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Pig kidney Na(+),K(+)-ATPase was studied by means of reaction-induced infrared difference spectroscopy. The reaction from E1Na(3)(+) to an E2P state was initiated by photolysis of P(3)-1-(2-nitrophenyl)ethyl ATP (NPE caged ATP) in samples that contained 3 mM free Mg(2+) and 130 mM NaCl at pH 7.5. Release of ATP from caged ATP produced highly detailed infrared difference spectra indicating structural changes of the Na(+),K(+)-ATPase. The observed transient state of the enzyme accumulated within seconds after ATP release and decayed on a timescale of minutes at 15 degrees C. Several controls ensured that the observed difference signals were due to structural changes of the Na(+),K(+)-ATPase. Samples that additionally contained 20 mM KCl showed similar spectra but less intense difference bands. The absorbance changes observed in the amide I region, reflecting conformational changes of the protein backbone, corresponded to only 0.3% of the maximum absorbance. Thus the net change of secondary structure was concluded to be very small, which is in line with movement of rigid protein segments during the catalytic cycle. Despite their small amplitude, the amide I signals unambiguously reveal the involvement of several secondary structure elements in the conformational change. Similarities and dissimilarities to corresponding spectra of the Ca(2+)-ATPase and H(+),K(+)-ATPase are discussed, and suggest characteristic bands for the E1 and E2 conformations at 1641 and 1661 cm(-1), respectively, for alphabeta heterodimeric ATPases. The spectra further indicate the participation of protonated carboxyl groups or lipid carbonyl groups in the reaction from E1Na(3)(+) to an E2P state. A negative band at 1730 cm(-1) is in line with the presence of a protonated Asp or Glu residue that coordinates Na(+) in E1Na(3)(+). Infrared signals were also detected in the absorption regions of ionized carboxyl groups.

Near-infrared excited Fourier-transform Raman spectra have been obtained from different spectral forms of Rhodobacter sulfidophilus light-harvesting II complexes. This complex, when isolated in lauryldimethylamine oxide, exists in a 805–828 nm form, which can be reversibly converted to the native 805–851 nm form upon addition of salt. The FT-Raman spectra predominantly show contributions of the carotenoid in the light-harvesting complex, with small but significant contributions of the bacteriochlorophylls excited in preresonance in the Q y transition. One strongly and one weakly interacting 2a acetyl C=O group as well as one moderately strong interacting and one non-interacting 9-keto C=O carbonyl modes of the bacteriochlorophylls can be discerned for the 805–828 nm form. Changes of relative band intensities caused by different resonance conditions for the different spectral forms lead to an assignment of the strongly interacting 2a acetyl C=O and the moderately strong interacting 9 keto C=O to bacteriochlorophylls organized in the 828 pigment moiety. Shifts of these bands to higher frequencies upon the salt-induced transition indicate a perturbation of the pigment-protein interaction, probably caused by a local protein conformational change.

Cells of Rhodobacter capsulatus wild-type strains (37b4, B 10) and mutant strains, lacking lightharvesting (LH) complex II (B800–850) and defective in formation of LH I (B870) complex [U 43 (pTXB 87), U43 (pTXA6-10)] were grown photosynthetically at high and low light intensities in a turbidostate. The mutant strain U43 (pTXA6-10), lacking any LH system, was able to grow at high and low light intensities with doubling times of 4.6 and 9.8 h, respectively. In this mutant the concentration of photochemical reaction centers (RC) per cell and per membrane protein was several times higher than in wild type cells, but the bacteriochlorophyll content, the size of the photosynthetic unit and the rate of photophosphorylation were lower than in wild type cells. Reversible bleaching of reaction center and photophosphorylation were measured under different excitation light intensities. The charge recombination in the RC between the primary donor and QB was very slow in the mutant strains. Two membrane fractions differing in absorption spectra and light saturation behaviour of reversible bleaching and photophosphorylation were isolated from the mutant strains. The experimental data indicate that photosynthetic units of different composition and/or organization are present in the mutant cells.

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 (

Light-induced intermediates in reaction centers of Rhodobacter sphaeroides have been investigated by time-resolved Fourier transform infrared (FTIR) difference spectroscopy with a time-resolution of 25 ms at a spectral resolution of 4 cm−1. Following photoexcitation, an electron is rapidly transferred from the primary donor (P) to acceptor quinones, QA and QB. Based on the different recombination lifetimes of the photoinduced intermediates (60–100 ms for P+Q−A → PQA and a few seconds for P+QAQ−B → PQAQB), a comparison between the P+Q−A-minus-PQA (P+Q−A/PQA) and P+QAQ−B-minus-PQAQB (P+QAQ−B/PQAQB) spectra becomes reliable, since both difference spectra are measured: (i) for the same sample, (ii) at the same temperature, (iii) in the same chemical environment, (iv) from the same actinic event, and (v) with the same background. For the first time, the small variations observed between the two difference spectra, P+Q−A/PQA and P+QAQ−B/PQAQB, have been interpreted in terms of contributions from only QA, Q−B, Q−A, QB and their amino acid partners without the interference from P and P+. Vibrational modes ascribed to neutral quinone carbonyls could not be singled out, instead several bands were related to changes in interaction of amino acid residues with QA and QB following photoexcitation. In particular, three bands (1670 cm−1, 1652 cm−1, 1630 cm−1) insensitive to 1H-2H exchange have been identified. The feature at 1670 cm−1 was not apparent in previous steady-state studies, the 1652−1 band has been associated to a conformational change of the peptide C=O of the conserved Ala M260 residue in the QA pocket. The 1493 cm−1, 1480 cm−1 (Q−B) and the 1460 cm−1 (Q−A) bands have been revealed and attributed to vibrations of the semiquinone anion without excluding the possibility of some C-O− contributions. The 1732 cm−1, 1555 cm−1, and 1533 cm−1 bands can be assigned to amino acid vibrations. The band at 1555 cm−1 could reflect the effect of the QA delocalized charge on the ring of Trp M252 in van der Waals contact with QA.

