Photothermal deflectometry enhanced by total internal reflection enables non-invasive glucose monitoring in human epidermis.

We present TIR-PTD spectroscopy, an IR-pump/VIS-probe method for the measurement of IR absorption spectra by means of photothermal deflectometry (PTD) enhanced by total internal reflection (TIR). It overcomes the limitations of IR spectroscopy for the study of opaque samples and allows molecular fingerprinting of IR-active liquids or solids. Another important advantage of the presented approach over traditional IR spectroscopy methods is the ability to obtain IR information by means of VIS detection, which is generally much cheaper and easier to handle than IR detection. By application of mid-IR TIR-PTD spectroscopy on human skin in vivo, we are demonstrating the correlation between epidermal- and blood-glucose levels on a type 1 diabetic patient.

Estimation of anisotropy coefficient and total attenuation of swine liver at 850 nm based on a goniometric technique: influence of sample thickness.

Estimation of optical properties of biologic tissue is crucial for theoretical modeling of laser treatments in medicine. Tissue highly absorbs and scatters the light between 650 nm and 1300 nm, where the laser provides therapeutic effects. Among other properties, the characteristic of biological tissues to scatter the light traveling trough, is described by the anisotropy coefficient (g). The relationship between g and the distribution of the scattered light at different angles is described by Henyey-Greenstein phase function. The measurement of angular distribution of scattered light is performed by the goniometric technique. This paper describes the estimation of g and attenuation coefficient, µt, of swine liver at 850 nm, performed by an ad hoc designed goniometric-based system, where a spectrometer measures intensities of scattered light at fixed angles (0°, 30°, 45°, 60, 120°, 135° and 150°). Both one-term and two-term Henyey-Greenstein phase function have been employed to estimate anisotropy coefficient for forward (gfs) and backward scattering (gbs). Measurements are performed on samples of two thicknesses (60 um and 30 urn) to investigate the influence of this factor on g, and repeated 6 times for each thickness. The estimated values of gfs were 0.947 and 0.951 for thickness of 60 µm and 30 µm, respectively; the estimations of gfs were -0.498 and -0.270 for thickness of 60 µm and 30 µm, respectively. Moreover, µt of liver has been estimated (i.e., 90±20 cm(1)), through Lambert-Beer equation. The comparison of our results with data reported in literature encourages the use of the ad hoc designed tool for performing experiments on other tissue, and at other wavelengths.

Infrared spectroscopic study of the structural and functional properties of the Na(+)/H(+) antiporter MjNhaP1 from Methanococcus jannaschii.

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 6.5; below and above it is inactive. The secondary structure analysis on the basis of ATR-IR spectra provides the first insights into the structural changes between inactive (pH 8) and active (pH 6) state of MjNhaP1. It results in decreased ordered structural elements with increasing the pH-value i.e. with inactivation of the protein. Analysis of temperature-dependent FTIR spectra indicates that MjNhaP1 in the active state exhibits a much higher unfolding temperature in the spectral region assigned to alpha-helical segments. In contrast, the temperature-induced structural changes for beta-sheet structure are similar for inactive and active state. Consequently, this structure element is not the part of the activation region of the protein. The surface accessibility of the protein was analyzed by following the extent of H/D exchange. Due to higher content of unordered structural elements a higher accessibility for amide protons is observed for the inactive as compared to the active state of MjNhaP1. Altogether, the results present the active state of MjNhaP1 as the state with ordered structural elements which exhibit high thermal stability and increased hydrophobicity.

Flexibility and dynamics of NhaA Na+/H+-antiporter of Escherichia coli studied by Fourier transform infrared spectroscopy.

NhaA (41,355Da) 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. Padan, S. Schuldiner, Biochemistry 36 (1997) 14572]. Protein dynamics and flexibility in the activated and inactivated state, respectively, was analysed by probing accessibility in (1)H/(2)H exchange experiments for the wild type and the mutant G338S which is constitutively active independent of pH [A. Rimon, Y. Gerchman, Z. Kariv, E. Padan, J. Biol. Chem. 273 (1998) 26470]. This was studied by ATR-FTIR difference spectroscopy using a home built microvolume (<5 microl) perfusion chamber [E. Agic, O. Klein, W. Mäntele, Proceedings of the 10th European Conference on the Spectroscopy of Biological Molecules, vol. 93, 2003, ISBN 9634826148; S. Gourion-Arsiquaud, S. Chevance, P. Bouyer, L. Garnier, J.-L. Montillet, A. Bondon, C. Berthomieu, Biochemistry 44 (2005) 8652]. The solution or suspension of the target protein is contained in a chamber with sample volumes of below 5 microl. It is in contact with the ATR crystal and separated from the flowing effector molecules by a dialysis membrane. The flow-ATR unit is characterised by high stability, fast response, and high sensitivity for the IR spectroscopic detection of binding-induced conformational changes and reactions. On the basis of (1)H-(2)H exchange of NhaA followed in the amide I and amide II region of the IR spectrum, it is concluded that the accessible fraction of the polypeptide chain of NhaA increases by more than 10% in the active state. For the mutant, no changes in accessibility were observed for different pH values. The increase of Na(+) concentration increases the extent of exchange. The stability of the wild type protein in the active and inactive form was analysed by measuring the temperature profiles of the IR spectra. A decrease of the structural stability of the protein with activation was observed. Together with the results from (1)H/(2)H exchange, the inactive state represents a more compact form whereas activation induces a more open conformation of the protein.

