Light-Mediated Hydrogen Generation in Photosystem I: Attachment of a Naphthoquinone–Molecular Wire–Pt Nanoparticle to the A_1A and A_1B 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 A_1A and A_1B sites of Photosystem I (PS I) in the <i>menB</i> variant of <i>Synechocystis</i> sp. PCC 6803. Transient electron paramagnetic resonance studies show that the naphthoquinone headgroup displaces plastoquinone-9 from the A_1A (and likely A_1B) 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 <i>c</i>₆. 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 F_A or F_B is sufficiently long to allow cytochrome <i>c</i>₆ 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

Reaction Mechanism of Adenylyltransferase DrrA from <i>Legionella pneumophila</i> Elucidated by Time-Resolved Fourier Transform Infrared Spectroscopy

Modulation of the function of small GTPases that regulate vesicular trafficking is a strategy employed by several human pathogens. <i>Legionella pneumophila</i> infects lung macrophages and injects a plethora of different proteins into its host cell. Among these is DrrA/SidM, which catalyzes stable adenylylation of Rab1b, a regulator of endoplasmatic reticulum to Golgi trafficking, and thereby alters the function and interactions of this small GTPase. We employed time-resolved FTIR-spectroscopy to monitor the DrrA-catalyzed AMP-transfer to Tyr77 of Rab1b. A transient complex between DrrA, adenylylated Rab1b, and the pyrophosphate byproduct was resolved, allowing us to analyze the interactions at the active site. Combination of isotopic labeling and site-directed mutagenesis allowed us to derive the catalytic mechanism of DrrA from the FTIR difference spectra. DrrA shares crucial residues in the ATP-binding pocket with similar AMP-transferring enzymes such as glutamine synthetase adenylyltransferase or kanamycin nucleotidyltransferase, but provides the complete active site on a single subunit. We determined that Asp112 of DrrA functions as the catalytic base for deprotonation of Tyr77 of Rab1b to enable nucleophilic attack on the ATP. The study provides detailed understanding of the <i>Legionella pneumophila</i> protein DrrA and of AMP-transfer reactions in general

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, Ni²⁺ 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

An ATR–FTIR Sensor Unraveling the Drug Intervention of Methylene Blue, Congo Red, and Berberine on Human Tau and Aβ

Alzheimer’s disease affects millions of human beings worldwide. The disease progression is characterized by the formation of plaques and neurofibrillary tangles in the brain, which are based on aggregation processes of the Aβ peptide and tau protein. Today there is no cure and even no <i>in vitro</i> assay available for the identification of drug candidates, which provides direct information concerning the protein secondary structure label-free. Therefore, we developed an attenuated total reflection Fourier transform infrared spectroscopy (ATR–FTIR) sensor, which uses surface bound antibodies to immobilize a desired target protein. The secondary structure of the protein can be evaluated based on the secondary structure sensitive frequency of the amide I band. Direct information about the effect of a drug candidate on the secondary structure distribution of the total target protein fraction within the respective body fluid can be detected by a frequency shift of the amide I band. Thereby, the extent of the amide I shift is indicative for the compound efficiency. The functionality of this approach was demonstrated by the quantification of the effect of the drug candidate methylene blue on the pathogenic misfolded tau protein as extracted from cerebrospinal fluid (CSF). Methylene blue induces a shift from pathogenic folded β-sheet dominated to the healthy monomeric state. A similar effect was observed for congo red on pathogenic Aβ isoforms from CSF. In addition, the effect of berberine on synthetic Aβ_1–42 is studied. Berberine seems to decelerate the aggregation process of synthetic Aβ_1–42 peptides

Unraveling the Phosphocholination Mechanism of the <i>Legionella pneumophila</i> Enzyme AnkX

The intracellular pathogen <i>Legionella pneumophila</i> infects lung macrophages and injects numerous effector proteins into the host cell to establish a vacuole for proliferation. The necessary interference with vesicular trafficking of the host is achieved by modulation of the function of Rab GTPases. The effector protein AnkX chemically modifies Rab1b and Rab35 by covalent phosphocholination of serine or threonine residues using CDP-choline as a donor. So far, the phosphoryl transfer mechanism and the relevance of observed autophosphocholination of AnkX remained disputable. We designed tailored caged compounds to make this type of enzymatic reaction accessible for time-resolved Fourier transform infrared difference spectroscopy. By combining spectroscopic and biochemical methods, we determined that full length AnkX is autophosphocholinated at Ser521, Thr620, and Thr943. However, autophosphocholination loses specificity for these sites in shortened constructs and does not appear to be relevant for the catalysis of the phosphoryl transfer. In contrast, transient phosphocholination of His229 in the conserved catalytic motif might exist as a short-lived reaction intermediate. Upon substrate binding, His229 is deprotonated and locked in this state, being rendered capable of a nucleophilic attack on the pyrophosphate moiety of the substrate. The proton that originated from His229 is transferred to a nearby carboxylic acid residue. Thus, our combined findings support a ping-pong mechanism involving phosphocholination of His229 and subsequent transfer of phosphocholine to the Rab GTPase. Our approach can be extended to the investigation of further nucleotidyl transfer reactions, which are currently of reemerging interest in regulatory pathways of host–pathogen interactions

Amyloid-β-Secondary Structure Distribution in Cerebrospinal Fluid and Blood Measured by an Immuno-Infrared-Sensor: A Biomarker Candidate for Alzheimer’s Disease

The misfolding of the Amyloid-beta (Aβ) peptide into β-sheet enriched conformations was proposed as an early event in Alzheimer’s Disease (AD). Here, the Aβ peptide secondary structure distribution in cerebrospinal fluid (CSF) and blood plasma of 141 patients was measured with an immuno-infrared-sensor. The sensor detected the amide I band, which reflects the overall secondary structure distribution of all Aβ peptides extracted from the body fluid. We observed a significant downshift of the amide I band frequency of Aβ peptides in Dementia Alzheimer type (DAT) patients, which indicated an overall shift to β-sheet. The secondary structure distribution of all Aβ peptides provides a better marker for DAT detection than a single Aβ misfold or the concentration of a specific oligomer. The discrimination between DAT and disease control patients according to the amide I frequency was in excellent agreement with the clinical diagnosis (accuracy 90% for CSF and 84% for blood). The amide I band maximum above or below the decisive marker frequency appears as a novel spectral biomarker candidate of AD. Additionally, a preliminary proof-of-concept study indicated an amide I band shift below the marker band already in patients with mild cognitive impairment due to AD. The presented immuno-IR-sensor method represents a promising, simple, robust, and label-free diagnostic tool for CSF and blood analysis