Werner Mäntele

Goethe University Frankfurt · Institut für Biophysik

CYBASC proteins are ascorbate (AscH⁻) reducible, diheme b containing membrane integral cytochrome b561 proteins (cytb561), which are proposed to be involved in AscH⁻ recycling and facilitation of iron absorption. Two distinct CYBASC paralogs from the plant Arabidopsis thaliana, Atcytb561-A (A-paralog) and Atcytb561-B (B-paralog), have been found to...

An apparatus based on the dialysis technique was specially designed for assessing in-vitro drug release of nanoparticles (NPs) by fluorescence measurement. The experimental setup provided continuous drug release profiles. A mathematical model was applied to process the data. With this method, the rate-limiting step caused by the transport of active...

Time-resolved studies were performed for tryptic proteolysis of β-casein in media containing 10–40 % (v/v) ethanol at 37 °C and pH 7.9. The peptide bond demasking, the process which implies the removal of steric obstacles shielding polypeptide sites against enzymatic attack, was quantitatively evaluated with fluorescence spectroscopy by monitoring...

Time-resolved studies were performed for tryptic proteolysis of β-casein. The accumulation and decay of the proteolysis products was shown to proceed in the form of nanoscale aggregates that exhibited considerable scattering cross sections for visible light. Monitoring the time course of degradation of these aggregates by static light scattering al...

Pluripotent mouse embryonic stem cells (mESCs) dif-ferentiate in vitro spontaneously to cardiomyocytes thereby providing a tool to study the effects of ionizing radiation on cardiac development and on cardiomyocytes themselves [1]. So far, the system has been used in the pharmacological industry to screen the embryotoxic po-tential of drugs or chem...

There is emerging epidemiological evidence of an in-creased risk of adverse cardiovascular effects at low or moderate doses of ionizing radiation occurring many years after the exposure [1]. However, essentially no in-formation is available on the potential cardiovascular ef-fects associated with the exposure to heavy ions. To ad-dress this issue,...

Human and bovine serum albumins are widely known proteins that can form amyloid fibrils under destabilizing conditions. Use of well-known proteins with easily-controlled aggregation process, and comparison of these processes for similar proteins from different species, could help elucidate the nature of the aggregation process implicated in many de...

A method for monitoring hemodialysis based on quantitative infrared spectroscopic determination of the molecules dialyzed from patient blood is reported. The measurements are reagent-free and aim at real-time and in-line monitoring of the hemodialysis patient. A flow cell using attenuated total reflection infrared spectroscopy is coupled downstream...

Aims: Current anticoagulation management in cardiac surgery is still strongly based on the intraoperative administration of unfractionated heparin. The restoring of blood coagulation is conducted by administration of protamine. However, unfractionated heparins often contain a considerable amount of fractionated heparin which cannot be completely ne...

The aim of this study was to explore the relationship of scanning parameters (clinical protocols), reconstruction kernels and slice thickness with image quality and radiation dose in a DSCT. The chest of an anthropomorphic phantom was scanned on a DSCT scanner (Siemens Somatom Definition flash) using different clinical protocols, including single-...

RUNX1/ETO, the fusion protein resulting from the chromosomal translocation t(8;21), is one of the most frequent translocation products in acute myeloid leukemia. Several in vitro and in vivo studies have shown that the homo-tetramerization domain of ETO, the nervy homology region 2 (NHR2), is essential for RUNX1/ETO oncogenic activity. We analyzed...

Nanoparticles represent promising carriers for controlled drug delivery. Particle size and size distribution of the particles are important parameters for the in vivo behaviour after intravenous injection and have to be characterised precisely. In the present study, the influence of lyophilisation on the storage stability of poly(d,l lactic-co-glyc...

In this study, structural, functional, and mechanistic properties of the Na(+)/H(+) antiporter MjNhaP1 from Methanococcus jannaschii were analyzed by infrared spectroscopic techniques. Na(+)/H(+) antiporters are generally responsible for the regulation of cytoplasmic pH and Na(+) concentration. MjNhaP1 is active in the pH range between pH 6 and pH...

