving activity. The MALDI-ToF mass spectrometry analysis of intact low molecular weight subunits (<10kDa) depicted wild type PSII with the absence of PsbJ. Relative quantification of the PsbA1/PsbA3 ratio by LC-ESI mass spectrometry using (15)N labeled PsbA3-specific peptides indicated the complete replacement of PsbA1 by the stress copy PsbA3 in the mutant, even under standard growth conditions (50µmol photons m(-2) s(-1)). This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Structural and functional alterations of cyanobacterial phycobilisomes induced by high-light stress.

Exposure of cyanobacterial or red algal cells to high light has been proposed to lead to excitonic decoupling of the phycobilisome antennae (PBSs) from the reaction centers. Here we show that excitonic decoupling of PBSs of Synechocystis sp. PCC 6803 is induced by strong light at wavelengths that excite either phycobilin or chlorophyll pigments. We further show that decoupling is generally followed by disassembly of the antenna complexes and/or their detachment from the thylakoid membrane. Based on a previously proposed mechanism, we suggest that local heat transients generated in the PBSs by non-radiative energy dissipation lead to alterations in thermo-labile elements, likely in certain rod and core linker polypeptides. These alterations disrupt the transfer of excitation energy within and from the PBSs and destabilize the antenna complexes and/or promote their dissociation from the reaction centers and from the thylakoid membranes. Possible implications of the aforementioned alterations to adaptation of cyanobacteria to light and other environmental stresses are discussed.

Reactive oxygen species target specific tryptophan site in the mitochondrial ATP synthase.

The release of reactive oxygen species (ROS) as side products of aerobic metabolism in the mitochondria is an unavoidable consequence. As the capacity of organisms to deal with this exposure declines with age, accumulation of molecular damage caused by ROS has been defined as one of the central events during the ageing process in biological systems as well as in numerous diseases such as Alzheimer's and Parkinson's Dementia. In the filamentous fungus Podospora anserina, an ageing model with a clear defined mitochondrial etiology of ageing, in addition to the mitochondrial aconitase the ATP synthase alpha subunit was defined recently as a hot spot for oxidative modifications induced by ROS. In this report we show, that this reactivity is not randomly distributed over the ATP Synthase, but is channeled to a single tryptophan residue 503. This residue serves as an intra-molecular quencher for oxidative species and might also be involved in the metabolic perception of oxidative stress or regulation of enzyme activity. A putative metal binding site in the proximity of this tryptophan residue appears to be crucial for the molecular mechanism for the selective targeting of oxidative damage.

Universal method for protein immobilization on chemically functionalized germanium investigated by ATR-FTIR difference spectroscopy.

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy allows a detailed analysis of surface attached molecules, including their secondary structure, orientation, and interaction with small molecules in the case of proteins. Here, we present a universal immobilization technique on germanium for all oligo-histidine-tagged proteins. For this purpose, new triethoxysilane derivates were developed: we synthesized a linker-silane with a succinimidyl ester as amine-reactive headgroup and a matrix-silane with an unreactive ethylene glycol group. A new methodology for the attachment of triethoxysilanes on germanium was established, and the surface was characterized by ATR-FTIR and X-ray photoelectron spectroscopy. In the next step, the succinimidyl ester was reacted with aminonitrilotriacetic acid. Subsequently, Ni(2+) was coordinated to form Ni-nitrilotriacetic acid for His-tag binding. The capability of the functionalized surface was demonstrated by experiments using the small GTPase Ras and photosystem I (PS I). The native binding of the proteins was proven by difference spectroscopy, which probes protein function. The function of Ras as molecular switch was demonstrated by a beryllium trifluoride anion titration assay, which allows observation of the "on" and "off" switching of Ras at atomic resolution. Furthermore, the activity of immobilized PS I was proven by light-induced difference spectroscopy. Subsequent treatment with imidazole removes attached proteins, enabling repeated binding. This universal technique allows specific attachment of His-tagged proteins and a detailed study of their function at the atomic level using FTIR difference spectroscopy.