More than two pyrrole tautomers of mesoporphyrin stabilized by a protein. High resolution optical spectroscopic study.

Mesoporphyrin IX substituted horseradish peroxidase was studied by fluorescence line narrowing and hole burning techniques at cryogenic temperatures. The spectral data reveal that four pyrrole tautomeric configurations of the chromophore are populated within the protein under the influence of irradiation and/or thermal treatment, and the existence of a fifth and a sixth tautomeric configuration is also likely. The relative population of the tautomers changes upon deuterium substitution through modification of the phototransition rate, and also depends on pH, which changes the protonation of neighboring amino acids in the heme pocket. The energy separation of the origins of the tautomers is approximately 100 cm-1. The distribution of barrier heights separating the different tautomeric forms in the ground state and their distribution was determined by temperature cycling hole burning. The distributions can be approximated by Gaussians. The experiment directly yields the distributions on a relative temperature scale, and a model is presented to transform the barrier heights into energy values. It is suggested that the energies for the tautomers are split partially due to the protein crystal field and that the trapping of the tautomeric forms is the consequence of interactions with neighboring amino acids within the heme pocket.     

Differences in the binding of aromatic substrates to horseradish peroxidase revealed by fluorescence line narrowing

The heme in horseradish peroxidase (HRP) isoenzyme C was replaced by mesoporphyrin (MP), and the binding effect of the aromatic substrates benzo-and naphthohydroxamic acid (BHA, NHA), resorcinol (RE), isomeric resorcylic acids (alpha-, beta-, gamma-RE), and hydroquinone (HQ) was studied at pH 5 by conventional and laser-excited fluorescence spectroscopy on the basis of the signal of the porphyrin. Under laser excitation at cryogenic temperatures site selection was demonstrated, and the fluorescence line narrowing data were used to characterize the HRP/substrate complexes by the inhomogeneous distribution function for the S0----S1 (0----0) transition energy and the vibrational energies in the S1 electronic state. A comparison with ground-state vibrational energies for MP in chloroform/ether showed a downward shift in vibrational energies for S1 by approximately 20 cm-1. The association characteristics of the substrates were in accordance with previous literature data indicating NHA to be of the strongest binding affinity. For BHA, spectral evidence was obtained for a second type of binding site where hydrophobic interactions with the porphyrin ring may be possible. The effect of the RE's was similar to each other, but only beta-RE showed saturation. Complexation in every case caused the strong reduction of the splitting in the 0----0 transition energy for the tautomeric forms of MP and an increase in the 0----0 energy by 100-200 cm-1 depending on the substrate. The substrate binding also affected the phonon coupling of vibronic transitions exciting into the delta v = 927- and 976-cm-1 modes; in the latter case, the vibrational energy was also increased to 983 cm-1 for beta-RE. In the same energy range, however, the transition into the delta nu = 958-960-cm-1 mode was not affected by binding. Both the magnitude of the energy shifting and the change in the strength of phonon coupling gave the same relation, BHA less than NHA less than HQ less than RE's, indicating a common conformational origin. A reduction of the fluctuational freedom of the protein chain at room temperature within the heme pocket was suggested on the basis of the reduction of the width of the inhomogeneous distribution of 0----0 energies (from 60-70 to approximately 30 cm-1 in case of HRP/HQ) upon substrate binding. Ways to relate the transition energy splitting and shifting effects to conformational changes are discussed by invoking the Jahn-Teller effect.     

Inhomogeneous broadening in spectral bands of carbonmonoxymyoglobin. The connection between spectral and functional heterogeneity.

