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Bimolecular processes involving exciton spin-state interactions gain attention for their deployment as wavelength-shifting tools. Particularly triplet–triplet annihilation induced photon energy up-conversion (TTA-UC) holds promise to enhance the performance of solar cell and photodetection technologies. Despite the progress noted, a correlation between the solid-state microstructure of photoactuating TTA-UC organic composites and their photophysical properties is missing. This lack of knowledge impedes the effective integration of functional TTA-UC interlayers as ancillary components in operating devices. We here investigate a solution-processed model green-to-blue TTA-UC binary composite. Solid-state films of a 9,10 diphenyl anthracene (DPA) blue-emitting activator blended with a (2,3,7,8,12,13,17,18-octaethyl-porphyrinato) PtII (PtOEP) green-absorbing sensitizer are prepared with a range of compositions and examined by a set of complementary characterization techniques. Grazing incidence X-ray diffractometry (GIXRD) measurements identify three PtOEP composition regions wherein the DPA:PtOEP composite microstructure varies due to changes in the packing motifs of the DPA and PtOEP phases. In Region 1 (≤2 wt%) DPA is semicrystalline and PtOEP is amorphous, in Region 2 (between 2 and 10 wt%) both DPA and PtOEP phases are amorphous, and in Region 3 (≥10 wt%) DPA remains amorphous and PtOEP is semicrystalline. GIXRD further reveals the metastable DPA-β polymorph species as the dominant DPA phase in Region 1. Composition dependent UV-vis and FT-IR measurements identify physical PtOEP dimers, irrespective of the structural order in the PtOEP phase. Time-gated photoluminescence (PL) spectroscopy and scanning electron microscopy imaging confirm the presence of PtOEP aggregates, even after dispersing DPA:PtOEP in amorphous poly(styrene). When arrested in Regions 1 and 2, DPA:PtOEP exhibits delayed PtOEP fluorescence at 580 nm that follows a power-law decay on the ns time scale. The origin of PtOEP delayed fluorescence is unraveled by temperature- and fluence-dependent PL experiments. Triplet PtOEP excitations undergo dispersive diffusion and enable TTA reactions that activate the first singlet-excited (S1) PtOEP state. The effect is reproduced when PtOEP is mixed with a poly(fluorene-2-octyl) (PFO) derivative. Transient absorption measurements on PFO:PtOEP films find that selective PtOEP photoexcitation activates the S1 of PFO within ∼100 fs through an up-converted 3(d, d*) PtII-centered state.

Marine diatoms contribute to oxygenic photosynthesis and carbon fixation and handle large changes under variable light intensity on a regular basis. The unique light-harvesting apparatus of diatoms are the fucoxanthin-chlorophyll a/c-binding proteins (FCPs). Here, we show the enhancement of chlorophyll a/c (Chl a/c), fucoxanthin (Fx), and diadinoxanthin (Dd) marker bands in the Raman spectra of the centric diatom T. pseudonana, which allows distinction of the pigment content in the cells grown under low- (LL) and high-light (HL) intensity at room temperature. Reversible LL-HL dependent conformations of Chl c, characteristic of two conformations of the porphyrin macrocycle, and the presence of five- and six-coordinated Chl a/c with weak axial ligands are observed in the Raman data. Under HL the energy transfer from Chl c to Chl a is reduced and that from the red-shifted Fxs is minimal. Therefore, Chl c and the blue-shifted Fxs are the only contributors to the energy transfer pathways under HL and the blue- to red-shifted Fxs energy transfer pathway characteristic of the LL is inactive. The results indicate that T. pseudonana can redirect its function from light harvesting to energy-quenching state, and reversibly to light-harvesting upon subsequent illumination to LL by reproducing the red-shifted Fxs and decrease the number of Dds. The LL to HL reversible transitions are accompanied by structural modifications of Chl a/c and the lack of the red-shifted Fxs.

The combined application of the structure sensitive techniques Fourier transform infrared μ-spectroscopy and Raman microscopy in conjunction with different approaches of laser-induced breakdown spectroscopy (LIBS) including the two-color double pulse (DP-LIBS) have been applied towards the characterization of whole ore copper-sulfide minerals. Discrete information from the surface of the whole ore minerals that lead to the establishment of infrared marker bands and from the surface of bioleached samples that allow the monitoring of jarosite and biofilm formation are provided by FTIR mapping experiments. Raman data can provide information related to the type of the mineral and of the secondary minerals formed on the surface of the ore. Of the four different LIBS approaches applied towards the characterization of the composition of the whole ore minerals, the DP-LIBS shows the highest sensitivity with increasing signals for both the Fe and Cu metals in the whole ore samples.