Molecular changes associated with the photoreduction of the pheophytin a intermediary electron acceptor in films of Photosystem II reaction center (D1D2 RC) were characterized by FTIR spectroscopy. Upon accumulation at 240 K of the photoreduced acceptor, three negative carbonyl bands are observed at 1739 cm−1, 1721 cm−1 and 1677 cm−1 in the light-minus-dark FTIR spectrum of D1D2 RC. The redox-induced FTIR spectrum of the pheophytin a anion generated electrochemically in tetrahydrofuran shows only two negative bands at 1743 cm−1 and 1706 cm−1 which are assigned to changes of absorption of the 10a-ester C=O and 9-keto C=O, respectively. These assignments are based upon the comparison between FTIR data obtained on radicals of pheophytin a and its pyroderivative lacking the 10a-ester C=O. Thus, the 1677 cm−1 band observed in vivo reflects an interacting 9-keto C=O in D1D2 RC. The close similarity observed between: (i) FTIR spectra obtained on Photosystem II and Rps. viridis reaction centers and (ii) amino-acid sequences of the L and D1 polypeptides leads to the assignment of the 1721 cm−1 band in D1D2 RC to a protein-bound 10a-ester C=O of the acceptor and the 1739 cm−1 band to a contribution from the protonated carboxylic group of Glu D1-130 which is proposed to be H-bonded to the 9-keto C=O of the pheophytin acceptor, in the same way as in the Rps. viridis reaction center, Glu L104 is interacting with the 9-keto C=O of HL. The FTIR data indicate that the interactions of the 9-keto C=O and of the 10a-ester of the intermediary acceptor with the protein are stronger in D1D2 RC than in Rps. viridis. These stronger interactions could account, at least in part, for the difference in accessibility to 1H-2H exchange of the H-bonded proton of the Glu D1-130 side-chain in D1D2 RC compared to Rps. viridis reaction center.

We have developed a new technique for the study of redox-linked conformational changes in proteins, by the combination of two established techniques. Fourier-transform infrared spectroscopy has been used together with direct electrochemistry of the protein at a modified metal electrode surface. The technique has been evaluated with cytochrome c, because of its well-characterized electrochemistry and because the availability of X-ray crystallographic and NMR studies of both redox states of the protein provides a reference against which our data can be compared. In electrochemical control experiments, it was confirmed that the spectroelectrochemical cell design allows fast, accurate and reproducible control of the redox poise of the protein. The resulting reduced-minus-oxidized infrared difference spectra show the changes in the frequencies and intensities of molecular vibrations which arise from the redox-linked conformational change. In contrast to the absolute infrared spectra of proteins, such difference spectra can be sufficiently straightforward to allow interpretation at the level of individual bonds. A complete interpretation of the spectra is beyond the scope of the present paper: however, on the basis of the data presented, we are able to suggest assignments for all except one of the major bands between 1500 cm-1 and 1800 cm-1.

The proton pumping mechanism of cytochrome c oxidase on a molecular level is highly disputed. Recently theoretical calculations and real time electron transfer measurements indicated the involvement of residues in the vicinity of the ring A propionate of heme a3, including Asp399 and the CuB ligands His 325, 326. In this study we probed the interaction of Asp399 with the binuclear center and characterize the protonation state of its side chain. Redox induced FTIR difference spectra of mutations at the site in direct comparison to wild type, indicate that below pH 5 Asp 399 displays signals typical for the deprotonation of the acidic residue with reduction of the enzyme. Interestingly at a pH higher than 5, no contributions from Asp 399 are evident. In order to probe the interaction of the site with the binuclear center we followed the rebinding of CO by infrared spectroscopy for mutations on residue Asp399 to Glu, Asn and Leu. Previously different CO conformers have been identified for bacterial cytochrome c oxidases, and its pH dependent behaviour discussed to be relevant for catalysis. Interestingly we observe the lack of this pH dependency and a strong influence on the observable conformers for all mutants studied here, clearly suggesting a communication of the site with the heme-copper center and the nearby histidine residues.

The tetrameric state of p53, p63, and p73 has been considered one of the hallmarks of this protein family. While the DNA binding domain (DBD) is highly conserved among vertebrates and invertebrates, sequences C-terminal to the DBD are highly divergent. In particular, the oligomerization domain (OD) of the p53 forms of the model organisms Caenorhabditis elegans and Drosophila cannot be identified by sequence analysis. Here, we present the solution structures of their ODs and show that they both differ significantly from each other as well as from human p53. CEP-1 contains a composite domain of an OD and a sterile alpha motif (SAM) domain, and forms dimers instead of tetramers. The Dmp53 structure is characterized by an additional N-terminal beta-strand and a C-terminal helix. Truncation analysis in both domains reveals that the additional structural elements are necessary to stabilize the structure of the OD, suggesting a new function for the SAM domain. Furthermore, these structures show a potential path of evolution from an ancestral dimeric form over a tetrameric form, with additional stabilization elements, to the tetramerization domain of mammalian p53.