NhaA (41,355 Da) is a Na+/H+ antiporter of Escherichia coli which plays a central role in regulation of intracellular pH, cellular Na+ content, and cell volume [E. Padan, S. Schuldiner, J. Exp. Biol. 196 (1994) 443]. Its activity is strongly regulated by pH and increases over 3 orders of magnitude between pH 7 and 8 [A. Rothman, Y. Gerchman, E. Pad...

Abstract— Molecular changes associated with the light-induced reduction of the intermediary electron acceptor I (bacteriopheophytin, BPh) in bacterial photosynthesis were studied by means of Fourier transform infrared (FTIR) difference spectroscopy. Chromatophore membranes and reconstituted reaction centers (RCs) of Rhodopseudomonas viridis were pr...

Es wurde eine neue, direkte und einfache Methode zur Bestimmung des Heparinspiegels im Blut entwickelt, die auf der Komplexbildung von Heparin mit Protamin beruht. Heparin bildet mit Protamin im Überschuss nanoskalige Partikel der Größe von ca. 100–200 nm, die mit Lichtstreuverfahren nachgewiesen werden können. Die Lichtstreuung ist proportional zu...

Ein alter Wunschtraum bei der Chemotherapie ist es, Cytostatika, die einen Tumor am Wachstum hindern können, in einer Art trojanischem Pferd zum Tumor zu bringen und dort ihre Wirkung entfalten zu lassen. Die Dosierung dieser Wirkstoffe könnte höher gewählt werden und die Nebenwirkungen der Wirkstoffe auf gesunde Organe wären geringer. Mit Hilfe vo...

Porins from outer membrane of Gram-negative bacteria have a highly stable structure. Our previous studies on porin from Paracoccus denitrificans showed that the outer membrane protein porin is extremely stable toward heat, pH, and chemical denaturants. The major question we have addressed in this paper is whether the high stability of porin is a co...

The biological activity of the double-ring chaperonin GroEL is regulated by complex allosteric interactions, which include positive intra-ring and negative inter-ring cooperativity. To further characterize inter-ring communication, the nucleotide-induced absorbance changes in the vibrational spectrum of the chaperonin GroEL, of two single-point mut...

The folding of membrane proteins was addressed using outer membrane protein porin from the soil bacterium Paracoccus denitrificans (P. den.). IR spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis were used to probe the effect of mutagenesis on the thermal stability of the protein. Secondary structure anal...

In the past 10 years infrared Spectroscopy has become a valuable tool for the analysis of the primary processes in reaction centers. Because it analyzes vibrational properties of the entire molecule, it can yield information on the protein which is otherwise difficult to obtain. In this chapter, the basic concepts of IR spectroscopy for macromolecu...

During the last decade, infrared spectroscopic techniques have entered the field of photosynthesis and they are meanwhile established methods for probing chlorophyll or quinone molecules in their binding sites and for the investigation of molecular processes in the protein upon electron transfer or proton transfer. This advancement has mainly becom...

In this work we present the separation of FTIR difference signals induced by electron transfer to/from the redox centers of the cytochrome c oxidase from P. denitrificans and compare electrochemically induced FTIR difference spectra with those induced by CO photolysis. FTIR difference spectra of rebinding of CO to the half reduced (mixed valence) f...

The proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, is the first energy-transducing complex of many respiratory chains. It couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. One FMN and up to nine iron-sulfur (FeS) clusters participate in the redox reaction. So far,...

The proton-pumping NADH:ubiquinone oxidoreductase is the first of the respiratory chain complexes in many bacteria and mitochondria of most eukaryotes. The bacterial complex consists of 14 different subunits. Seven peripheral subunits bear all known redox groups of complex I, namely one FMN and five EPR-detectable iron-sulfur (FeS) clusters. The re...