Cytochrome c oxidase (CcO) couples the oxidation of cytochrome c to the reduction of molecular oxygen to water and links these electron transfers to proton translocation. The redox-driven CcO conserves part of the released free energy generating a proton motive force that leads to the synthesis of the main biological energy source ATP. Cytochrome ba3 oxidase is a B-type oxidase from the extremely thermophilic eubacterium Thermus thermophilus with high O2 affinity, expressed under elevated temperatures and limited oxygen supply and possessing discrete structural, ligand binding, and electron transfer properties. The origin and the cause of the peculiar, as compared to other CcOs, thermodynamic and kinetic properties remain unknown. Fourier transform infrared (FTIR) and time-resolved step-scan FTIR (TRS2-FTIR) spectroscopies have been employed to investigate the origin of the binding and electron transfer properties of cytochrome ba3 oxidase in both the fully reduced (FR) and mixed valence (MV) forms. Several independent and not easily separated factors leading to increased thermostability and high O2 affinity have been determined. These include (i) the increased hydrophobicity of the active center, (ii) the existence of a ligand input channel, (iii) the high affinity of CuB for exogenous ligands, (iv) the optimized electron transfer (ET) pathways, (v) the effective proton-input channel and water-exit pathway as well the proton-loading/exit sites, (vi) the specifically engineered protein structure, and (vii) the subtle thermodynamic and kinetic regulation. We correlate the unique ligand binding and electron transfer properties of cytochrome ba3 oxidase with the existence of an adaption mechanism which is necessary for efficient function. These results suggest that a cascade of structural factors have been optimized by evolution, through protein architecture, to ensure the conversion of cytochrome ba3 oxidase into a high O2-affinity enzyme that functions effectively in its extreme native environment. The present results show that ba3-cytochrome c oxidase uses a unique structural pattern of energy conversion that has taken into account all the extreme environmental factors that affect the function of the enzyme and is assembled in such a way that its exclusive functions are secured. Based on the available data of CcOs, we propose possible factors including the rigidity and nonpolar hydrophobic interactions that contribute to the behavior observed in cytochrome ba3 oxidase.

We report the μm-FTIR mapping and Raman microspectroscopic detection of bornite [Cu5FeS4]-, chalcocite [Cu2S]-, and covelitte [CuS]- bacterial interactions by a consortium of microorganisms consisted of Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, Acidithiobacillus caldus, Leptospirillum ferriphilum, Leptospirillum ferroodiazotrophum and Sulfobacillus thermosulfidooxidans. The absorption signals of amide I, K+-jarosite [KFe3(SO4)2(OH)6] and of the produced extracellular polymeric substances (EPS) from the mixed culture as a function of position on the surfaces of the bioleached bornite, chalcocite and covellite demonstrated their heterogeneity within the surface of the minerals. This reveals the high level of biofilm, EPS, and jarosite biosynthesis on the surface of the minerals and might explain why they associate. To our knowledge this is the first combined application of μm-FTIR mapping and Raman microspectroscopy for the bioleaching behaviour of bornite, chalcocite and covellite and the comparison with other bioleached systems such as chalcopyrite [CuFeS2] provides valuable information on the whole bio-hydrometallurgy Cu/Fe/S system. Both techniques provide spectrally rich, label-free, nondestructive visualizations of the bio-hydrometallurgy dynamics of copper sulfide minerals for processing and storage of large spectral data sets which are valuable for evaluation of copper containing minerals.

Raman, fluorescence and FTIR experiments of prestine Sagittula stellata and Sagittula stellata-metal ion complexes grown in light and in dark were performed to probe the photosensitivity response of the cellular components in the marine bacterium. In the presence of Cu(ii) and Zn(ii) the frequency shifts of PO2-, C-O-C and C-O-P vibrations indicate metal binding to nucleic acids, carbohydrates and polysaccharides. We assign the observed bands in the 514.1 nm Raman spectra of the prestine S. Stellata and of the extracted carotenoids to the CC and C-C stretching vibrations. The fluorescence excitation-emission matrix (EEM) of S. stellata in light, dark and in the presence of metal ions are reported and compared with the Raman and FTIR data. The novel ability of S. stellata although heterotrophic, to show light-dependent metal binding ability may be an important feature property that maintains a stable heterotroph-prototroph interaction and a dynamic system.

peer-reviewed A reversible temperature-dependent high-spin to low-spin transition with T1/2¼ 60 C has been observed in the resonance Raman spectra of the equilibrium reduced and photoreduced heme a3 of the thermophilic ba3 heme–copper oxidoreductase. The transition is based on the frequency shifts of the spin-state marker bands n2 (CbCb) and n10 (CaCm) and is attributed to the displacement of the heme iron along the heme normal as a consequence of the Fe–Np repulsion at temperature below 40 C which will increase the ligand field strength forcing the pairing of d electrons into the lower energy orbitals. PUBLISHED peer-reviewed