The rebinding kinetics of CO to myoglobin after flash photolysis is nonexponential in time below approximately 180 K; the kinetics is governed by a distribution of enthalpic barriers. This distribution results from inhomogeneities in the protein conformation, referred to as conformational substates. Hole-burning experiments on the Soret and IR CO-stretch bands test the assumption that an inhomogeneous distribution of conformational substates results in inhomogeneously broadened spectra. CO was slowly photolyzed at different wavelengths in the Soret band at 10 K. Both the Soret band and the CO-stretch band A1, centered at 1,945 cm-1, shift during photolysis, demonstrating that different wavelengths excite different parts of the distributed population. We have also done kinetic hole-burning experiments by measuring peak shifts in the Soret and A1 bands as the CO molecules rebind. The shifts indicate that the spectral and enthalpic distributions are correlated. In the A1 band, the spectral and enthalpic distributions are highly correlated while in the Soret the correlation is weak. From the peak shifts in the spectral and kinetic hole-burning experiments the inhomogeneous broadening is estimated to be approximately 15% of the total width in the Soret band and approximately 60% in A1. We have previously measured the tilt angle alpha between the bound CO and the heme normal (Ormos, P., D. Braunstein, H. Frauenfelder, M. K. Hong, S.-L. Lin, T. B. Sauke, and R. D. Young. 1988. Proc. Natl. Acad. Sci. USA. 85:8492-8496) and observed a wave number dependence of the tilt angles within the CO-stretch A bands. Thus the spectral and enthalpic distributions of the A bands are coupled to a heterogeneity of the structure.     

Trehalose effect on low temperature protein dynamics: fluctuation and relaxation phenomena

We performed spectral diffusion experiments in trehalose-enriched glycerol/buffer-glass on horseradish peroxidase where the heme was replaced by metal-free mesoporphyrin IX, and compared them with the respective behavior in a pure glycerol/buffer-glass (Schlichter et al., J. Chem. Phys. 2000, 112:3045-3050). Trehalose has a significant influence: spectral diffusion broadening speeds up compared to the trehalose-free glass. This speeding up is attributed to a shortening of the correlation time of the frequency fluctuations most probably by preventing water molecules from leaving the protein interior. Superimposed to the frequency fluctuation dynamics is a relaxation dynamics that manifests itself as an aging process in the spectral diffusion broadening. Although the trehalose environment speeds up the fluctuations, it does not have any influence on the relaxation. Both relaxation and fluctuations are governed by power laws in time. The respective exponents do not seem to change with the protein environment. From the spectral dynamics, the mean square displacement in conformation space can be determined. It is governed by anomalous diffusion. The associated frequency correlation time is incredibly long, demonstrating that proteins at low temperatures are truly nonergodic systems.     

Energy selection is not correlated in the Qx and Qy bands of a Mg-porphyrin embedded in a protein

The Qx-Qy splitting observed in the fluorescence excitation spectra of Mg-mesoporphyrin-IX substituted horseradish peroxidase (MgMP-HRP) and of its complex with naphthohydroxamic acid (NHA) was studied by spectral hole burning techniques. The width of a hole directly burnt in the Qy band and that of a satellite hole indirectly produced in Qy as a result of hole burning in Qx was compared. We also studied the dependence of the satellite hole in the Qy band on the burning frequency used in the Qx band. Both the directly and indirectly burnt holes were very broad in the (higher energy) Qy band. The width of the satellite hole in the Qy band was equal to the entire width of the inhomogeneously broadened band, independently from the position of hole burning in Qx. This is indicative of a clear lack of correlation between the electronic transition energies of the Qx and Qy bands. A photoproduct was produced by laser irradiation of the MgMP-HRP/NHA complex and was identified as a species with lowered Q-splitting. Conversion of the photoproduct could be achieved by thermal activation measured in temperature-cycling experiments, with a characteristic temperature of 25 K. We attribute the phototransformation to a conformational change of MgMP.     

Ultrafast infrared spectroscopy of bacteriorhodopsin.

Picosecond infrared spectroscopy is developed and used for the first time to study the dynamics of photoexcited bacteriorhodopsin (BR). Both spectral and time-resolved data are obtained. The results open an entirely new approach to investigations of the BR photocycle. The infrared difference spectrum (K minus BR570) recorded at ambient temperature between 1,560 and 1,700 cm-1 is not identical with the spectrum reported for a frozen sample. Three bands of the K state at 1,622, 1,610, and 1,580 cm-1 and the bleaching at 1,637 cm-1 (C = NH stretch) are seen. These new spectral lines appear in less than 10 ps.     

