FT-IR study of pyridoxamine 5' phosphate

Aqueous solutions of pyridoxamine 5' phosphate (PMP) at several pH conditions have been studied using FT-IR spectroscopy using the attenuated total reflection (ATR) technique. In spite of the strong intense OH stretching and bending bands of water, most of the vibrational structure of solute can be observed from 900 to 1500 cm(-1). With increasing pH, very intense changes in the spectra have been observed due to concentration changes of the hydrogen bonded species. Spectra of the different ionic species have been calculated from the mathematical fitting of experimental absorption spectra as a function of pH. Spectra are characterized by the presence of broad band-like structures in the 2400-3500 cm(-1) region, with extended continua that indicate very large proton polarizability of hydrogen bonds. Contributions of the phosphate group to the total absorption have been analyzed by comparison with pyridoxamine spectra.     

Methods to probe protein transitions with ATR infrared spectroscopy

We describe techniques that can be used in conjunction with modern attenuated total reflection (ATR) infrared micro-prisms to allow proteins to be manipulated cyclically between different states whilst simultaneously monitoring both mid-IR and UV/visible/near IR changes. These methods provide increased flexibility of the types of changes that can be induced in proteins in comparison to transmission methods. Quantitative measurements can be made of vibrational changes associated with conversion between stable catalytic reaction intermediates, ligand binding and oxidation-reduction. Both hydrophobic and soluble proteins can be analysed and the ability to induce transitions repetitively allows IR difference spectra to be acquired at a signal/noise sufficient to resolve changes due to specific cofactors or amino acids. Such spectra can often be interpreted at the atomic level by standard IR methods of comparisons with model compounds, by isotope and mutation effects and, increasingly, by ab initio simulations. Combination of such analyses with atomic 3D structural models derived from X-ray and NMR studies can lead to a deeper understanding of molecular mechanisms of enzymatic reactions.     

Infrared evidence of azide binding to iron, copper, and non-metal sites in heart cytochrome c oxidase.

Interactions of azide ion with bovine heart cytochrome c oxidase (CcO) at five redox levels (IV) to (0), obtained by zero to four electron reduction of fully oxidized enzyme CcO(IV), were monitored by infrared and visible/Soret spectra. Partially reduced CcO gave three azide asymmetric stretch band at 2040, 2016, and 2004 cm-1 for CcO(III)N3 and two at 2040 and 2016 cm-1 for CcO(II)N3 and CcO(I)N3. Resting CcO(IV) reacts with N3- to give one band at 2041 cm-1 assigned to CuB2+N3 and another at 2051 cm-1 to N3- that is associated with protein but is not bound to a metal ion. At high azide concentrations the weak association of many azide molecules with non-metal protein sites was observed at all redox levels. These findings provide direct evidence for 1) N3- binding to CuB as well as Fea3 in partially reduced enzyme, but no binding to Fea3 in fully oxidized enzyme and no binding to either metal in fully reduced enzyme; 2) a long range effect of the oxidation state of Fea or CuA on ligand binding at heme a3, but not at CuB; and 3) an insensitivity of either Fea3 or CuB ligand site to changes in ligand or oxidation state at the other site. The observed independence of the Fea3 and CuB sites provides further support for Fea3(3)+ OOH, rather than Fea3(3)+ OOCuB2+, as an intermediate in the reduction of O2 to water by the oxidase.     

A technique for monitoring mammalian cell growth and inhibition in situ via Fourier transform infrared spectroscopy

A single culture of Chinese hamster ovary cells was grown on germanium attenuated total reflectance (ATR) crystals and continuously monitored in situ via ATR/Fourier transform infrared (FT-IR) spectroscopy for approximately 60 h. The cells were seeded into a specially designed flow cell which controlled physiological conditions, flow rate, and addition of growth medium or metabolic inhibitors. Infrared spectra were taken at 20-min intervals until a confluent monolayer was formed. Several strong bands are evident in the spectra which can be generally ascribed to molecular features of cellular components. Cell growth kinetics were measured as a function of infrared band intensity over time and exhibited the normal lag phase, logarithmic growth, and stationary phase on reaching confluence. Spectra of growing cells, normalized to the area under the spectral region 1800-1000 cm-1, were subtracted from reference spectra of confluent cells at 60 h. Difference spectra showed that the largest differences were observed between confluent cells and cells in early growth stages. Differences may reflect cell morphological changes, biochemical activity, and degree of ATR crystal exposure to the bulk medium. ATR/FT-IR spectroscopy of living Chinese hamster ovary cells was also used in a toxicological study to monitor the effects of hydroxyurea, an inhibitor of DNA synthesis. Delayed growth was observed in the cell growth curve of the hydroxyurea-treated cells during the course of treatment as compared to the control culture.     