Heme-copper oxidases, in particular cytochrome ba 3, is capable of reducing NO to N2O and oxidizing CO to CO2, although with a low activity. Despite the very low (20%) overall sequence homology to other structurally known cytochrome c oxidases, the four redox centers of cytochrome ba 3 display high similarity with those of other oxidases. Extensive studies, at ambient temperatures, have shown that the heme a 3-CuB binuclear center of fully reduced (FR) and mixed valence (MV) cytochrome ba 3 is subjected to peculiar thermodynamic and kinetic properties, with significant deviations, relative to the mesophilic aa 3-type oxidases. These properties include an increased hydrophobicity, a high affinity of CuB for exogenous ligands such as CO (K > 104 M-1) and a slow intramolecular ligand transfer to heme a 3 (k = 8 s-1). As a result, cytochrome ba 3 is the only documented oxidase where the binding of CO to CuB is exergonic and accounts for 25-30% of the total enzyme concentration. Furthermore, the proximal and the distal environment of heme a 3 shows a high structural flexibility evidenced from the multiple heme a 3 Fe2+-CO conformers detected in both the FR and MV forms of the enzyme. Time-resolved step-scan FTIR studies showed that after photolysis of CO from heme a 3 2+ the ligand is almost quantitatively transferred to CuB 1+ and the transiently formed CuB 1+-CO complex exhibits an unusual long lifetime of 20 ms (pD 8.50). In addition, the midpoint redox potentials of hemes b and a 3 have been reported in three different studies being inverted, with values of 210/213 and 430/285 mV (versus Normal Hydrogen Electrode (NHE)), respectively. Resonance Raman and FTIR studies of the ligand binding properties of heme-copper oxidases will be presented. References. Discrete Ligand Binding and Electron Transfer Properties of ba 3-Cytochrome c Oxidase from Thermus thermophilus: Evolutionary Adaption to Low Oxygen and ...C Koutsoupakis, T Soulimane, C. Varotsis Accounts of chemical research 52 (5), 1380-139, 2019. The structure of a ferrous heme-nitro species in the binuclear heme a3/CuB center of ba 3-cytochrome c oxidase as determined by resonance Raman spectroscopy Loullis, MR Noor, T Soulimane, E Pinakoulaki Chemical Communications 51 (2), 286-289, 2015.

Maillard reaction products (MRPs) participate in reactions of carbohydrate intermediates with proteins, resulting in the formation of advanced glycation end-products (AGEs). Dietary Maillard reaction products are recognized as potential chemical modifiers of human proteins. We have investigated the reaction of isolated MRPs from an asparagine-glucose model system with hemoglobin (Hb) to elucidate the binding effect of the MRPs in hemoglobin by fluorescence spectrophotometry. The tryptophan-specific fluorescence obtained for glycated hemoglobin exhibited a Stokes effect since the wavelength of the emission peak was shifted to a higher wavelength than that of native Hb. The formation of new fluorescence emission features indicates the formation of modified hemoglobin species. Fluorescence spectroscopic studies provide evidence that the conformational changes in the β-Trp 37 moiety induce motion of the distal His 64 (E7) in the heme binding pocket. This results in the formation of inactive hemichrome forms of hemoglobin which are related to blood disorders.

Raman, fluorescence and FTIR experiments of prestine

In this work we report Raman and FTIR evidence for extracellular electron uptake by

In this work we report Raman and FTIR evidence for extracellular electron uptake by Sulfobacillus thermosulfidooxidans from the solid phase carbon-based π-electron donor surface of graphite sheets. The primary step in the reaction is the intercalation of water on the surface of graphite followed by the formation of EPS and proceeds to form graphite oxide (GO) with a Raman ID/IG = 0.3 ratio which represents the highest defect content in the carbon lattice reported by bio-oxidation process. We propose and discuss a direct extracellular electron transfer mechanism via outer membrane redox proteins for the electron transfer.

The glycation of hemoglobin to form stable Amadori products and advanced glycation end products causes structural perturbation in the protein. We have applied Fourier-transform infrared (FTIR) spectroscopy and a coupled HPLC-ATR-FTIR system toward the characterization of the reaction products (Rt = 5.5–6.5 min) at pH 8 in the T = 25–140°C range of the Glucose/Asparagine model system. The initial reaction species of the Asn-Gluc reaction were characterized by detection of the vibrational marker bands of the CAsn N CGluc moiety of the Schiff base. The UV-vis spectrum of the reaction product of Hb-Schiff base showed the formation of a 423 nm species and the FTIR spectrum modifications in the AmideIvibration. The FTIR band observed at 1611 cm− 1 indicates the formation of beta-sheet structures. Taken together, these observations suggest the formation of a stable six-coordinated low-spin His64-Fe(III)-His93 species, which is known as hemichrome in which the distal His64 (F6) is coordinated to the heme Fe, thereby blocking irreversibly the O2 binding site.

The whole ore chalcopyrite-bacteria interaction and the formation of the extracellular polymeric substances (EPS) during the bioleaching process by microorganisms found in the mine of Hellenic Copper Mines in Cyprus were investigated. Raman and FTIR microspectroscopies have been applied towards establishing a direct method for monitoring the formation of secondary minerals and the newly found vibrational marker bands were used to monitor the time evolution of the formation of covellite, and the K(+) and NH4(+)-jarosites from the chalcopyrite surfaces. The Raman data indicate that the formation of K(+)-jarosite is followed by the formation of NH4(+)-jarosite. The variation in color in the FTIR imaging data and the observation of the amide I vibration at 1637cm(-1) indicate that the microorganisms are attached on the mineral surface and the changes in the frequency/intensity of the biofilm marker bands in the 900-1140cm(-1) frequency range with time demonstrate the existence of biofilm conformations.