Structure-function correlation of outer membrane protein porin from Paracoccus denitrificans.

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 consequence of the beta-barrel structure and whether it is required for its function. To explain this we have analyzed two cases: first, we used porin wild-type and mutants and compared their structure and function; second, we compared the activity of porin preheated to different temperatures. Structural changes were monitored by infrared spectroscopy. We observed that the structural stability of porin is not equivalent to functional activity as minor alteration in the structure can result in drastic differences in the activity of porins.

Investigation of the thermal stability of porin from Paracoccus denitrificans by site-directed mutagenesis and Fourier transform infrared spectroscopy.

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 analysis by amide I ir spectroscopy showed that the wild-type protein was predominantly composed of beta-sheet, which supports the x-ray crystal structure information (A. Hirsch, J. Breed, K. Saxena, O.-M. H. Richter, B. Ludwig, K. Diederichs, and W. Welte, FEBS Letters, 1997, Vol. 404, pp. 208-210). The mutants E81Q, W74C, and E81Q/D148N were shown to have similar secondary structure composition as the wild type. Wild-type protein and the mutants in detergent micelles underwent irreversible denaturation as a result of heating. Transition temperature calculated from the amide I analysis revealed that mutant porins were slightly less stable compared to the wild type. The protein in micelles showed complete monomerization of the trimer above 85 degrees C. In native-like conditions (provided by liposomes), no change was observed in the secondary structure of the protein until 95 degrees C. This is supported by SDS-PAGE as no change in quaternary structure was observed, proving that the proteins are structurally thermostable in liposomes as compared to micelles. Our studies demonstrated that porins resistant to detergents and proteases are highly thermostable as well.

Redox dependent conformational changes in the mixed valence form of the cytochrome c oxidase from p. The reorganization of glutamic acid 278 is coupled to the electron transfer from/to heme a and the binuclear center. denitrificans.

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) form of the cytochrome c oxidase after photolysis reflect the conformational changes induced by the rebinding of CO and by electron transfer reactions from heme a3 to heme a and further on to CUA. During this process, heme a3 (and CUB) are oxidized, whereas heme a and CuA are reduced. By subtracting these difference spectra from an electrochemically induced FTIR difference spectrum, where all four cofactors are reduced, the contributions for heme a3 (and CuB) could be separated. Correspondingly, the spectral contributions of heme a and CuA have been separated. The comparison of these spectra with the spectra calculated for the hemes on the basis of their redox dependent changes previously published in Hellwig et al., (Biochemistry 38, (1999) 1685-1694) show a high degree of similarity, except for additional signals coupled to the reorganization of the binuclear center upon CO rebinding. The separated spectra clearly show that the signals attributed to Glu278, an amino acid discussed to be crucial for proton pumping, is coupled to electron transfer to/from heme a and the binuclear heme a3-CuB center.

Characterization of two novel redox groups in the respiratory NADH:ubiquinone oxidoreductase (complex I).

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 remaining seven subunits are hydrophobic proteins predicted to fold into 54 alpha-helices across the membrane. Little is known about their function, but they are most likely involved in proton translocation. The mitochondrial complex contains in addition to the homologues of these 14 subunits at least 29 additional proteins that do not directly participate in electron transfer and proton translocation. A novel redox group has been detected in the Neurospora crassa complex, in an amphipathic fragment of the Escherichia coli complex I and in a related hydrogenase and ferredoxin by means of UV/Vis spectroscopy. This group is made up by the two tetranuclear FeS clusters located on NuoI (the bovine TYKY) which have not been detected by EPR spectroscopy yet. Furthermore, we present evidence for the existence of a novel redox group located in the membrane arm of the complex. Partly reduced complex I equilibrated to a redox potential of -150 mV gives a UV/Vis redox difference spectrum that cannot be attributed to the known cofactors. Electrochemical titration of this absorption reveals a midpoint potential of -80 mV. This group is believed to transfer electrons from the high potential FeS cluster to ubiquinone.