Thermal fluctuations between conformational substates of the Fe(2+)-HisF8 linkage in deoxymyoglobin probed by the Raman active Fe-N epsilon (HisF8) stretching vibration.

We have measured the VFe-His Raman band of horse heart deoxymyoglobin dissolved in an aqueous solution as a function of temperature between 10 and 300 K. The minimal model to which these data can be fitted in a statistically significant and physically meaningful way comprises four different Lorentzian bands with frequencies at 197, 209, 218, and 226 cm-1, and a Gaussian band at 240 cm-1, which exhibit halfwidths between 10 and 12.5 cm-1. All these parameters were assumed to be independent of temperature. The temperature dependence of the apparent total band shape's frequency is attributed to an intensity redistribution of the subbands at omega 1 = 209 cm-1, omega 2 = 218 cm-1, and omega 3 = 226 cm-1, which are assigned to Fe-N epsilon (HisF8) stretching modes in different conformational substrates of the Fe-HisF8 linkage. They comprise different out-of-plane displacements of the heme iron. The two remaining bands at 197 and 240 cm-1 result from porphyrin modes. Their intensity ratio is nearly temperature independent. The intensity ratio I3/I2 of the vFe-His subbands exhibits a van't Hoff behavior between 150 and 300 K, bending over in a region between 150 and 80 K, and remains constant between 80 and 10 K, whereas I2/I1 shows a maximum at 170 K and approaches a constant value at 80 K. These data can be fitted by a modified van't Hoff expression, which accounts for the freezing into a non-equilibrium distribution of substates below a distinct temperature Tf and also for the linear temperature dependence of the specific heat of proteins. The latter leads to a temperature dependence of the entropic and enthalpic differences between conformational substates. The fits to the intensity ratios of the vFe-His subbands yield a freezing temperature of Tf = 117 K and a transition region of delta T = 55 K. In comparison we have utilized the above thermodynamic model to reanalyze earlier data on the temperature dependence of the ratio Ao/A1 of two subbands underlying the infrared absorption band of the CO stretching vibration in CO-ligated myoglobin (A. Ansari, J. Berendzen, D. Braunstein, B. R. Cowen, H. Frauenfelder, M. K. Kong, I. E. T. Iben, J. Johnson, P. Ormos, T. B. Sauke, R. Scholl, A. Schulte, P. J. Steinbach, R. D. Vittitow, and R. D. Young, 1987, Biophys. Chem. 26:237-335).(ABSTRACT TRUNCATED AT 400 WORDS)     

Kinetics of formation of antibody-ferric porphyrin complex with peroxidase activity

The antibody 2B4 combines with ferric mesoporphyrin to form an antibody-ferric mesoporphyrin complex which has a peroxidase activity. Formation of the complex was investigated by measuring the absorption in the Soret region after mixing the antibody and ferric mesoporphyrin. A rapid increase and a gradual decrease in the absorption were observed, and the respective first-order rate constants were obtained. From the dependence of values of the rate constants on the concentration of ferric mesoporphyrin, the complex formation was explained by a plausible mechanism, in which the antibody associated with ferric mesoporphyrin to form the first complex followed by a conformational change to the second complex. The first complex had almost the same peroxidase activity as that of the second complex. Our results suggests that the antibody acquires the peroxidase activity as soon as ferric mesoporphyrin is incorporated into its binding site, and that there will be no protein ligand to the iron center of ferric mesoporphyrin in the complex.     

Influence of static and dynamic disorder on the visible and infrared absorption spectra of carbonmonoxy horseradish peroxidase.