The Aerobic anoxygenic phototrophic bacteria (AAPB) Roseobacter denitrificans and Roseobacter litoralis are widespread in the bacterioplankton community with a particular role in the marine carbon cycle. Measurements of carotenoids isolated from dark-grown cells indicated the presence of spheroidenone (SO, N = 11) and of 3,4 dihydrospheroidenone (N = 10) in the carotenoids isolated from illuminated cells. Time-dependent Raman 514 nm excitation experiments of R. denitrificans and R. litoralis cells grown under illumination demonstrated that v1 (C=C) of SO exhibits a time-dependent substantial frequency upshift relative to its frequency in the dark-grown cells, in a manner resembling shorting the conjugation length (N). We suggest that the irreversible dark-SO to light- 3,4 dihydrospheroidenone transition observed in the intact carotenoids of R. denitrificans and R. litoralis cells is an operative photoreduction strategy of SO containing AAPB that affects the energy transfer mechanism.

Biofilm formation is a molecular assembly process occurring at interfaces, such as in bioleaching processes. The real time monitoring of the marker bands of amide I/amide II by FTIR microspectroscopy during Acidithiobacillus ferrooxidans colonization on chalcopyrite surfaces revealed the central role of lipids, proteins and nucleic acids in bacterial cell attachment to copper sulfide surfaces. The Raman and FTIR spectra of the interactions of Acidithiobacillus ferrooxidans with bornite are also reported.

A reversible temperature-dependent high-spin to low-spin transition with

This study aimed to develop hydrogenated amorphous carbon thin films with embedded metallic nanoparticles (a–C:H:Me) of controlled size and concentration. Towards this end, a novel hybrid deposition system is presented that uses a combination of Plasma Enhanced Chemical Vapor Deposition (PECVD) and Physical Vapor Deposition (PVD) technologies. The a–C:H matrix was deposited through the acceleration of carbon ions generated through a radio-frequency (RF) plasma source by cracking methane, whereas metallic nanoparticles were generated and deposited using terminated gas condensation (TGC) technology. The resulting material was a hydrogenated amorphous carbon film with controlled physical properties and evenly dispersed metallic nanoparticles (here Ag or Ti). The physical, chemical, morphological and mechanical characteristics of the films were investigated through X-ray reflectivity (XRR), Raman spectroscopy, Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), Transmission Electron Microscopy (TEM) and nanoscratch testing. The resulting amorphous carbon metal nanocomposite films (a–C:H:Ag and a–C:H:Ti) exhibited enhanced nanoscratch resistance (up to +50%) and low values of friction coefficient (

Nitric oxide reductase (Nor) is the third of the four enzymes of bacterial denitrification responsible for the catalytic formation of laughing gas (N 2 O). Here we report the detection of the hyponitrite (HO–N = N–O − ) species (ν N–N = 1332 cm − 1 ) in the heme b 3 Fe–Fe B dinuclear center of Nor from Paracoccus denitrificans . We have also applied density functional theory (DFT) to characterize the bimetallic-bridging hyponitrite species in the reduction of NO to N 2 O by Nor and compare the present results with those recently reported for the N–N bond formation in the ba 3 and caa 3 oxidoreductases from Thermus thermophilus .

All rights reserved.Organic compounds used in solvent extraction (SX) processes severely inhibit copper bioleaching microorganisms. In this experimental study, it was found that bioleaching microorganisms at 0.5% and 0.75% organic (SX) resulted in lower Cu recovery from low grade sulphide ore. Two new Candida species OR3 and OR6 were isolated from the liquid phase in solvent extraction process and these isolated strains were tolerant and capable of growing on 1% organic phase. The inhibition of organic phase to bioleaching microorganisms was alleviated by the addition of the 2 isolates and as a result the Cu recovery remained at high level. This is the first study which shows that the organic SX mixture inhibition to acidophilic microorganism can be overcome by the addition of other microorganisms.

The protonation/deprotonation of active carboxyl side chains by water networks forming the proton loading and exit sites in proteins are important steps in protein catalysis. An excellent system to study such basic principles is the heme-copper ba3 from T. thermophilus because it utilizes one proton input channel and it delivers protons to the active site for both O2 chemistry and proton pumping. We report the interaction of the heme a3 Fe propionate-A and the Asp372-His376 pair which forms the valve for the exit pathway for the protons with internal water molecules in ba3 oxidoreductase by light minus dark FTIR spectroscopy in conjunction with H2O/H218O/D2O exchange. The proton loading site consists of several water molecules including w941/w946 which are H-bonded to propionate-A-H+, acting as the Zundel cation. The detection of two H218O sensitive bands at 3640 and 3634 cm-1 shows the existence of weakly H-bonded water molecules. This journal is

High performance liquid chromatography (HPLC) coupled with a Fraction Collector was employed to isolate Maillard reaction products (MRPs) formed in model systems comprising of asparagine and monosaccharides in the 60–180°C range. The primary MRP which is detected at 60°C is important for Acrylamide content and color/aroma development in foods and also in the field of food biotechnology for controlling the extent of the Maillard reaction with temperature. The discrete fractions of the reaction products were reacted with Hemoglobin (Hb) and Myoglobin (Mb) at physiological conditions and the reaction adducts were monitored by UV-vis and Attenuated Total Reflection-Fourier transform infrared (FTIR) spectrophotometry. The UV-vis kinetic profiles revealed the formation of a Soret transition characteristic of a low-spin six-coordinated species and the ATR-FTIR spectrum of the Hb-MRP and Mb-MRP fractions showed modifications in the protein Amide I and II vibrations. The UV-vis and the FTIR spectra of the Hb-MRPs indicate that the six-coordinated species is a hemichrome in which the distal E7 Histidine is coordinated to the heme Fe and blocks irreversibly the ligand binding site. Although the Mb-MRPs complex is a six-coordinated species, the 1608 cm-1 FTIR band characteristic of a hemichrome was not observed.