Omp85 proteins form a ubiquitous protein family, members of which are found in all Gram-negative bacteria. Omp85 of Neisseria meningitidis and YaeT of Escherichia coli are shown to be essential for outer membrane biogenesis. Interestingly, there exists a homologue to YaeT in E. coli and many proteobacteria, denoted YtfM, the function of which has not been described yet. Like YaeT, YtfM is predicted to consist of an amino-terminal periplasmic domain and a membrane-located carboxy-terminal domain. In this study, we present a first characterisation of YtfM by comparison to YaeT concerning structural, biochemical and electrophysiological properties. Furthermore, a knockout strain revealed that ytfM is a non-essential gene and lack of the protein had no effect on outer membrane composition and integrity. The only observable phenotype was strongly reduced growth, indicating an important role of YtfM in vivo.

The ϵ-proteobacteria Helicobacter pylori and Campylobacter jejuni are both human pathogens. They colonize mucosal surfaces causing severe diseases. The membrane protein complex QFR (quinol:fumarate reductase) from H. pylori has previously been established as a potential drug target, and the same is likely for the QFR from C. jejuni. In the present paper, we describe the cloning of the QFR operons from the two pathogenic bacteria H. pylori and C. jejuni and their expression in Wolinella succinogenes, a non-pathogenic ϵ-proteobacterium. To our knowledge, this is the first documentation of heterologous membrane protein production in W. succinogenes. We demonstrate that the replacement of the homologous enzyme from W. succinogenes with the heterologous enzymes yields mutants where fumarate respiration is fully functional. We have isolated and characterized the heterologous QFR enzymes. The high quality of the enzyme preparation enabled us to determine unequivocally by analytical ultracentrifugation the homodimeric state of the three detergent-solubilized heterotrimeric QFR enzymes, to accurately determine the different oxidation–reduction (‘redox’) midpoint potentials of the six prosthetic groups, the Michaelis constants for the quinol substrate, maximal enzymatic activities and the characterization of three different anti-helminths previously suggested to be inhibitors of the QFR enzymes from H. pylori and C. jejuni. This characterization allows, for the first time, a detailed comparison of the QFR enzymes from C. jejuni and H. pylori with that of W. succinogenes.

In the past 10 years infrared Spectroscopy has become a valuable tool for the analysis of the primary processes in reaction centers. Because it analyzes vibrational properties of the entire molecule, it can yield information on the protein which is otherwise difficult to obtain. In this chapter, the basic concepts of IR spectroscopy for macromolecules and the reaction-induced difference techniques using Fourier transform and single-wavelength techniques are briefly reviewed. The strategies used for the assignment of vibrational modes are described. Light-induced Fourier transform difference spectra monitoring charge-separated states and electrochemically-induced spectra monitoring the redox transitions of individual cofactors are reported. A major section deals with the vibrational spectra of the quinones in the RC, and information on the modes of the quinones in situ and of their protein sites is discussed. Finally, time-resolved IR experiments at individual wavelengths showing quinone reduction and concomitant protonation processes are reviewed.

During the last decade, infrared spectroscopic techniques have entered the field of photosynthesis and they are meanwhile established methods for probing chlorophyll or quinone molecules in their binding sites and for the investigation of molecular processes in the protein upon electron transfer or proton transfer. This advancement has mainly become possible with the rapid development and availability of Fourier transform infrared (FTIR) spectrophotometers, but also with the development of sensitive infrared semiconducor detectors and tunable IR lasers or picosecond IR laser systems. This chapter introduces the basic concepts for using IR Spectroscopy to study such complex systems as a reaction center or even complete photosynthetic membranes. The basic principles of FTIR spectroscopy for steady-state or time-resolved investigations will be described, and examples from the study of primary electron transfer in reaction centers will be given. Single-wavelength time-resolved techniques for the millisecond-to-nanosecond and pump-probe techniques for the picosecond time domain are reported. A separate section deals with the strategies for the assignment of IR bands, an essential issue for the use of IR techniques in studies of macromolecules. Furthermore, techniques for the preparation of IR samples from photosynthetic membranes or reaction centers are described.

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.

Reconciliation of apparently contradictory experimental results obtained on the quinol:fumarate reductase, a diheme-containing respiratory membrane protein complex from Wolinella succinogenes , was previously obtained by the proposal of the so-called “E pathway hypothesis.” According to this hypothesis, transmembrane electron transfer via the heme groups is strictly coupled to cotransfer of protons via a transiently established pathway thought to contain the side chain of residue Glu-C180 as the most prominent component. Here we demonstrate that, after replacement of Glu-C180 with Gln or Ile by site-directed mutagenesis, the resulting mutants are unable to grow on fumarate, and the membrane-bound variant enzymes lack quinol oxidation activity. Upon solubilization, however, the purified enzymes display ≈1/10 of the specific quinol oxidation activity of the wild-type enzyme and unchanged quinol Michaelis constants, K m . The refined x-ray crystal structures at 2.19 Å and 2.76 Å resolution, respectively, rule out major structural changes to account for these experimental observations. Changes in the oxidation–reduction heme midpoint potential allow the conclusion that deprotonation of Glu-C180 in the wild-type enzyme facilitates the reoxidation of the reduced high-potential heme. Comparison of solvent isotope effects indicates that a rate-limiting proton transfer step in the wild-type enzyme is lost in the Glu-C180 → Gln variant. The results provide experimental evidence for the validity of the E pathway hypothesis and for a crucial functional role of Glu-C180.