Changes in the vibrational spectrum of the sarcoplasmic reticulum Ca2+-ATPase upon nucleotide binding were recorded in H2O and 2H2O at −7°C and pH 7.0. The reaction cycle was triggered by the photochemical release of nucleotides (ATP, ADP, and AMP-PNP) from a biologically inactive precursor (caged ATP, P3-1-(2-nitrophenyl) adenosine 5′-triphosphate...

By specific (13)C labeling of the heme propionates, four bands in the reduced-minus-oxidized FTIR difference spectrum of cytochrome c oxidase from Paracoccus denitrificans have been assigned to the heme propionates [Behr, J., Hellwig, P., Mäntele, W., and Michel, H. (1998) Biochemistry 37, 7400-7406]. To attribute these signals to the individual pr...

In bacterial reaction centers the charge separation process across the photosynthetic membrane is predominantly driven by the excited state of the bacteriochlorophyll dimer (D). An X-ray structure analysis of the Phe M197-->Tyr mutant reaction center from Rhodobacter sphaeroides at 2.7 A resolution suggests the formation of a hydrogen bond as postu...

In this study we present the infrared spectroscopic characterization of the bound ubiquinone in cytochrome bo(3) from Escherichia coli, Electrochemically induced Fourier transform infrared (FTIR) difference spectra of Delta UbiA (an oxidase devoid of bound ubiquinone) and Delta UbiA reconstituted with ubiquinone 2 and with isotopically labeled ubiq...

The cytochrome bc(1) complex from Rhodobacter capsulatus was investigated by protein electrochemistry and visible/IR spectroscopy. Infrared difference spectra, which represent redox-induced conformational changes of cofactors and their protein environments, show signals of the hemes, the quinone Q(i), and small conformational changes of the protein...

The conformational changes associated with the redox transition of plastocyanin (PC) were investigated by absorption and reaction-induced infrared spectroscopy. In addition to spectral features readily ascribed to beta and turn protein secondary structures, the amide I band shows a major component band at 1647 cm(-1) in both redox states of the pro...

The ba3 cytochrome c oxidase from Thermus thermophilus has been studied with a combined electrochemical, UV/VIS, and FTIR spectroscopic approach. Oxidative electrochemical redox titrations yielded midpoint potentials of Em1= -0.02 +/- 0.01 V and Em2 = 0.16 +/- 0.04 V for heme b and Em1 = 0.13 +/- 0.04 V and Em2 = 0.22 +/- 0.03 V for heme a(3) (vs A...

The rebinding of CO to cytochrome c oxidase from Paracoccus denitrificans in the fully reduced and in the half-reduced (mixed valence) form as a function of temperature was investigated using time-resolved rapid-scan FT-IR spectroscopy in the mid-IR (1200-2100 cm-1). For the fully reduced enzyme, rebinding was complete in approximately 2 s at 268 K...

Changes in the vibrational spectrum of the chaperonin GroEL in the presence of ADP and ATP have been followed as a function of time using rapid scan Fourier transform infrared spectroscopy. The interaction of nucleotides with GroEL was triggered by the photochemical release of the ligands from their corresponding biologically inactive precursors (c...

Cytochrome c oxidase from Paracoccus denitrificans was studied with a combined electrochemical and ultraviolet/visible/infrared (UV/vis/IR) spectroscopic approach. Global fit analysis of oxidative electrochemical redox titrations was used to separate the spectral contributions coupled to heme a and a3 redox transitions, respectively. Simultaneous a...

In order to study the role of subunits III and IV of the cytochrome c oxidase from P. denitrificans for electron and proton transfer, electrochemically induced FT-IR difference spectra of the two- and of the four-subunit enzyme have been compared. These spectra reflect the alterations in the protein upon electron and proton transfer. Since the spec...

Cytochrome oxidase catalyses the stepwise reduction of oxygen to water and efficiently couples e-/H+-transfer. Electrochemical IR and UV/vis spectroscopic properties of the different heme centers in the cytochrome oxidases from P. denitriflcans, T. thermophilus, E. coli and bovine heart have been studied by combination of protein electrochemistry a...