FT-IR spectroscopic characterization of NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli: oxidation of FeS cluster N2 is coupled with the protonation of an aspartate or glutamate side chain.

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, complex I has been described mainly by means of EPR- and UV-vis spectroscopy. Here, we report for the first time an infrared spectroscopic characterization of complex I. Electrochemically induced FT-IR difference spectra of complex I from Escherichia coli and of the NADH dehydrogenase fragment of this complex were obtained for critical potential steps. The spectral contributions of the FMN in both preparations were derived from a comparison using model compounds and turned out to be unexpectedly small. Furthermore, the FT-IR difference spectra reveal that the redox transitions of the FMN and of the FeS clusters induce strong reorganizations of the polypeptide backbone. Additional signals in the spectra of complex I reflect contributions induced by the redox transition of the high-potential FeS cluster N2 which is not present in the NADH dehydrogenase fragment. Part of these signals are attributed to the reorganization of protonated/deprotonated Asp or Glu side chains. On the basis of these data we discuss the role of N2 for proton translocation of complex I.

Structural changes of the sarcoplasmic reticulum Ca(2+)-ATPase upon nucleotide binding studied by fourier transform infrared spectroscopy.

Changes in the vibrational spectrum of the sarcoplasmic reticulum Ca(2+)-ATPase upon nucleotide binding were recorded in H(2)O and (2)H(2)O at -7 degrees 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, P(3)-1-(2-nitrophenyl) adenosine 5'-triphosphate, and related caged compounds). Infrared absorbance changes due to ATP release and two steps of the Ca(2+)-ATPase reaction cycle, ATP binding and phosphorylation, were followed in real time. Under the conditions used in our experiments, the rate of ATP binding was limited by the rate of ATP release (k(app) congruent with 3 s(-1) in H(2)O and k(app) congruent with 7 s(-1) in (2)H(2)O). Bands in the amide I and II regions of the infrared spectrum show that the conformation of the Ca(2+)-ATPase changes upon nucleotide binding. The observation of bands in the amide I region can be assigned to perturbations of alpha-helical and beta-sheet structures. According to similar band profiles in the nucleotide binding spectra, ATP, AMP-PNP, and ADP induce similar conformational changes. However, subtle differences between ATP and AMP-PNP are observed; these are most likely due to the protonation state of the gamma-phosphate group. Differences between the ATP and ADP binding spectra indicate the significance of the gamma-phosphate group in the interactions between the Ca(2+)-ATPase and the nucleotide. Nucleotide binding affects Asp or Glu residues, and bands characteristic of their protonated side chains are observed at 1716 cm(-1) (H(2)O) and 1706 cm(-1) ((2)H(2)O) and seem to depend on the charge of the phosphate groups. Bands at 1516 cm(-1) (H(2)O) and 1514 cm(-1) ((2)H(2)O) are tentatively assigned to a protonated Tyr residue affected by nucleotide binding. Possible changes in Arg, Trp, and Lys absorption and in the nucleoside are discussed. The spectra are compared with those of nucleotide binding to arginine kinase, creatine kinase, and H-ras P21.

Functional properties of the heme propionates in cytochrome c oxidase from Paracoccus denitrificans. Evidence from FTIR difference spectroscopy and site-directed mutagenesis.

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 propionates, we have constructed seven cytochrome coxidase variants using site-directed mutagenesis of subunit I. The mutant enzymes W87Y, W87F, W164F, H403A, Y406F, R473K, and R474K were characterized by measurement of enzymatic turnover, proton pumping activity, and Vis and FTIR spectroscopy. Whereas the mutant enzymes W164F and Y406F were found to be structurally altered, the other cytochrome c oxidase variants were suitable for band assignment in the infrared. Reduced-minus-oxidized FTIR difference spectra of the mutant enzymes were used to identify the ring D propionate of heme a as a likely proton acceptor upon reduction of cytochromic oxidase. The ring D propionate of heme a(3) might undergo conformational changes or, less likely, act as a proton donor.

Essential role of Glu-C66 for menaquinol oxidation indicates transmembrane electrochemical potential generation by Wolinella succinogenes fumarate reductase.