In pyruvate oxidase (POX) from Lactobacillus plantarum, valine 265 participates in binding the cofactor FAD and is responsible for the strained conformation of its isoalloxazine moiety that is visible in the crystal structure of POX. The contrasting effects of the conservative amino acid exchange V265A on the enzyme's catalytic properties, cofactor affinity, and protein structure were investigated. The most prominent effect of the exchange was observed in the 2.2 A crystal structure of the mutant POX. While the overall structures of the wild-type and the variant are similar, flavin binding in particular is clearly different. Local disorder at the isoalloxazine binding site prevents modeling of the complete FAD cofactor and two protein loops of the binding site. Only the ADP moiety shows well-defined electron density, indicating an "anchor" function for this part of the molecule. This notion is corroborated by competition experiments where ADP was used to displace FAD from the variant enzyme. Despite the fact that the affinity of FAD binding in the variant is reduced, the catalytic properties are very similar to the wild-type, and the redox potential of the bound flavin is the same for both proteins. The rate of electron transfer toward the flavin during turnover is reduced to one-third compared to the wild-type, but k(cat) remains unchanged. Redox-triggered FTIR difference spectroscopy of free FAD shows the nu(C(10a)=N(1)) band at 1548 cm(-)(1). In POX-V265A, this band is found at 1538 cm(-)(1) and thus shifted less strongly than in wild-type POX where it is found at 1534 cm(-)(1). Taking these observations together, the conservative exchange V265A in POX has a surprisingly small effect on the catalytic properties of the enzyme, whereas the effect on the three-dimensional structure is rather big.

Lipid protein interactions play a key role in the stability and function of various membrane proteins. Earlier we have reported the extreme thermal stability of porin from Paracoccus denitrificans reconstituted into liposomes. Here, we used Fourier transform infrared spectroscopy for a label free analysis of the global secondary structural changes and local changes in the tyrosine microenvironment. Our results show that a mixed lipid system (non-uniform bilayer) optimizes the thermal stability of porin as compared to the porin in pure lipids (uniform bilayer) or detergent micelles. This is in line with the fact that the bacterial outer membrane is a dynamic system made up of lipids of varying chain lengths, head groups and the barrel wall height contacting the membrane is uneven.

The caa3-oxidase from Thermus thermophilus has been studied with a combined electrochemical, UV/VIS and Fourier-transform infrared (FT-IR) spectroscopic approach. In this oxidase the electron donor, cytochrome c, is covalently bound to subunit II of the cytochrome c oxidase. Oxidative electrochemical redox titrations in the visible spectral range yielded a midpoint potential of -0.01 +/- 0.01 V (vs. Ag/AgCl/3m KCl, 0.218 V vs. SHE') for the heme c. This potential differs for about 50 mV from the midpoint potential of isolated cytochrome c, indicating the possible shifts of the cytochrome c potential when bound to cytochrome c oxidase. For the signals where the hemes a and a3 contribute, three potentials, = -0.075 V +/- 0.01 V, Em2 = 0.04 V +/- 0.01 V and Em3 = 0.17 V +/- 0.02 V (0.133, 0.248 and 0.378 V vs. SHE', respectively) could be obtained. Potential titrations after addition of the inhibitor cyanide yielded a midpoint potential of -0.22 V +/- 0.01 V for heme a3-CN- and of Em2 = 0.00 V +/- 0.02 V and Em3 = 0.17 V +/- 0.02 V for heme a (-0.012 V, 0.208 V and 0.378 V vs. SHE', respectively). The three phases of the potential-dependent development of the difference signals can be attributed to the cooperativity between the hemes a, a3 and the CuB center, showing typical behavior for cytochrome c oxidases. A stronger cooperativity of CuB is discussed to reflect the modulation of the enzyme to the different key residues involved in proton pumping. We thus studied the FT-IR spectroscopic properties of this enzyme to identify alternative protonatable sites. The vibrational modes of a protonated aspartic or glutamic acid at 1714 cm-1 concomitant with the reduced form of the protein can be identified, a mode which is not present for other cytochrome c oxidases. Furthermore modes at positions characteristic for tyrosine vibrations have been identified. Electrochemically induced FT-IR difference spectra after inhibition of the sample with cyanide allows assigning the formyl signals upon characteristic shifts of the nu(C=O) modes, which reflect the high degree of similarity of heme a3 to other typical heme copper oxidases. A comparison with previously studied cytochrome c oxidases is presented and on this basis the contributions of the reorganization of the polypeptide backbone, of individual amino acids and of the hemes c, a and a3 upon electron transfer to/from the redox active centers discussed.

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-Å 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.

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.

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 ν(C=O) and ν(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.

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.

The α-amylase inhibitor tendamistat (74 amino acid residues, approx. 8 kDa) consists of two three-stranded anti-parallel β-sheets (69% of the amino acid residues) completed by loops and irregular structure [1]. Tendamistat is thus well suitable for investigating the unfolding and refolding process of β-sheet structure. Here, we present FTIR data of the temperature dependent denaruration of tendamistat.

Time-resolved infrared difference spectra of the ATP-induced phosphorylation of the sarcoplasmic reticulum Ca2+-ATPase have been recorded in H2O and 2H2O at pH 7.0 and 1 degrees C. The reaction was induced by ATP release from P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate (caged ATP) and from [gamma-18O3]caged ATP. A band at 1546 cm-1, not observed with the deuterated enzyme, can be assigned to the amide II mode of the protein backbone and indicates that a conformational change associated with ATPase phosphorylation takes place after ATP binding. This is also indicated between 1700 and 1610 cm-1, where bandshifts of up to 10 cm-1 observed upon protein deuteration suggest that amide I modes of the protein backbone dominate the difference spectrum. From the band positions it is deduced that alpha-helical, beta-sheet, and probably beta-turn structures are affected in the phosphorylation reaction. Model spectra of acetyl phosphate, acetate, ATP, and ADP suggest the tentative assignment of some of the bands of the phosphorylation spectrum to the molecular groups of ATP and Asp351, which participate directly in the phosphate transfer reaction: a positive band at 1719 cm-1 to the C==O group of aspartyl phosphate, a negative band at 1239 cm-1 to the nuas(PO2-) modes of the bound ATP molecule, and a positive band at 1131 cm-1 to the nuas(PO32-) mode of the phosphoenzyme phosphate group, the latter assignment being supported by the band's sensitivity toward isotopic substitution in the gamma-phosphate of ATP. Band positions and shapes of these bands indicate that the alpha- and/or beta-phosphate(s) of the bound ATP molecule become partly dehydrated when ATP binds to the ATPase, that the phosphoenzyme phosphate group is unprotonated at pH 7.0, and that the C==O group of aspartyl phosphate does not interact with bulk water. The Ca2+ binding sites seem to be largely undisturbed by the phosphorylation reaction, and a functional role of the side chains of Asn, Gln, and Arg residues was not detected.