Time-resolved infrared difference spectra of the ATP-induced phosphorylation of the sarcoplasmic reticulum Ca2+-ATPase have been recorded in H2O and 2H2O at pH 7.0 and 1°C. The reaction was induced by ATP release from P3-1-(2-nitro)phenylethyladenosine 5′-triphosphate (caged ATP) and from [γ-18O3]caged ATP. A band at 1546 cm−1, not observed with th...

The molecular processes concomitant with the redox reactions of wild-type and mutant cytochrome c oxidase from Paracoccus denitrificans were analyzed by a combination of protein electrochemistry and Fourier transform infrared (FTIR) difference spectroscopy. Oxidized-minus-reduced FTIR difference spectra in the mid-infrared (4000-1000 cm-1) reflecti...

Specific isotope labeling at the carboxyl groups of the four heme propionates of cytochrome c oxidase from Paracoccus denitrificans was used in order to assign signals observed in electrochemically induced redox Fourier transform infrared (FTIR) difference spectra of this enzyme. For this purpose, the hemA gene of the P. denitrificans strain PD1222...

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Structural changes of the sarcoplasmic reticulum Ca2+-ATPase occurring in the reaction step involving phosphoenzyme conversion and Ca2+ release (Ca2E1-P --> E2-P) were followed using time-resolved infrared spectroscopy in H2O and 2H2O. The difference spectra measured between 1800 and 1500 cm-1 were almost identical to those of Ca2+ release from the...

Rapid scan Fourier transform infrared (FTIR) spectroscopy and time-resolved single wavelength infrared (IR) spectroscopy have been applied to study the mechanism of photochemical release of adenosine 5‘-triphosphate (ATP) from its P3-[1-(2-nitrophenyl)ethyl] ester (caged ATP). Bands arising from phosphate and non-phosphate vibrations characteristic...

In order to achieve fast, quantitative and reversible electrochemistry for UV-vis-IR- spectroscopic studies of redox proteins in electrochemical cells[1], surface modification of the electrode is essential to prevent denaturation of the protein on bare metallic surfaces. However, the electrochemistry of proteins is still limited and the application...

Cytochrome c oxidase, the terminal complex of the respiratory chain of aerobic organisms, catalyses the stepwise reduction of oxygen to water. The redox reactions of the enzyme are coupled with proton translocation across the membrane. This process was investigated by combination of protein electrochemistry and FTIR difference spectroscopy in a thi...

The cytochrome c oxidase is the terminal enzyme in the respiratory chain of eucaryotic plants and animals. Its binuclear heme-copper center is capable to bind oxygen and reduce it to water 1. The enzyme can be poisened with IR-active ligands like CO and CN−. Photoexcitation of the heme knocks out the ligand and the rebinding after photolysis can be...

We have studied changes in the vibrational spectrum of Sarcoplasmic reticulum Ca2+-ATPase upon nucleotide binding, using ATP and the non-hydrolyzable ATP-analogue AMP-PNP (Adenylyl-imido-diphosphate) that stops the reaction cycle at the nucleotide binding state.

Electron transport in Photosystem I leads to characteristic changes in the vibrational spectra of P700 and its surrounding protein [1, 2]. Redox triggering by the use of electrochemistry allows to adjust defined redox states of PS I. In combination with vis/IR spectroscopy, information about the electronic and molecular changes of one cofactor and...

The redox reactions of P700, the primary electron donor of photosystem I (PS I), have been analyzed by electrochemistry and Fourier transform infrared (FTIR) spectroscopy in two different preparations from the cyanobacterium Synechocystis PCC 6803 yielding monomeric and trimeric PS I complexes, respectively. Reversible and quantitative oxidation an...

In order to investigate the changes of protonation or environment of carboxylic residues occurring upon photoreduction of the secondary quinone acceptor (QB) in the reaction center (RC) of the photosynthetic bacteria Rhodobacter sphaeroides 2.4.1., we have performed light-induced Fourier transform infrared (FTIR) spectroscopy on RCs from wild-type...