© 2017 Elsevier B.V. The isolation of reaction products of asparagine with reducing sugars at alkaline pH and high temperature has been probed by a combination of high performance liquid chromatography (HPLC) coupled with a Fraction Collector. The UV–vis and FTIR spectra of the isolated Maillard reaction products showed structure-sensitive changes as depicted by deamination events and formation of asparagine-saccharide conjugates. The initial reaction species of the Asn-Gluc reaction were also characterized by Density Functional Theory (DFT) methods. Evidence for Cu (II) metal ion complexation with the Maillard reaction products is supported by UV–vis and FTIR spectroscopy.

Time-resolved step-scan FTIR spectroscopy has been employed to probe the dynamics of the ba3 oxidoreductase from Thermus thermophilus in the ns-μs time range and in the pH/pD 6–9 range. The data revealed a pH/pD sensitivity of the D372 residue and of the ring-A propionate of heme a3. Based on the observed transient changes a model in which the protonic connectivity of w941-w946-927 to the D372 and the ring-A propionate of heme a3 is described.

Nanosecond time-resolved step-scan FTIR spectroscopy (nTRS (2) -FTIR) has been applied to literally probe the active site of the carbon monoxide (CO)-bound thermophilic ba3 heme-copper oxidoreductase as it executes its function. The nTRS (2) - snapshots of the photolysed heme a3 Fe-CO/CuB species captured a "transition state" whose side chains prevent the photolysed CO to enter the docking cavity. There are three sets of ba3 photoproduct bands of docked CO with different orientation exhibiting different kinetics. The trajectories of the "docked" CO at 2122, 2129 and 2137cm(-1) is referred to in the literature as B2, B1 and B0 intermediate states, respectively. The present data provided direct evidence for the role of water in controlling ligand orientation in an intracavity protein environment.

Maillard reaction products in the food-, nutrition- and pharmaceutical related processes are of great interest in cases where the substances involved are chemically reactive or unstable. Detailed data using vibrational FTIR spectroscopy of the Maillard-type reaction products are limited and needs more experimental evidences with explicit mechanisms of the reaction to demonstrate how the reducing sugars be avoided in formulating the amine-containing substances. The combination of high performance liquid chromatography (HPLC) with Fourier transform infrared (FTIR) spectrometry is a powerful instrumental separation-structure sensitive technique that allows characterization in real time of the separated chemical species. In this short review we demonstrate the benefits of the HPLC-FTIR coupling technique in studying the Maillard reaction model Fructose/Asparagine system.

The complete understanding of a molecular mechanism of action requires the thermodynamic and kinetic characterization of different states and intermediates. Cytochrome c oxidase reduces O2 to H2O, a reaction coupled to proton translocation across the membrane. Therefore, it is necessary to undertake a thorough characterization of the reduced form of the enzyme and the determination of the electron transfer processes and pathways between the redox-active centers. In this study Fourier transform infrared (FTIR) and time-resolved step-scan FTIR spectroscopy have been applied to study the fully reduced and mixed valence states of cytochrome ba3 from Thermus thermophilus. We used as probe carbon monoxide (CO) to characterize both thermodynamically and kinetically the cytochrome ba3-CO complex in the 5.25–10.10 pH/pD range and to study the reverse intramolecular electron transfer initiated by the photolysis of CO in the two-electron reduced form. The time-resolved step-scan FTIR data revealed no pH/pD dependence in both the decay of the transient CuB1+-CO complex and rebinding to heme a3 rates, suggesting that no structural change takes place in the vicinity of the binuclear center. Surprisingly, photodissociation of CO from the mixed valence form of the enzyme does not lead to reverse electron transfer from the reduced heme a3 to the oxidized low-spin heme b, as observed in all the other aa3 and bo3 oxidases previously examined. The heme b-heme a3 electron transfer is guaranteed, and therefore, there is no need for structural rearrangements and complex synchronized cooperativities. Comparison among the available structures of ba3- and aa3-cytochrome c oxidases identifies possible active pathways involved in the electron transfer processes and key structural elements that contribute to the different behavior observed in cytochrome ba3.

Dynamics and reactivity in heme proteins include direct and indirect interactions of the ligands/substrates like CO, NO and O(2) with the environment. Direct electrostatic interactions result from amino acid side chains in the inner cavities and/or metal coordination in the active site, whereas indirect interactions result by ligands in the same coordination sphere. Interactions play a crucial role in stabilizing transition states in catalysis or altering ligation chemistry. We have probed, by Density Functional Theory (DFT), the perturbation degree in the stretching vibrational frequencies of CO, NO and O(2) molecules in the presence of electrostatic interactions or hydrogen bonds, under conditions simulating the inner cavities. Moreover, we have studied the vibrational characteristics of the heme bound form of the CO and NO ligands by altering the chemistry of the proximal to the heme ligand. CO, NO and O(2) molecules are highly polarizable exerting vibrational shifts up to 80, 200 and 120 cm(-1), respectively, compared to the non-interacting ligand. The importance of Density Functional Theory (DFT) methodology in the investigation of the heme-ligand-protein interactions is also addressed.