Quinol:fumarate reductase (QFR) is a membrane protein complex that couples the reduction of fumarate to succinate to the oxidation of quinol to quinone, in a reaction opposite to that catalyzed by the related enzyme succinate:quinone reductase (succinate dehydrogenase). In the previously determined structure of QFR from Wolinella succinogenes, the site of fumarate reduction in the flavoprotein subunit A of the enzyme was identified, but the site of menaquinol oxidation was not. In the crystal structure, the acidic residue Glu-66 of the membrane spanning, diheme-containing subunit C lines a cavity that could be occupied by the substrate menaquinol. Here we describe that, after replacement of Glu-C66 with Gln by site-directed mutagenesis, the resulting mutant is unable to grow on fumarate and the purified enzyme lacks quinol oxidation activity. X-ray crystal structure analysis of the Glu-C66-->Gln variant enzyme at 3.1-A resolution rules out any major structural changes compared with the wild-type enzyme. The oxidation-reduction potentials of the heme groups are not significantly affected. We conclude that Glu-C66 is an essential constituent of the menaquinol oxidation site. Because Glu-C66 is oriented toward a cavity leading to the periplasm, the release of two protons on menaquinol oxidation is expected to occur to the periplasm, whereas the uptake of two protons on fumarate reduction occurs from the cytoplasm. Thus our results indicate that the reaction catalyzed by W. succinogenes QFR generates a transmembrane electrochemical potential.

Mutation of Arg-54 strongly influences heme composition and rate and directionality of electron transfer in Paracoccus denitrificans cytochrome c oxidase.

The effect of a single site mutation of Arg-54 to methionine in Paracoccus denitrificans cytochrome c oxidase was studied using a combination of optical spectroscopy, electrochemical and rapid kinetics techniques, and time-resolved measurements of electrical membrane potential. The mutation resulted in a blue-shift of the heme a alpha-band by 15 nm and partial occupation of the low-spin heme site by heme O. Additionally, there was a marked decrease in the midpoint potential of the low-spin heme, resulting in slow reduction of this heme species. A stopped-flow investigation of the reaction with ferrocytochrome c yielded a kinetic difference spectrum resembling that of heme a(3). This observation, and the absence of transient absorbance changes at the corresponding wavelength of the low-spin heme, suggests that, in the mutant enzyme, electron transfer from Cu(A) to the binuclear center may not occur via heme a but that instead direct electron transfer to the high-spin heme is the dominating process. This was supported by charge translocation measurements where Deltapsi generation was completely inhibited in the presence of KCN. Our results thus provide an example for how the interplay between protein and cofactors can modulate the functional properties of the enzyme complex.

Identification of a hydrogen bond in the phe M197-->Tyr mutant reaction center of the photosynthetic purple bacterium Rhodobacter sphaeroides by X-ray crystallography and FTIR spectroscopy.

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 postulated by Wachtveitl et al. [Biochemistry 32, 12875-12886, 1993] between the Tyr M197 hydroxy group and one of the 2a-acetyl carbonyls of D. In combination with electrochemically induced FTIR difference spectra showing a split band of the pi-conjugated 9-keto carbonyl of D, there is clear evidence for the existence of such a hydrogen bond.

Vibrational modes of ubiquinone in cytochrome bo(3) from Escherichia coli identified by Fourier transform infrared difference spectroscopy and specific (13)C labeling.

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 DeltaUbiA (an oxidase devoid of bound ubiquinone) and DeltaUbiA reconstituted with ubiquinone 2 and with isotopically labeled ubiquinone 2, where (13)C was introduced either at the 1- or at the 4-position of the ring (C=O groups), have been obtained. The vibrational modes of the quinone bound to the discussed high-affinity binding site (Q(H)) are compared to those from the synthetic quinones in solution, leading to the assignment of the C=O modes to a split signal at 1658/1668 cm(-)(1), with both carbonyls similarly contributing. The FTIR spectra of DeltaUbiA reconstituted with the labeled quinones indicate an essentially symmetrical and weak hydrogen bonding of the two C=O groups from the neutral quinone with the protein and distinct conformations of the 2- and 3-methoxy groups. Perturbations of the vibrational modes of the 5-methyl side groups are discussed for a signal at 1452 cm(-)(1). Only negligible shifts of the aromatic ring modes can be reported for the reduced and the protonated form of the quinone. Alterations of the protein upon quinone binding are reflected in the electrochemically induced FTIR difference spectra. In particular, difference signals at 1640-1633 cm(-)(1) and 1700-1670 cm(-)(1) indicate variations of beta-sheet secondary structure elements and loops, bands at 1706 and 1678 cm(-)(1) are tentatively attributed to individual amino acids, and a difference signal a 1540 cm(-)(1) is discussed to reflect an influence on C=C modes of the porphyrin ring or on deprotonated propionate groups of the hemes. Further tentative assignments are presented and discussed. The (13)C labeling experiments allow the assignment of the vibrational modes of a bound ubiquinone 8 in the electrochemically induced FTIR difference spectra of wild-type bo(3).