A phospholipid bilayer supported under a polymerized Langmuir film

Phospholipid single bilayers supported on a hydrophilic solid substrate are extensively used in the study of the interaction between model membranes and proteins or polypeptides. In this article, the formation of a single dimyristoylphosphatidylcholine (DMPC) bilayer under an octadecyltrimethoxysilane (OTMS) polymerized Langmuir monolayer at the air-water interface is followed by Brewster angle microscopy (BAM) and polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). The formation of the bilayer is initiated by injection of dimyristoylphosphatidylcholine small unilamellar vesicles into the aqueous subphase. Brewster angle microscopy allows visualization of the kinetics of formation and the homogeneity of the bilayer. Spectral simulations of the polarization-modulated infrared reflection absorption spectroscopy spectra reveal that the bilayer thickness is 39 +/- 5 A. This system constitutes the first example of a phospholipid bilayer on a "nanoscopic" support and opens the way to studies involving supported bilayers using powerful experimental techniques such as x-ray reflectivity, vibrational spectroscopies, or Brewster angle microscopy.     

ATR-FTIR difference spectroscopy of the P(M) intermediate of bovine cytochrome c oxidase

Perfusion-induced attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used to investigate changes induced in protein and cofactors of bovine cytochrome c oxidase when it was converted from the oxidised state to the catalytic P(M) intermediate. The transition was induced in a film of detergent-depleted 'fast' oxidase with a buffer containing CO and O(2). The extent of formation of the P(M) state was quantitated simultaneously by monitoring formation of its characteristic 607-nm band with a scanned visible beam reflected off the top surface of the prism. The P(M) minus O FTIR difference spectrum is distinctly different from the redox spectra reported to date and includes features that can be assigned to changes of haem a(3) and surrounding protein. Tentative assignments are made based on vibrational data of related proteins and model compounds.     

Attenuated total reflection Fourier transform infrared studies of redox changes in bovine cytochrome c oxidase: resolution of the redox Fourier transform infrared difference spectrum of heme a(3).

Attenuated total reflection Fourier transform infrared (ATR-FTIR) difference spectroscopy has been performed on samples of bovine cytochrome c oxidase that have been deposited as a thin film on the surface of a silicon microprism. The technique has several advantages over transmission methods in terms of amount of material required, the time required to reach sufficient optical stability, and the range of reactants that can be repetitively added and removed. The ATR-FTIR method has been used to record redox difference spectra of cytochrome c oxidase in the unligated and cyanide-ligated states. By subtraction of the spectra, the redox FTIR difference spectrum of heme a(3) can be resolved from those of the other metal centers. This difference spectrum is compared with available vibrational and Raman data on homologous oxidases and on heme A model compounds.     

Transient Binding of CO to CuB in Cytochrome c Oxidase Is Dynamically Linked to Structural Changes around a Carboxyl Group: A Time-Resolved Step-Scan Fourier Transform Infrared Investigation

The redox-driven proton pump cytochrome c oxidase is that enzymatic machinery of the respiratory chain that transfers electrons from cytochrome c to molecular oxygen and thereby splits molecular oxygen to form water. To investigate the reaction mechanism of cytochrome c oxidase on the single vibrational level, we used time-resolved step-scan Fourier transform infrared spectroscopy and studied the dynamics of the reduced enzyme after photodissociation of bound carbon monoxide across the midinfrared range (2300-950 cm⁻¹). Difference spectra of the bovine complex were obtained at -20°C with 5 μs time resolution. The data demonstrate a dynamic link between the transient binding of CO to Cu_B and changes in hydrogen bonding at the functionally important residue E(I-286). Variation of the pH revealed that the pK_a of E(I-286) is >9.3 in the fully reduced CO-bound oxidase. Difference spectra of cytochrome c oxidase from beef heart are compared with those of the oxidase isolated from Rhodobacter sphaeroides. The bacterial enzyme does not show the environmental change in the vicinity of E(I-286) upon CO dissociation. The characteristic band shape appears, however, in redox-induced difference spectra of the bacterial enzyme but is absent in redox-induced difference spectra of mammalian enzyme. In conclusion, it is demonstrated that the dynamics of a large protein complex such as cytochrome c oxidase can be resolved on the single vibrational level with microsecond Fourier transform infrared spectroscopy. The applied methodology provides the basis for future investigations of the physiological reaction steps of this important enzyme.     