Visible and UV-resonance Raman spectroscopy was employed to investigate the reaction of NO with cytochrome caa3 from Thermus thermophilus. We show the formation of the hyponitrite (HO–N[double bond, length as m-dash]N–O)− bound to the heme a3 species (νN[double bond, length as m-dash]N = 1330 cm−1) forming a high spin complex in the oxidized heme a3 Fe/CuB binuclear center of caa3-oxidoreductase. In the absence of heme a3 Fe2+–NO formation, the electron required for the formation of the N[double bond, length as m-dash]N bond originates from the autoreduction of CuB by NO, producing nitrite. With the identification of the hyponitrite intermediate the hypothesis of a common phylogeny of aerobic respiration and bacterial denitrification is fully supported and the mechanism for the 2e−/2H+ reduction of NO to N2O can be described with more certainty.

Understanding of the chemical nature of the dioxygen and nitric oxide moiety of ba3-cytochrome c oxidase from Thermus thermophilus is crucial for elucidation of its physiological function. In the present work, direct resonance Raman (RR) observation of the Fe-C-O stretching and bending modes and the C-O stretching mode of the CuB-CO complex unambiguously establishes the vibrational characteristics of the heme-copper moiety in ba3-oxidase. We assigned the bands at 507 and 568 cm(-1) to the Fe-CO stretching and Fe-C-O bending modes, respectively. The frequencies of these modes in conjunction with the C-O mode at 1973 cm(-1) showed, despite the extreme values of the Fe-CO and C-O stretching vibrations, the presence of the alpha-conformation in the catalytic center of the enzyme. These data, distinctly different from those observed for the caa3-oxidase, are discussed in terms of the proposed coupling of the alpha-and beta-conformations that occur in the binuclear center of heme-copper oxidases with enzymatic activity. The CuB-CO complex was identified by its nu(CO) at 2053 cm(-1) and was strongly enhanced with 413.1 nm excitation indicating the presence of a metal-to-ligand charge transfer transition state near 410 nm. These findings provide, for the first time, RR vibrational information on the EPR silent CuB(I) that is located at the O2 delivery channel and has been proposed to play a crucial role in both the catalytic and proton pumping mechanisms of heme-copper oxidases.

The description of reaction regulation in enzymes responsible for activating and catalyzing small molecules (O(2), NO) requires identification of ligand movement into the binding site and out of the enzyme through specific channels and docking sites. We have used time-resolved step-scan Fourier transform infrared spectroscopy on CO-photolyzed cytochrome c oxidase ba(3) from T. thermophilus, which is responsible for the activation and reduction of both O(2) and NO, to gain insight into the structure of ligand-binding intermediates at ambient temperature. We show that, upon dissociation, the photolyzed CO becomes trapped within a ligand docking site located near the ring A propionate of heme a(3). The 2131 cm(-1) mode of the "docked" CO we have detected corresponds to the B(1) state of Mb and persists for 35 micros. The release of CO from the docking site is not followed by recombination to the heme a(3) Fe. Our analysis indicates that this behavior reflects a mechanism in which the protein near ring A of heme a(3) propionate reorganizes about the released CO from the docking site, and establishes a transient barrier that inhibits the recombination process to the heme a(3) Fe for a few milliseconds. Rebinding to heme a(3) occurs with k(2) = 29.5 s(-1). These results have implications for understanding the role of ligand binding/escape through docking sites and channels in heme-copper oxidases and, thus, in respiration.

Ligand trajectories trapped within a docking site or within an internal cavity near the active site of proteins are important issues toward the elucidation of the mechanism of reaction of such complex systems, in which activity requires the shuttling of oriented ligands to and from their active site. The ligand motion within ba3-cytochrome c oxidase from Thermus thermophilus has been investigated by measuring time-resolved step-scan Fourier transform infrared difference spectra of photodissociated CO from heme a3 at ambient temperature. Upon photodissociation, 15-20% of the CO is not covalently attached to CuB but is trapped within a docking site near the ring A of heme a3 propionate. Two trajectories of CO that are distinguished spectroscopically and kinetically (vCO = 2131 cm-1, td = 10-35 micros and vCO = 2146 cm-1, td = 85 micros) are observed. At later times (td = 110 micros) the docking site reorganizes about the CO and quickly establishes an energetic barrier that facilitates equilibration of the ligand with the protein solvent. The time-dependent shift of the CO trajectories we observe is attributed to a conformational motion of the docking site surrounding the ligand. The implications of these results with respect to the ability of the docking site to constrain ligand orientation and the reaction dynamics of the docking site are discussed herein.

6,7-Dioxabicyclo[3.2.2]non-8-ene 2 and 1-isopropyl-4-methyl-2,3- dioxabicyclo[2.2.2]oct-5-ene (ascaridol) 3 were prepared as simplified, endoperoxide versions of clinically used antimalarial drugs. Fourier transform infrared (FTIR) technique in conjunction with 18O 2- enriched compound 2 has been applied in probing the bonds of the endoperoxide moiety and the bonds of the rings owing to the presence of the O-O, the C-O, the O-O-C as well as the C=O modes in the spectrum. The endoperoxide moiety is especially useful in this regard because the homolytic cleavage of the O-O bond can be characterized and hence can be used to assess the vibrational properties of the O- and C-centered radicals and subsequently that of the C-C bond cleavage. The cleavage of the O-O bond, and the ability to correlate vibrational properties of the reaction products with structural properties of the isolated products suggest that infrared spectroscopy is an appropriate tool to study the mode of action of antimalarial endoperoxides.