Rapid scan Fourier transform infrared (FTIR) spectroscopy and time-resolved single wavelength infrared (IR) spectroscopy have been used to follow the photochemical release of adenosine 5'-triphosphate (ATP) from P-3-(1-(2-nitrophenyl)ethyl) adenosine 5'-triphosphate (caged ATP). Vibrational difference spectra for the formation first of the aci-nitr...

A series of transmetalated bacteriochlorophyll a, [M] BCHla (M = Mn, Zn, Co, Ni, Cu, Pd), and the corresponding 13²-hydroxy derivatives, [M]-OH-BChla, were investigated by low-temperature cyclic voltammetry and by spectroelectrochemistry in the vis/near-IR range. This is the first systematical investigation of bacteriochlorin macrocycles with elec...

The monoanions (the terms monoanions (anions) and monocations (cations) used in this paper refer to Ï-monoanion radical and Ï-monocation radical, respectively) and monocations of transmetalated bacteriochlorophyll a [M]-BChla (M = Mn, Zn, Cd, Co, Ni, Cu, Pd) and the corresponding 13²-hydroxy derivatives [M]-OH-BChla were investigated by a combinat...

Time-resolved pump-and-probe experiments of reaction centers of the purple bacterium Rhodobacter sphaeroides (R26) in the mid-IR region between 1000 and 1800 cm-1 are recorded with a time resolution of 300-400 fs. The difference spectra of the states P*, P+HA-, and P+QA- with respect to the ground state P predominantly reflect changes of the specia...

The protonation events that occur upon QA-QB-->QAQB- electron transfer in photosynthetic reaction centers from Rhodobacter sphaeroides were investigated by time-resolved infrared spectroscopy using tunable diode lasers as previously described [Mäntele, W., Hienerwadel, R., Lenz, F., Riedel, E. J., Grisar, R., & Tacke, M. (1990) Spectrosc. Int. 2, 2...

Infrared absorbance changes of the sarcoplasmic reticulum (SR) Ca2+-ATPase arising from 3 partial reactions of its Ca2+-pumping cycle were triggered by the photochemical release of ATP from caged ATP (P 3-1-(2-nitro)phenylethyladenosine 5′-triphosphate) and were followed in real time using rapid scan FTIR spectroscopy. Each reaction was investigate...

Halocyanin is a recently discovered archaebacterial copper protein classified as "type I" small blue copper protein (Scharf, B., Ph.D. Thesis, University of Bochum, Germany). Its redox properties were investigated by a combination of protein electrochemical and spectroscopic techniques. Using electrochemical reactions in an ultrathin-layer electroc...

Electron transfer in photosynthetic reaction centers from Rhodobacter sphaeroides was investigated by time-resolved infrared spectroscopy using tunable lead salt diode lasers. In the spectral region between approximately 1690 and 1770 cm−1, transient µsec to msec signals could be characterized upon electron transfer from the primary quinone accepto...

Infrared difference spectroscopy was used to get more insight into the transport mechanism of sarcoplasmic reticulum Ca2+-ATPase. Several partial reactions were induced directly in the infrared cuvette via photolytic release of ligands or substrates from photolabile precursors (1,2). From the spectra before and after the release a difference spectr...

The conformational change coupled to the redox processes of two tetraheme cytochromes c3 from bacteria of the genus Desulfovibrio have been studied by UV-vis and FTIR difference spectroscopy combined with protein electrochemistry. Two pairs of equivalent hemes were found in Desulfovibrio desulfuricans Norway 4 cytochrome c3 by UV-vis spectroelectro...

Infrared spectroscopic methods have been developed in the past decade to a sensitivity and selectivity which renders them useful for the study of enzyme function and enzyme reaction mechanisms. Originally developed as difference techniques for the investigation of light-induced reactions of photoreactive proteins, and matured in the field of bacter...