On the production of glycogen by Pseudomonas fluorescens during biofilm development: an in situ study by attenuated total reflection-infrared with chemometrics

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used to monitor Pseudomonas fluorescens biofilms in situ, non-destructively, in real time, and under fully hydrated conditions. Changes accompanying the metabolic evolution of the sessile bacterial cells from the nascent biofilm monolayer to the beginning of the multi-layered structure in the presence of nutrients were identified via the ATR-FTIR fingerprints of the young biofilm on the ATR crystal. The ATR-FTIR spectra were analysed by classical methods (time evolution of integrated intensities and profile evolution of specific bands), and also by a multivariate curve resolution, Bayesian positive source separation, to extract the pure component spectra and their change of concentration over time occurring during biofilm settlement. This work showed clearly the overproduction of glycogen by sessile P. fluorescens, which had not previously been described by other research groups.     

Membrane molecule reorientation in an electric field recorded by attenuated total reflection Fourier-transform infrared spectroscopy

Electric fields play an important role in the physiological function of macromolecules. Much is known about the role that electric fields play in biological systems, but membrane molecule structure and orientation induced by electric fields remain essentially unknown. In this paper, we present a polarized attenuated total reflection (ATR) experiment we designed to study the effect of electric fields on membrane molecule structure and orientation by Fourier-transform infrared (FTIR) spectroscopy. Two germanium crystals used as the internal reflection element for ATR-FTIR experiments were coated with a thin layer of polystyrene as insulator and used as electrodes to apply an electric field on an oriented stack of membranes made of dioleylphosphatidylcholine (DOPC) and melittin. This experimental set up allowed us for the first time to show fully reversible orientational changes in the lipid headgroups specifically induced by the electric potential difference.     

Complexation of U(VI) with highly phosphorylated protein, phosvitin: A vibrational spectroscopic approach

The complexation of uranium(VI) to variant functional groups of the highly phosphorylated protein phosvitin in aqueous solution was investigated by attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy. For the verification of the affinity of the actinyl ions to carboxyl and phosphate groups of the amino acid side chains, samples with different phosphate to uranium(VI) (P/U) ratios were investigated under denaturing conditions as well as in aqueous medium. From a comparative study with other heavy metal ions, i.e. Ba²⁺ and Pb²⁺, a strong coordination of U(VI) to carboxyl and phosphoryl groups can be derived. Furthermore, with increasing P/U ratios, a preferential binding of U(VI) to phosphoryl groups is indicated by the spectra of the batch samples. These findings are confirmed by spectra of aqueous U(VI)-phosvitin complexes reflecting an explicit coordination of the uranyl ions to phosphate groups at a high P/U ratio. Our study provides a deeper insight into the molecular interactions between actinyl ions and protein, and can be conferred to other basic biomolecules such as polysaccharides and nucleic acids.     

Conformational changes in bacteriorhodopsin studied by infrared attenuated total reflection.