We report the first study of O2 migration in the putative O2 channel of cytochrome ba 3 and its effect to the properties of the binuclear hemea 3-CuB center of cytochromeba 3 from Thermus thermophilus. The Fourier transform infrared spectra of theba 3-CO complex demonstrate that in the presence of 60–80 μm O2, the ν(C-O) of CuB 1+-C-O at 2053 cm−1 (complex A) shifts to 2045 cm−1 and remains unchanged in H2O/D2O exchanges and in the pH 6.5–9.0 range. The frequencies but not the intensities of the C-O stretching modes of heme a 3-CO (complex B), however, remain unchanged. The change in the ν(C-O) of complex A results in an increase of k −2, and thus in a higher affinity of CuB for exogenous ligands. The time-resolved step-scan Fourier transform infrared difference spectra indicate that the rate of decay of the transient CuB 1+-CO complex at pH 6.5 is 30.4 s−1 and 28.3 s−1 in the presence of O2. Similarly, the rebinding to hemea 3 is slightly affected and occurs withk 2 = 26.3 s−1 and 24.6 s−1 in the presence of O2. These results provide solid evidence that in cytochrome ba 3, the ligand delivery channel is located at the CuB site, which is the ligand entry to the heme a 3pocket. We suggest that the properties of the O2 channel are not limited to facilitating ligand diffusion to the active site but are extended in controlling the dynamics and reactivity of the reactions of ba 3 with O2 and NO.

We report the first evidence for the existence of the equilibrium Cu(B)1+-CO species of CO-bound reduced cytochrome ba(3) from Thermus thermophilus at room temperature. The frequency of the C-O stretching mode of Cu(B)1+-CO is located at 2053 cm(-1) and remains unchanged in H(2)O/D(2)O exchanges and, between pD 5.5 and 9.7, indicating that the chemical environment does not alter the protonation state of the Cu(B) histidine ligands. The data and conclusions reported here are in contrast to the changes in protonation state of Cu(B)-His-290, reported recently (Das, T. K., Tomson, F. K., Gennis, R. B., Gordon, M., and Rousseau, D. L. (2001) Biophys. J. 80, 2039-2045 and Das, T. P., Gomes, C. M., Teixeira, M., and Rousseau, D. L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9591-9596). The time-resolved step-scan FTIR difference spectra indicate that the rate of decay of the transient Cu(B)1+-CO complex is 34.5 s(-1) and rebinding to heme a(3) occurs with k(2) = 28.6 s(-1). The rate of decay of the transient Cu(B)1+-CO complex displays a similar time constant as the absorption changes at 1694(+)/1706(-), attributed to perturbation of the heme a(3) propionates (COOH). The nu(C-O) of the transient Cu(B)1+-CO species is the same as that of the equilibrium Cu(B)1+-CO species and remains unchanged in the pD range 5.5-9.7 indicating that no structural change takes place at Cu(B) between these states. The implications of these results with respect to proton pathways in heme-copper oxidases are discussed.

We have applied resonance Raman spectroscopy to investigate the properties of the dinuclear center of oxidized, reduced, and NO-bound nitric-oxide reductase from Paracoccus denitrificans. The spectra of the oxidized enzyme show two distinct νas(Fe-O-Fe) modes at 815 and 833 cm−1 of the heme/non-heme diiron center. The splitting of the Fe-O-Fe mode suggests that two different conformations (open and closed) are present in the catalytic site of the enzyme. We find evidence from deuterium exchange experiments that in the dominant conformation (833 cm−1mode, closed), the Fe-O-Fe unit is hydrogen-bonded to distal residue(s). The ferric nitrosyl complex of nitric-oxide reductase exhibits the ν(Fe3+-NO) and ν(N-O) at 594 and 1904 cm−1, respectively. The nitrosyl species we detect is photolabile and can be photolyzed to generate a new form of oxidized enzyme in which the proximal histidine is ligated to hemeb 3, in contrast to the resting form. Photodissociation of the NO ligand yields a five-coordinate high-spin heme b 3. Based on the findings reported here, the structure and properties of the dinuclear center of nitric- oxide reductase in the oxidized, reduced, and NO-bound form as well as its photoproduct can be described with certainty.

We report the photobiochemical production of carbon monoxide by a terminal ba3 -cytochrome c oxidase from T. thermophilus HB8. FTIR and time-resolved step-scan FTIR spectroscopies were combined to probe this process and also monitor the concomitant binding of the produced gas to other intact ba3 molecules forming the ba3 -CO complex. The activation of this mechanism by ba3 -oxidase under visible excitation raises the question as to whether such a mechanism is physiologically relevant to the extreme environment in which it operates.