The molecular processes concomitant with the redox reactions of wild-type and mutant cytochrome c oxidase from Paracoccus denitrificans were analyzed by a combination of protein electrochemistry and Fourier transform infrared (FTIR) difference spectroscopy. Oxidized-minus-reduced FTIR difference spectra in the mid-infrared (4000-1000 cm-1) reflecting full or stepwise oxidation and reduction of the respective cofactor(s) were obtained. In the 1800-1000 cm-1 range, these FTIR difference spectra reflect changes of the polypeptide backbone geometry in the amide I (ca. 1620-1680 cm-1) and amide II (ca. 1560-1540 cm-1) region in response to the redox transition of the cofactor(s). In addition, several modes in the 1600-1200 cm-1 range can be tentatively attributed to heme modes. A peak at 1746 cm-1 associated with the oxidized form and a peak at 1734 cm-1 associated with the reduced form were previously discussed by us as proton transfer between Asp or Glu side chain modes in the course of the redox reaction of the enzyme [Hellwig, P., Rost, B., Kaiser, U., Ostermeier, C., Michel, H., and Mantele, W. (1996) FEBS Lett. 385, 53-57]. These signals were resolved into several components associated with the oxidation of different cofactors. For a stepwise potential titration from the fully reduced state (-0.5 V) to the fully oxidized state (+0.5 V), a small component at 1738 cm-1 develops in the potential range of approximately +0.15 V and disappears at more positive potentials while the main component at 1746 cm-1 appears in the range of approximately +0.20 V (all potentials quoted vs Ag/AgCl/3 M KCl). This observation clearly indicates two different ionizable residues involved in redox-induced proton transfer. The major component at 1746 cm-1 is completely lost in the FTIR difference spectra of the Glu 278 Gln mutant enzyme. In the spectrum of the subunit I Glu 278 Asp mutant enzyme, the major component of the discussed difference band is lost. In contrast, the complete difference signal of the wild-type enzyme is preserved in the Asp 124 Asn, Asp 124 Ser, and Asp 399 Asn variants, which are critical residues in the discussed proton pump channel as suggested from structure and mutagenesis experiments. On the basis of these difference spectra of mutants, we present further evidence that glutamic acid 278 in subunit I is a crucial residue for the redox reaction. Potential titrations performed simultaneously for the IR and for the UV/VIS indicate that the signal related to Glu 278 is coupled to the electron transfer to/from heme a; however, additional involvement of CuB electron transfer cannot be excluded.

The redox reaction of proteins are essential steps in biological energy conversion. Electron transfer to a redox cofactor and subsequent protein reactions can be monitored by combination of electrochemical and spectroscopic techniques using thin transparent electrodes prepared by sputtering techniques. Here the electrochemical reactions of cytochrome c (cyt c) at 4, 4′-dithiodipyridine coated gold electrodes are characterized.

In order to investigate the changes of protonation or environment of carboxylic residues occurring upon photoreduction of the secondary quinone acceptor (QB) in the reaction center (RC) of the photosynthetic bacteria Rhodobacter sphaeroides 2.4.1., we have performed light-induced Fourier transform infrared (FTIR) spectroscopy on RCs from wild-type (Wt) and several site-directed mutants. The FTIR QB-/QB spectra have been obtained at pH 7 upon single-saturating flash excitation for native RCs and RC mutants containing either a single-site mutation, with Gln at L212 (EQ L212), Asn at L213 (DN L213), or Asn at L210 (DN L210), or a double-site mutation with both Gln at L212 and Asn at L213 (EQ L212 + DN L213). The assignment of an IR band to the protonation/deprotonation of a particular carboxylic side chain was analyzed by combining the effects of site-directed mutagenesis and 1H/2H isotope exchange. A positive band at 1728 cm-1 in the QB-/QB spectra was observed in Wt, DN L213, and DN L210 and was absent in the mutants EQ L212 and EQ L212 + DN L213. The intensity of the 1728 cm-1 band was significantly reduced in 2H2O, and a new feature appears at 1717 +/- 1 cm-1. Furthermore, the amplitude of the 1728 cm-1 band was similar in native and DN L210 RCs but was increased in DN L213. This band is attributed to partial proton uptake by Glu L212 estimated to be 0.3-0.4 H+/QB- in native and DN L210 RCs and O.5-0.6 H+/QB- in DN L213 RCs. In contrast, the FTIR QB-/QB spectra show no evidence for change of protonation or environment of Asp L213 upon QB- formation. The increased protonation of Glu L212 in DN L213 RCs is explained by a decreased Glu L212 pKa value due to the loss of a negatively charged Asp L213. Part of a small differential signal at 1732 (+)/1740 (-) cm-1 that is affected by 1H/2H exchange is tentatively assigned to an environmental shift of the protonated Asp L210. A negative signal at 1685 cm-1 is propose to arise from the absorption change of the amide I carbonyl mode of Glu L212.(ABSTRACT TRUNCATED AT 400 WORDS)