We report on a new method based on Fourier transform infrared (FTIR)-difference spectroscopy for studying the conformational changes occurring during the photocycle of bacteriorhodopsin. Previous studies have been made by measuring the absorbance of an infrared (IR) beam transmitted through a thin hydrated purple membrane film. In contrast, the present study utilizes the technique of attenuated total reflection (ATR). Purple membrane is fixed on the surface of a germanium internal reflection crystal and immersed in a buffer whose pH and ionic composition can be varied. Measurements of the amide I and II absorbance with light polarized parallel and at 45 degrees to the crystal surface reveals that the membrane is highly oriented. An ATR-FTIR-difference spectrum of the light to dark (bR570 to bR548) transition is similar but not identical to the transmittance FTIR-difference spectrum. This disagreement between the two methods is shown to be due in the ATR case to the absorption of transition moments oriented predominantly out of the membrane plane. Raising the pH of La3+ substituted purple membrane films from 6.8 to 8.0 slows the M-decay rate sufficiently so that a bR570 to M412 difference spectrum can be obtained with steady state illumination at room temperature. A comparison of this difference spectrum with that obtained at -23 degrees C using the transmittance method reveals several changes that cannot be attributed to out-of-plane transition moments.(ABSTRACT TRUNCATED AT 250 WORDS)     

In situ monitoring of the catalytic activity of cytochrome C oxidase in a biomimetic architecture

Cytochrome c oxidase (CcO) from Paracoccus denitrificans was immobilized in a strict orientation via a his-tag attached to subunit I on a gold film and reconstituted in situ into a protein-tethered bilayer lipid membrane. In this orientation, the cytochrome c (cyt c) binding site is directed away from the electrode pointing to the outer side of the protein-tethered bilayer lipid membrane architecture. The CcO can thus be activated by cyt c under aerobic conditions. Catalytic activity was monitored by impedance spectroscopy, as well as cyclic voltammetry. Cathodic and anodic currents of the CcO with cyt c added to the bulk solution were shown to increase under aerobic compared to anaerobic conditions. Catalytic activity was considered in terms of repeated electrochemical oxidation/reduction of the CcO/cyt c complex in the presence of oxygen. The communication of cyt c bound to the CcO with the electrode is discussed in terms of a hopping mechanism through the redox sites of the enzyme. Simulations supporting this hypothesis are included.     

Critical self-association of bile lipids studied by infrared spectroscopy and viscometry

Fourier transform infrared (FTIR)-attenuated total reflection (ATR) spectroscopy and viscometry were applied to study the micellization of two bile lipids, sodium taurochenodeoxycholate (NaTCDC) and sodium glycocholate (NaGC), in aqueous solutions. The CH2 stretching bands of the bile lipid hydrocarbon region were shifted to higher frequencies suggesting initial critical micellization at 2.5 mM for NaTCDC and 9 mM for NaGC. An abrupt enhancement of the absorption intensity of the CH3 groups of the sterol rings in bile lipids were under conformational strain at 3.5 mM NaTCDC and 9 mM NaGC. Viscometry measurements showed abrupt changes in viscosities in the region of critical micellar concentration (CMC) of both bile lipids. Both infrared and viscometry studies confirmed the onset of conformational strains in tightly packed lipid micelles at their CMC. In addition, FTIR/ATR spectroscopy has defined the specific hydrophobic interactions which bring about critical micellization of bile lipids.     

Two-dimensional infrared population transfer spectroscopy for enhancing structural markers of proteins

We propose the possibility of using vibrational population transfer to enhance the structural markers for protein motifs that occur in two-dimensional infrared spectroscopy. We demonstrate the potential of this method by calculating the spectrum of the trpzip2 β-hairpin peptide, a system that is small enough to allow accurate simulation of its two-dimensional infrared spectra, including vibrational population transfer induced by a fluctuating solvent. The results show that under selected experimental conditions, in particular by using perpendicular polarization and finite waiting times, the cross peaks that constitute the well-known Z-shape marker for β-sheet structure in two-dimensional spectra are strongly enhanced. This enhancement is shown to result from vibrational population transfer. It should be possible to use the same technique for enhancing cross peaks in other structures and generally improve structure determination by two-dimensional infrared spectroscopy. The simulated population transfer times are in good agreement with those observed in experiments on typical proteins.     

Probing conformational changes in the nicotinic acetylcholine receptor by Fourier transform infrared difference spectroscopy.