Density functional theory (DFT) and combined Molecular Mechanics/Quantum Mechanics (MM/QM-MD) calculations have been applied to models of the cytochrome c oxidase (CcO) including the Fe–CuB binuclear center, where dioxygen is bound and subsequently reduced to water. The properties of several intermediates of the CcO dioxygen reaction have been investigated by theoretical approaches. In this chapter, we investigate the dynamics of the binuclear heme Fe–CuB throughout the O2 catalytic cycle. We are focused on the effects of the protein matrix and proton/water motion exerted on the heme a 3 group. For this, we have built models of CcO, which vary at the heme a 3 environment. This variability is based on hydrogen bonding interactions and amino acid protonation states. Different control points have been identified for the transition from one intermediate to the next. The hydrogen bonding networks in the proximity of heme a 3 area also have consequences for the characteristics of the binuclear center. A theoretical framework for the direct link between an H+ delivery channel (termed D) and an accumulation of waters, termed ‘water pool’ close to the active site, has been achieved at the QM/MM level of theory. Two proton valves (E278 and His403) and an electron/proton coupling site (propionate-A/Asp399) exist in this pathway for the aa 3 CcO from P. denitrificans. The ferryl intermediate, produced subsequent to the O–O bond scission, is found to have characteristics highly dependent on the basicity of the proximal His411, in contrast to the hydroxyl intermediate that is sensitive to distal effects.

Fourier transform infrared spectra are reported for the Fe(III)- and Fe(II)-mediated activation of the antimalarial agents artemisinin 1 and its simplified synthetic analogue, trioxane alcohol 2. By monitoring the frequencies of the newly established marker lines in the FTIR spectra, the products of the Fe(II) and Fe(III) reactions have been characterized. In both reactions, artemisinin is activated giving a product mixture of a ring-contracted tetrahydrofuran acetatal 3, C(4)-hydroxy deoxyartemisinin 4, and deoxyartemisinin 5. These data illustrate that the oxidation state of the iron places no restrictions on the endoperoxide reduction mechanism. The FTIR difference (light - dark) spectra indicate that the endoperoxide moiety of artemisinin is photolabile and that the resulted products have the same vibrational characteristics as those observed in the reactions with Fe(II) and Fe(III). The use of 18O-18O enriched endoperoxide in 2 has allowed us to identify two oxygen sensitive modes in the reactions with Fe(II). The reduction of the peroxide bond by Fe(II) in trioxane alcohol 2 follows both the C-C cleavage and 1,5-H shift pathways and produces a ring-contracted tetrahydrofuran acetal 6 which is converted to tetrahydrofuran aldehyde 7 and C(4)-hydroxy deoxytrioxane alcohol 8, respectively. The cleavage of the O-O bond in 1 and 2 by iron and the ability to correlate vibrational properties of the reaction products with structural properties of the isolated products suggest that infrared spectroscopy is an appropriate tool to study the mode of action of antimalarial endoperoxides.

Resonance Raman and FTIR spectra are reported for the oxidized azide-bound derivative of the quinol cytochrome bo3 oxidase from Escherichia coli. The resonance Raman spectra display three isotope-dependent vibrational modes at 419, 2040, and 2061 cm-1. The FTIR spectra display two isotope-dependent bands at 2040 and 2061 cm-1. We assign the band at 419 cm-1 to v(Fe-N3-CuB) and the bands at 2040 and 2061 cm-1 to Vas(N3). The observation of two vas(N3) modes suggests that the azide ion binds to two different enzyme conformations, both forming bridging complexes with the binuclear center. Comparison of the FTIR data of the azide-bound cytochrome bo3 and cytochrome aa3 complexes reveal that there are quantitative differences in the structure of the heme o3-CuB and heme a3-CuB binuclear pockets upon azide binding. The present data on the vibrational frequencies of the azide-bound cytochrome bo3 complex do not support the recent proposal that azide binds as a terminal ligand to CuB (Little, R. H.; Cheesman, M. R.; Thomson, A. J.; Greenwood, C.; Watmough, N. J. Biochemistry 1996, 35, 13780-13787) but are more reasonably interpreted to conclude that azide functions as a bridge between heme o3 and CuB

Resonance Raman and FTIR spectra are reported for the fully reduced carbon monoxy derivative of the quinol aa3-600 oxidase from Bacillus subtilis. The resonance Raman spectra display two isotope-dependent vibrational modes at 520 and 575 cm-1. The FTIR spectrum displays a single vibrational mode at 1963 cm-1. We assign the band at 520 cm-1 to the Fe-CO stretching mode, the band at 575 cm-1 to the Fe-C-O bending mode, and the band at 1963 cm-1 to the C-O stretching mode. The frequencies of these modes are similar to those that have been reported for the CO-bound mammalian cytochrome c oxidase. Despite the fact that two different heme-protein conformations that affect the iron-his bond strength are present in the ferrous ligand-free form of aa3-600, the CO-bound adduct has a single conformation in which the His-Fe-CO CUB moiety has the same structure as the α form found in the mammalian cytochrome c oxidase. The present and previous data on the vibrational frequencies of ferrous ligand-free and ferrous CO-bound forms of terminal oxidases show that an inverse linear relationship exists between the frequencies of the Fe-his and Fe-CO stretching modes. We suggest that the frequencies of both the Fe-CO and C-O modes found in heme-CUB oxidases are affected by the proximal His376, which is H-bonded to the peptide carbonyl of Gly351, and by distal effects on the heme a3-bound CO exerted by CuB