The protonation events that occur upon QA-QB-->QAQB- electron transfer in photosynthetic reaction centers from Rhodobacter sphaeroides were investigated by time-resolved infrared spectroscopy using tunable diode lasers as previously described [Mantele, W., Hienerwadel, R., Lenz, F., Riedel, E. J., Grisar, R., & Tacke, M. (1990) Spectrosc. Int. 2, 29-35; Hienerwadel, R., Thibodeau, D. L., Lenz, F., Nabedryk, E., Breton, J., Kreutz, W., & Mantele, W. (1992) Biochemistry 31, 5799-5808]. In the mid-infrared region between 1695 and 1780 cm-1, transient signals associated with QA-QB-->QAQB- electron transfer were observed and characterized. The dominant transient absorbance changes are three positive signals at 1732, 1725, and 1706 cm-1 and two negative signals at 1716 and at 1698 cm-1. The 1725 cm-1-signal disappears upon 1H-->2H exchange as expected for an accessible COOH group and is absent in Glu L212 Gln mutant reaction centers. On this basis, we propose an assignment of this signal to the COOH group of Glu L212. The other signals could correspond to intensity changes and/or shifts of other carboxylic residues, although contributions from ester C = O groups of bacteriopheophytins cannot be ruled out. In native reaction centers at pH 7 and at 4 degrees C, biphasic kinetics of the transient components were observed at most frequencies. The major signal at 1725 cm-1 exhibits a fast kinetic component of t 1/2 = 0.18 ms (25% of the total amplitude) and a slow one of t1/2 = 1 ms (75% of the total amplitude). A global fit analysis of the signals between 1695 and 1780 cm-1 revealed that the spectral distributions of the fast and the slow components are different. Biphasic kinetics with comparable half-times were also observed for the Glu L212 to Gln mutant. The simplest model to explain these results is that the fast phase represents electron transfer and the slow phase represents proton transfer and/or conformational changes coupled to electron transfer. The difference spectra of the slow component from native reaction centers show that the 1725 cm-1 band corresponds to an absorbance increase and not to a shift of an existing band. The signal is therefore proposed to arise from the protonation of Glu L212. The amplitude of the 1725 cm-1 signal varies distinctly with pH as expected for protonation of a COO- group. With increasing pH, the amplitude of the slow component increases while that of the fast component decreases slightly.(ABSTRACT TRUNCATED AT 400 WORDS)

Ca 2+ binding to sacoplasmic reticulum Ca 2+ -ATPase was investigated by Fourier transform infrared (FTIR) spectroscopy using the photolytic release of Ca 2+ from the photolabile Ca 2+ chelator 1-(2-nitro-4,5-dimethoxy)- N , N , N ′, N ′,-tetrakis[(oxy-carbonyl)]methyl-1,2-ethandiamine (DM-nitrophen). IR absorbance changes in 1 H 2 O and 2 H 2 O were detected in the spectral region from 1800 cm −1 to 1200 cm −1 , reflecting photolysis of DM-nitrophen and Ca 2+ binding to the Ca 2+ -ATPase. As an independent probe for protein conformational changes, intrinsic fluorescence changes upon Ca 2+ release were monitored simultaneously to the FTIR measurements. Both the IR absorbance changes and the fluorescence intensity changes correlated well with the Ca 2+ binding activity of the ATPase in this specific step. Ca 2+ binding caused IR difference bands mainly in the region of amide I absorption of the polypeptide backbone, reflecting conformational changes of the protein. The small amplitude of the signals indicates that only a few residues perform local structural changes such as changes of bond angles or hydrogen bonding. Other absorbance changes appearing above 1700 cm −1 can be assigned to Ca 2+ binding to Glu or Asp side chain carboxyl groups and concomitant deprotonation of these residues. This assignment is strengthened by downshifts of these bands by 4 cm −1 to 6 cm −1 upon 1 H 2 O/ 2 H 2 O exchange. This is in line with results of mutagenesis studies where such residues containing carboxyl groups were associated with the high affinity Ca 2+ binding site (Clarke, D.M., Loo, T.W. and MacLennan, D.H. (1990) J. Biol. Chem. 265, 6262–6267).

Infrared difference spectroscopy was used to get more insight into the transport mechanism of sarcoplasmic reticulum Ca2+-ATPase. Several partial reactions were induced directly in the infrared cuvette via photolytic release of ligands or substrates from photolabile precursors (1,2). From the spectra before and after the release a difference spectrum can be calculated that reflects only those changes of infrared absorption which are associated with the catalytic reactions and the triggering photolysis reaction. The accuracy of this method is extremely high, since it avoids possible inaccuracies when spectra of two samples prepared in different enzyme states are compared.

Electron transfer in photosynthetic reaction centers from Rhodobacter sphaeroides was investigated by time-resolved infrared spectroscopy using tunable lead salt diode lasers. In the spectral region between approximately 1690 and 1770 cm−1, transient µsec to msec signals could be characterized upon electron transfer from the primary quinone acceptor QA to he secondary acceptor QB. The dominant transient absorbance changes are three positive sig. s at 1725, 1735, and 1706 cm−1 and two negative signals at ca. 1716 and at 1698 cm−1. They are interpreted in terms of the C=O frequencies of aspartic or glutamic acid side chain residues affected by electron transfer. The major signal at 1725 cm−1 exhibits a fast kinetic component of t1/2 = 0.2 ms and a slow one oft t1/2 = 1 ms at pH 7. Analysis of its time course and amplitude under different conditions (pH, 1H/2H echange, mutations) allows its assignment to glutamic acid L212 near QB and indicates the uptake of a significant amount of a proton upon \( {{Q}_{{{{B}^{ - }}}}} \) formation. We propose that this proton transfer leads to a favorable electrostatic interaction in the site since it lowers the interaction energy of Glu L212 with \( {{Q}_{{{{B}^{ - }}}}} \).