Spectroscopy of horseradish peroxidase with and without the substrate analog, benzohydroxamic acid, was monitored in a glycerol/water solvent as a function of temperature. It was determined from the water infrared (IR) absorption that the solvent has a glass transition at 170-180 K. In the absence of substrate, both the heme optical Q(0,0) absorption band and the IR absorption band of CO bound to heme broaden markedly upon heating from 10-300 K. The Q(0,0) band broadens smoothly in the whole temperature interval, whereas the IR bandwidth is constant in the glassy matrix and increases from 7 to 16 cm(-1) upon heating above the glass transition. Binding of substrate strongly diminishes temperature broadening of both the bands. The results are consistent with the view that the substrate strongly reduces the amplitude of motions of amino acids forming the heme pocket. The main contribution to the Q(0,0) bandwidth arises from the heme vibrations that are not affected by the phase transition. The CO band thermal broadening stems from the anharmonic coupling with motions of the heme environment, which, in the glassy state, are frozen in. Unusually strong temperature broadening of the CO band is interpreted to be caused by thermal population of a very flexible excited conformational substrate. Analysis of literature data on the thermal broadening of the A(0) band of Mb(CO) (Ansari et al., 1987. Biophys. Chem. 26:337-355) shows that such a state presents itself also in myoglobin.     

An FT-IR study of DNA and RNA conformational transitions at low temperatures

Fourier Transform Infrared (FT-IR) spectra of solid samples of DNA and RNA obtained from freeze-drying at solid CO2 and liquid nitrogen temperatures, have been recorded and correlation between the conformational transitions and spectral changes is proposed. It is concluded that an equilibrium exists between A, B and Z conformations at low temperatures for the DNA molecule, which is temperature dependent, whereas the RNA molecule exhibits only the A conformation. The results have been compared with the metal-adducts of DNA and RNA, where one of the conformations is predominant. Marker infrared bands for the B conformer have been found to be the strong band at 825 cm-1 (sugar conformer mode) and a band with medium intensity at 690 cm-1 (guanine breathing mode). The A conformation showed characteristic bands at 810 and 675 cm-1. The B to Z conformational transition was characterized by the strong absorption bands near 820-810 cm-1 and at 665-600 cm-1.     

Time-resolved FT-IR studies on the CO adduct of Paracoccus denitrificans cytochrome c oxidase: comparison of the fully reduced and the mixed valence form.

The rebinding of CO to cytochrome c oxidase from Paracoccus denitrificans in the fully reduced and in the half-reduced (mixed valence) form as a function of temperature was investigated using time-resolved rapid-scan FT-IR spectroscopy in the mid-IR (1200-2100 cm-1). For the fully reduced enzyme, rebinding was complete in approximately 2 s at 268 K and showed a biphasic reaction. At 84 K, nonreversible transfer of CO from heme a3 to CuB was observed. Both photolysis at 84 K and photolysis at 268 K result in FT-IR difference spectra which show similarities in the amide I, amide II, and heme modes. Both processes, however, differ in spectral features characteristic for amino acid side chain modes and may thus be indicative for the motional constraint of CO at low temperature. Rebinding of photodissociated CO for the mixed-valence enzyme at 268 K is also biphasic, but much slower as compared to the fully reduced enzyme. FT-IR difference spectra show band features similar to those for the fully reduced enzyme. Additional strong bands in the amide I and amide II range indicate local conformational changes induced by electron and coupled proton transfer. These signals disappear when the temperature is lowered to 84 K. At 268 K, a difference signal at 1746 cm-1 is observed which is shifted by 6 cm-1 to 1740 cm-1 in 2H2O. The absence of this signal for the mutant Glu 278 Gln allows assignment to the COOH stretching mode of Glu 278, and indicates changes of the conformation, proton position, or protonation of this residue upon electron transfer.     

Pigment spectra and intermolecular interaction potentials in glasses and proteins