Protein electrochemistry in an ultra-thin-layer electrochemical cell suitable for UV/vis and IR spectroscopy has been used to characterize the vibrational modes of the primary electron donors of Rhodobacter sphaeroides and Rhodopseudomonas viridis reaction centers in their neutral and cation radical states (P and P+, respectively). The P-->P+ redox...

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IR difference spectra between the oxidized and the reduced state of horse heart cytochrome c were obtained for different temperature and pH conditions at various surface-modified electrodes using an optically transparent thin-layer electrochemical cell. These difference spectra reflect changes in protein conformation, side-chain geometries, and pro...

PS I reaction center is a membrane-bound pigment-protein complex in the thylakoid membranes of higher plants, green algae and cyanobacteria. The photoactive components include the primary electron donor, P700, a primary electron acceptor Ao, an intermediate quinone acceptor A1 and three iron-sulphur centers Fx, FA and FB. Absorption of light brings...

Biochemical redox reactions are typically studied by either chemical or electrochemical [1,2] titrations of redox-sensitive electronic or vibrational [3] transitions.

The nicotinic acetylcholine receptor (n-AChR) is a ligand gated ion channel (type-1 receptor) transducing extracellular signals through plasma membranes (1–3). Because of the lack of appropriate crystals, the three dimensional structure of the AChR as determined by X-ray chrystallography is not yet known. Secondary structure prediction, based on hy...

Light induced charge separation in the reaction center (RC) of Rb. sphaeroides leads to the formation of the oxidized primary donor P+ and the semiquinone anion QA‒ on a picosecond time scale. Electron transfer to the secondary acceptor QB, if present, proceeds in ≈100 µs. Charge recombination between P+ and QB‒ occurs in 1 to 5 s. The electron tra...

Infrared spectroscopy has proven to be a valuable tool for the study of enzyme structure and function. Using differential techniques, molecular changes in the course of photoreactions and redox reactions have been studied (for a review see [1]). Recently, the use of photolabile substrate analogues, which can be released by a UV flash, has been repo...

Ca2+ transport from the cytoplasm of muscle cells into SR, necessary for muscle relaxation, is performed by the Ca2+-ATPase, an intrinsic membrane protein of about 110 kDa molecular mass. The energy required for this active transport is provided by hydrolysis of ATP. Although the catalytic cycle of the ATPase has been subject of detailed investigat...

The photosynthetic reaction center (RC) of Cf. aurantiacus contains a multiheme cytochrome subunit which is responsible for the rereduction of the photooxidized primary electron donor. In contrast to the hemes of the cytochrome subunit of Rps.vihdis, all hemes exhibit the same α-band absorption at 554 nm, thus rendering an individual titration of t...

Reaction-induced infrared difference spectroscopy has long been used to study the molecular basis of light-induced reactions in pigment-protein complexes. The techniques used are either light-minus-dark Fourier transform infrared (FTIR) spectroscopy for steady states or flash-induced time-resolved IR and FTIR techniques for transient reactions, wit...

Ca2+ transport from the cytoplasm of muscle cells into SR, necessary for muscle relaxation, is performed by the Ca2+- ATPase, an intrinsic membrane protein of about 110 kDa molecular mass. The energy required for this active transport is provided by hydrolysis of ATP. Although the catalytic cycle of the ATPase has been subject of detailed investiga...

This chapter discusses a biosensor for triazine herbicides based on chlorophyll fluorescence in photosystem 2. Several commercially available herbicides have been found to be significant pollutants of ground and drinking water. Existing and planned legislation aimed at controlling this type of pollution leads to a requirement for convenient and sen...

The vibrational infrared absorption changes associated with the oxidation of cytochrome b559 (Cyt b559) have been characterized. In photosystem II (PS II) enriched membranes, low-potential (LP) and high-potential (HP) Cyt b559 were investigated by light-induced FTIR difference spectroscopy. The redox transition of isolated Cyt b559 is characterized...