Infrared spectroscopic methods have been developed in the past decade to a sensitivity and selectivity which renders them useful for the study of enzyme function and enzyme reaction mechanisms. Originally developed as difference techniques for the investigation of light-induced reactions of photoreactive proteins, and matured in the field of bacteriorhodopsin and rhodopsin, they can now be used for the study of redox proteins by the use of electrochemical cells, or for the study of many different enzymes by the use of photolabile effector molecules. This brief review summarizes the currently available methods of infrared difference spectroscopy, the technical prerequisites, achievements and limitations.

The nicotinic acetylcholine receptor (n-AChR) is a ligand gated ion channel (type-1 receptor) transducing extracellular signals through plasma membranes (1–3). Because of the lack of appropriate crystals, the three dimensional structure of the AChR as determined by X-ray chrystallography is not yet known. Secondary structure prediction, based on hydrophobicity analysis according to Kyte and Doolittle (4) and Fourier analysis of the hydrophobicities of the four different receptor subunit sequences (5,6) led to several proposals as to the transmembrane folding of the polypeptide chains. In order to obtain reliable information on predicting the secondary structure FTIR spectroscopy has been used (7). Furthermore the binding and interaction of charbamoylcholine as an agonist with the n-AChR was investigated using photolytically released carbamoylcholine (“caged” carbamoylcholine) and difference FTIR spectroscopy (8).

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

Reaction-induced infrared difference spectroscopy has long been used to study the molecular basis of light-induced reactions in pigment-protein complexes. The techniques used are either light-minus-dark Fourier transform infrared (FTIR) spectroscopy for steady states or flash-induced time-resolved IR and FTIR techniques for transient reactions, with the major focus on bacteriorhodopsin, rhodopsin, and the photosynthetic reaction center (for reviews, see [1–3]). Upon development of protein electrochemical techniques and ultra-thin-layer IR spectroelectrochemical cells, the basic concepts of IR difference spectroscopy could be extended to the large class of redox proteins and enzymes [4–6]. Yet, enzyme-substrate reactions in general, ATPases, and many other protein reactions started by an effector molecule could only be studied for very few cases by IR difference techniques, since the mixing of the effector to the enzyme presents too large a perturbation for the sensitivity needed in these experiments.

Ca2+ transport from the cytoplasm of muscle cells into SR, necessary for muscle relaxation, is performed by the Ca2+- ATPase, an intrinsic membrane protein of about 110 kDa molecular mass. The energy required for this active transport is provided by hydrolysis of ATP. Although the catalytic cycle of the ATPase has been subject of detailed investigation, the molecular nature of the transport mechanism is still unknown. The reaction cycle involves successive Ca2+ binding (E2/E1→Ca2E1), formation of an ADP-sensitive phosphoenzyme with occluded Ca2+, formation of an ADP-insensitive phosphoenzyme and concomitant Ca2+ release to the SR lumen and deposphorylation of the ATPase [1].

This chapter focuses on the infrared vibrational spectroscopy of the photosynthetic reaction center. IR vibrational spectroscopy in photosynthesis has provided direct evidence for the participation of the protein in charge separation and stabilization. It provides information not only on the neutral, quiescent state of the RC but also on the active state and, with time resolution available, on the dynamic processes that occur on photoactivation. For the Quinone environment, combined steady-state and time-resolved IR spectroscopy in conjunction with structural information may soon provide mechanistic details of electron transfer and protonation. An important aspect of IR difference spectroscopy is its applicability to sample forms ranging from an intact membrane to isolated or even crystallized reaction centers. The applicability of IR difference spectroscopy to intact membranes also facilitates studies of site-directed mutants as a fairly quick test of structure–function relationships. Rapid progress in site-directed mutagenesis will lead to significant advances. By combining the rapidly advancing IR techniques with a solid band assignment, it is possible to provide a consistent picture of the intraprotein molecular mechanisms of primary photosynthetic electron transport.

The bacterial photosynthetic reaction center (RC) of Rhodobacter (Rb.) sphaeroides performs light-induced charge separation by transferring an electron from a donor P (a bacteriochlorophyll a-dimer) to the acceptors QA and QB (quinones). With X-ray analysis of RC crystals [1] it has been possible to obtain information on the level of individual bonds. Since only the ‘resting’ state of the protein is observed, and atom positions should be considered as average values, other methods are necessary to study the small changes the RC undergoes upon charge-separation.

Infrared spectroscopy has proven to be a valuable tool for the study of enzyme structure and function. Using differential techniques, molecular changes in the course of photoreactions and redox reactions have been studied (for a review see [1]). Recently, the use of photolabile substrate analogues, which can be released by a UV flash, has been reported for the study of ATPases [2,3]. This technique of photo-chemo-triggering a reaction can be used for many different enzymes (see Barth et al., these proceedings), provided that a suitable photolabile compound is available.

Light induced charge separation in the reaction center (RC) of Rb. sphaeroides leads to the formation of the oxidized primary donor P+ and the semiquinone anion QA ‒ on a picosecond time scale. Electron transfer to the secondary acceptor QB, if present, proceeds in ≈100 µs. Charge recombination between P+ and QB ‒ occurs in 1 to 5 s. The electron transfer between QA and QB leads to proton uptake by ionizable residues in the vicinity of QB. These residues have been identified to be most likely carboxylates [1]. Proton uptake is expected either to occur as fast as the electron transfer or on a slightly slower time scale, depending on the mechanisms involved in the proton transfer to the QB binding pocket.