Electrochemical and spectroscopic investigations of the cytochrome bc1 complex from Rhodobacter capsulatus.

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 backbone. Furthermore, band features were tentatively assigned to protonated aspartic or glutamic acids involved in the redox transition of each of the b hemes, a proline in that of the [2Fe-2S] protein, and an arginine in that of cytochrome b(H). The midpoint potential of the [2Fe-2S] protein was determined for the first time at physiological temperature to be +290 mV at pH 8.7. The reduced minus oxidized difference extinction coefficients of the alpha-bands of the cytochromes were calculated as 11.5, 19, and 6.7 mM(-1) cm(-1) for cytochromes c(1), b(H), and b(L), respectively. A novel method has been developed to quantify protonation reactions of the complex during the redox reactions of its cofactors by evaluation of the buffer signals in the midinfrared region. Values will be discussed in relation to the pH dependence of the midpoint potentials.

Similarities and dissimilarities in the structure-function relation between the cytochrome c oxidase from bovine heart and from Paracoccus denitrificans as revealed by FT-IR difference spectroscopy.

The redox dependent changes in the cytochrome c oxidase from bovine heart were studied with a combined electrochemical and FT-IR spectroscopic approach. A direct comparison to the electrochemically induced FT-IR difference spectra of the cytochrome c oxidase from Paracoccus denitrificans reveals differences in the structure and intensity of vibrational modes. These differences are partially attributed to interactions of subunits influencing the heme and protein modes. In the spectral regions characteristic for v(C=O) and v(COO-)s/as modes of protonated and deprotonated Asp and Glu residues, additional signals at 1736, 1602 and 1588 cm-1 are observed. On this basis, the possible involvement of Asp-51, a residue specifically conserved in mammalian oxidase and previously proposed to show redox depended conformational changes in the respective X-ray structures, is critically discussed.

Redox-induced conformational changes in plastocyanin: an infrared study.

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 protein. The sensitivity of this component to deuteration and increasing temperature suggests that PC adopts an unusual secondary structure in solution, which differs from those described for other type I copper proteins, such as azurin and halocyanin. The conformations of oxidized and reduced PC are different, as evidenced (1) by analysis of their amide I band contour and the electrochemically induced oxidized-minus-reduced difference spectrum and (2) by their different thermal stability. The redox-induced difference spectrum exhibits a number of difference bands within the conformationally sensitive amide I band that could be assigned to peptide C=O modes, in light of their small shift upon deuteration, and to signals attributable to side chain vibrational modes of Tyr residues. Lowering the pH to 4.8 induces destabilization of both redox states of the protein, more pronounced for reduced PC, without significantly affecting their secondary structure. Besides the conformational differences obtained at neutral pH, the oxidized-minus-reduced difference spectrum shows two broad and strong negative bands at 1405 and 1571 cm(-1), assigned to COO(-) vibrations, and a broad positive band at 1710 cm(-1), attributed to the C=O vibration of a COOH group(s). These bands are indicative of a protonation of (an) Asp or Glu side chain(s) upon plastocyanin oxidation at acidic pH.

Electrochemical, FTIR, and UV/VIS spectroscopic properties of the ba(3) oxidase from Thermus thermophilus.

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 Ag/AgCl/3 M KCl). Fully reversible electrochemically induced UV/VIS and FTIR difference spectra were obtained for the full potential step from -0. 5 to 0.5 V as well as for the critical potential steps from -0.5 to 0.1 V (heme b is fully oxidized and heme a3 remains essentially reduced) and from 0.1 to 0.5 V (heme b remains oxidized and heme a3 becomes oxidized). The difference spectra thus allow to us distinguish modes coupled to heme b and heme a3. Analogous difference spectra were obtained for the enzyme in D2O buffer for additional assignments. The FTIR difference spectra reveal the reorganization of the polypeptide backbone, perturbations of single amino acids and of hemes b and a3 upon electron transfer to/from the four redox-active centers heme b and a3, as well as CuB and CuA. Proton transfer coupled to redox transitions can be expected to manifest in the spectra. Tentative assignments of heme vibrational modes, of individual amino acids, and of secondary structure elements are presented. Aspects of the uncommon electrochemical and spectroscopic properties of the ba3 oxidase from T. thermophilus are discussed.