We have developed a Fourier transform infrared (FTIR) difference method for probing conformational changes that occur upon the binding of ligands to the nicotinic acetylcholine receptor (nAChR). Our approach is to deposit reconstituted nAChR membranes in a thin film on the surface of a germanium internal reflection element, acquire FTIR spectra in the presence of bulk aqueous solution using attenuated total reflection, and then trigger conformational changes by sequentially flowing a buffer either with or without an agonist past the film surface. Using the fluorescent probe, ethidium bromide, it is demonstrated that the method of nAChR film deposition does not affect the ability of the receptor to undergo the resting-to-desensitized state transition. The difference of FTIR spectra of nAChR films recorded in the presence and absence of agonists reveal highly reproducible infrared bands that are not observed in the difference of spectra recorded with only buffer flowing past the film surface. Some of the bands are assigned to changes in protein secondary structure and to changes in the structure of individual amino acid residues. Bands arising from the vibrations of the agonist bound to the receptor are also observed. The results demonstrate that FTIR difference spectroscopy can detect structural changes in the nAChR that occur upon the binding of ligands. The technique will be an effective method for investigating nAChR structure and function as well as receptor-drug interactions.     

FTIR spectroscopic evidence for the involvement of an acidic residue in quinone binding in cytochrome bd from Escherichia coli

In this work, FTIR difference spectroscopy is used to search for possible binding partners and protonable groups involved in the binding of the quinol to cytochrome bd from Escherichia coli. In addition, the electrochemically induced FTIR difference spectra are compared for preparations of the enzyme isolated from cells grown at different oxygen levels in which the quinone content of the membrane is altered. On this basis, difference signals can be tentatively attributed to the vibrational modes of the different quinones types that are associated with the enzyme depending on growth conditions. Furthermore, vibrational modes due to the redox-dependent reorganization of the protein vary depending on the quinone associated with the isolated enzyme. Of particular interest are the observations that a mode at 1738 cm(-1) is decreased and a mode at 1595 cm(-1) is increased as observed in direct comparison to the data obtained from samples grown anaerobically. These signals indicate a change in the protonation state of an aspartic or glutamic acid. Since these changes are observed when the ubiquinone ratio in the preparation increases, the data provide evidence for the modulation of the binding site by the interacting quinone and the involvement of an acidic group in the binding site. The tentative assignments of the vibrational modes are supported by electrochemically induced FTIR difference spectra of cytochrome bd in the presence of the specific quinone binding site inhibitors heptylhydroxyquinoline-N-oxide (HQNO) or 2-methyl-3-undecylquinolone-4. Whereas HQNO leads to strong shifts in the FTIR redox difference spectrum, 2-methyl-3-undecylquinolone-4 induces a specific shift of a mode at 1635 cm(-1), which likely originates from the displacement of the C=O group of the bound quinone.     

2D-SEIRA spectroscopy to highlight conformational changes of the cytochrome c oxidase induced by direct electron transfer

Potentiometric titrations of the cytochrome c oxidase (CcO) immobilized in a biomimetic membrane system were followed by two-dimensional surface-enhanced IR absorption spectroscopy (2D SEIRAS) in the ATR-mode. Direct electron transfer was employed to vary the redox state of the enzyme. The CcO was shown to undergo a conformational transition from a non-activated to an activated state after it was allowed to turnover in the presence of oxygen. Differences between the non-activated and activated state were revealed by 2D SEIRA spectra recorded as a function of potential. The activated state was characterized by a higher number of correlated transitions as well as a higher number of amino acids associated with electron transfer.     

Redox-induced transitions in bovine cytochrome bc1 complex studied by perfusion-induced ATR-FTIR spectroscopy

Redox transitions in a film of detergent-purified bovine cytochrome bc(1) complex were investigated by perfusion-induced attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. The technique provides a flexible method for generating redox-induced IR changes of components of bovine cytochrome bc(1) complex at a high signal:noise ratio. These IR redox difference spectra arise from perturbations of prosthetic groups and surrounding protein. Visible difference spectra were recorded synchronously using a light beam reflected from the exposed prism surface and provided a quantitative means of determining the redox transitions that were occurring. IR and visible redox difference spectra of iron-sulfur protein/cytochrome c(1), heme b(H), and heme b(L) were separated by selective reduction and/or oxidation that extends published data on the homologous bacterial enzyme. Several bands could be tentatively assigned to redox-sensitive modes of hemes and ubiquinone and changes in the surrounding protein by comparison with available data for bacterial bc(1) complex, other related heme proteins, and model compounds. Some tentative assignments of further signals to specific amino acids are made on the basis of known crystal structures.