Light-induced forward electron transfer in the bacterial photosynthetic reaction center from Rhodobacter sphaeroides was investigated by time-resolved infrared spectroscopy. Using a highly sensitive kinetic photometer based on a tunable IR diode laser source [Mantele, W., Hienerwadel, R., Lenz, F., Riedel, W. J., Grisar, R., & Tacke, M. (1990a) Spectrosc. Int. 2, 29-35], molecular processes concomitant with electron-transfer reactions were studied in the microsecond-to-second time scale. Infrared (IR) signals in the 1780-1430-cm-1 spectral region, appearing within the instrument time resolution of about 0.5 microseconds, could be assigned to molecular changes of the primary electron donor upon formation of a radical cation and to modes of the primary quinone electron acceptor QA and its environment upon formation of QA-. These IR signals are consistent with steady-state FTIR difference spectra of the P+Q- formation [Mantele, W., Nabedryk, E., Tavitian, B. A., Kreutz, W., & Breton, J. (1985) FEBS Lett. 187, 227-232; Mantele, W., Wollenweber, A., Nabedryk, E., & Breton, J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8468-8472; Nabedryk, E., Bagley, K. A., Thibodeau, D. L., Bauscher, M., Mantele, W., & Breton, J. (1990) FEBS Lett. 266, 59-62] and with time-resolved FTIR studies [Thibodeau, D. L., Nabedryk, E., Hienerwadel, R., Lenz, F., Mantele, W., & Breton, J. (1990) Biochim. Biophys. Acta 1020, 253-259]. At given wavenumbers, kinetic components with a half-time of approximately 120 microseconds were observed and attributed to QA----QB electron transfer. The time-resolved IR signals, in contrast to steady-state experiments where full protein relaxation after electron transfer can occur, allow us to follow directly the modes of QA and QB and their protein environment under conditions of forward electron transfer. Apart from signals attributed to the primary electron donor, signals are proposed to arise not only from the C = O and C = C vibrational modes of the neutral quinones and from the C-O and C-C vibrations of their semiquinone anion form but also from amino acid groups forming their binding sites. Some of the signals appearing with the instrument rise time as well as the transient 120-microseconds signals are interpreted in terms of binding and interaction of the primary and secondary quinone electron acceptor in the Rb. sphaeroides reaction center and of the conformational changes in their binding site.(ABSTRACT TRUNCATED AT 400 WORDS)

Primary photosynthetic reactions feature rapid kinetics with high quantum yield of stable charge separation occurring in basic units called reaction centers (RC). The X-ray structure of bacterial RC [1,2] suggests that the efficiency and the stability of the charge separation rely at least partly on the protein environment to optimize the orientation and the localization of the various redox components involved in electron transport. The differences in the nature and organization of the aminoacids forming the binding pocket of the two quinones, QA and QB, are thought to be responsible for their different properties in JRC. Indeed, the two quinones are unequivalent, QA (menaquinone for Rps. viridis and ubiquinone for Rb. sphaeroides) is more tightly bound to the protein than QB (ubiquinone for both RC), and they display different redox properties i.e. QA is a one-electron acceptor while QB can accept two electrons under physiological conditions.

Light-induced electron transport in bacterial photosynthetic reaction centers leads to the creation of a charge-separated state stable for milliseconds to seconds. The structures provided by X-ray crystallography (Michel et aL, 1986; Allen et al., 1988; Deisenhofer & Michel, 1989; El-Kabbani et al, 1991) constitute a unique guideline to address questions on how the function may be related to the arrangement of the cofactors and of specific amino acid residues in their vicinity. The sequence of electron transfer reactions, the identity of the reaction partners, and the reaction mechanisms have been characterized from static and time-resolved absorbance measurements (for a review, see Parson & Ke, 1982). Transfer of the first electron to the primary (QA) and secondary (QB) quinone electron acceptors has received considerable attention, since it is associated with intraprotein protolytic reactions (for a recent review, see Okamura & Feher, 1992), which have a potential role in electrostatic charge stabilization.

A sensitive and quick assay for redox proteins based on electrochemical titrations in a thin-layer electrochemical cell is described. Using a combination of modified-electrode and "mediator-enhanced" electrochemistry, equilibration of the cell volume (4 microliters) with the applied potential allows series of spectra as a function of the potential to be recorded rapidly. A complete redox titration between +500 and -600 mV (vs Ag/AgCl/3 M KCl) in 30-mV intervals takes approximately 2 h. The detection limit of the assay, evaluated for cytochrome c at the alpha-band absorption, is quoted to approximately 100 pmol. The use of this redox assay for the detection of redox-active contaminants in biochemical preparations, for the determination of midpoint potentials of redox enzymes, and for the characterization of complex membrane-bound or soluble redox systems is described.

Fourier transform infrared spectroscopy was used to investigate ligand binding and conformational changes in the Ca2(+)-ATPase of sarcoplasmic reticulum during the catalytic cycle. The ATPase reaction was started in the infrared sample by release of ATP from the inactive, photolabile ATP derivative P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate (caged ATP). Absorption spectroscopy in the visible spectral region using the Ca2(+)-sensitive dye Antipyrylazo III ensured that the infrared samples were able to transport Ca2+ in spite of their low water content, which is required for mid-infrared measurements (1800-950 cm-1). Small, but characteristic and highly reproducible infrared absorbance changes were observed upon ATP release. These infrared absorbance changes exhibit different kinetic properties. Comparison with model compound infrared spectra indicates that they are related to photolysis of caged ATP, hydrolysis of ATP in consequence of ATPase activity and to molecular changes in the active ATPase. The absorbance changes due to alterations in the ATPase were observed mainly in the region of Amide I and Amide II protein absorbance and presumably reflect the molecular processes upon phosphoenzyme formation. Since the absorbance changes were small compared to the overall ATPase absorbance, no major rearrangement of ATPase conformation as the result of catalysis could be detected.