A model is proposed for chromophore optical spectra in solids over a wide range of temperatures and pressures. Inhomogeneous band shapes and their pressure dependence, as well as baric shift coefficients of spectral lines, selected by the frequency, were derived using Lennard-Jones potentials of the ground and excited states. Quadratic electron-phonon coupling constants, describing the thermal shift and broadening of zero-phonon lines, were also calculated. Experimentally, thermal shift and broadening of spectral holes were studied between 5 and 40 K for a synthetic pigment, chlorin, embedded in polymer hosts. The baric effects on holes were determined by applying hydrostatic He gas pressure up to 200 bar, at 6 K. Absorption spectra of pheophytin a, chlorophyll a, and beta-carotene in polymers and plant photosystem II CP47 complex were measured between 5 (or 77) and 300 K, and subject to Voigtian deconvolution. A narrowing of inhomogeneous bandwidth with increasing temperature, predicted on the basis of hole behavior, was observed as the shrinking of Gaussian spectral component. The Lorentzian broadening was ascribed to optical dephasing up to 300 K in transitions with weak to moderate linear electron-phonon coupling strength. The thermal broadening is purely Gaussian in multiphonon transitions (S(2) band of beta-carotene, Soret bands of tetrapyrrolic pigments), and the Lorentz process appears to be suppressed, indicating a lack of exponential dephasing. Density, polarity, polarizability, compressibility, and other local parameters of the pigment binding sites in biologically relevant systems can be deduced from spectroscopic data, provided that sufficient background information is available.     

Low temperature optical absorption spectroscopy: an approach to the study of stereodynamic properties of hemeproteins

In this short review we show how suitable analysis of the temperature dependence of the optical absorption spectra of metalloproteins can give insight into their stereodynamic properties in the region of the chromophore. To this end, the theory of coupling between an intense allowed electronic transition of a chromophore and Franck-Condon active vibrations of the nearby atoms is applied to the Soret band of hemeproteins to obtain an analytical expression suitable for fitting the spectral profile at various temperatures. The reported approach enables one to separate the various contributions to the overall bandwidth together with the parameters that characterize the vibrational coupling. The thermal behavior of these quantities gives information on the dynamic properties of the active site and on their dependence upon protein structure and ligation state. The Soret band of hemeproteins appears to be coupled to high frequency vibrational modes of the heme group (as already shown by resonance Raman spectroscopy) and to a bath of low frequency modes most likely deriving from the bulk of the protein. For the deoxy derivatives inhomogeneous broadening arising from conformational heterogeneity appears to contribute substantially to the linewidth. The data indicate the onset; at temperatures near 180 K, of large scale anharmonic motions that can be attributed to jumping among different conformational substates of the protein.Abbreviations MbCO Carbonmonoxy-myoglobin - Mb Deoxymyoglobin - Mb³⁺ Aquomet-myoglobin - SWMbCO Spermwhale carbonmonoxy-myoglobin - SWMb Spermwhale deoxy-myoglobin Correspondence to: A. Cupane     

Hydration of single-stranded phosphodiester and phosphorothioate oligodeoxyribonucleotides.

Infrared spectroscopy was used to identify hydration-sensitive structural differences between single- stranded phosphorothioate (PS) and phosphodiester (PO) oligodeoxyribonucleotides. Spectra were recorded in the mid-infrared region, 500-1800 cm-1, at relative humidities between 0 and 98%; the PS and PO spectra are substantially different. The hydration effects on spectral bands in these single-stranded oligodeoxyribonucleotides is markedly different from such behavior in double- and triple-stranded oligodeoxyribonucleotides. A strong absorption occurs at 656 cm-1 in the phosphorothioate sample which is completely absent from the PO spectra. Gravimetric measurements were carried out on one PS and one PO sample to monitor and confirm hydration. The calculated BET adsorption constants [Brunauer, S., Emmett, RH. and Teller, E. (1938) J. Am. Chem. Soc., 60, 309-319] are 1.2 and 1.4 water molecules per nucleotide in the first hydration layer of PS and PO respectively. While the gravimetric data indicate that the single-stranded oligodeoxyribonucleotides hydrate very similarly to duplex DNA, the mid-infrared conformational marker bands are strikingly different from those observed for duplex DNA. In particular, the Vas of the phosphate group (PO2) at 1222 cm-1 in the single-stranded PO spectra is independent of relative humidity.     

Variable temperature infrared spectroscopy of cytosine-guanine base pairs: tautomerism versus polarization.

This report describes an infrared (IR) spectroscopic study of a model cytosine-guanine base pair. This base pair is part of a self-consistent experimental system based on lipophilic ribose derivatives of cytidine (C), guanosine (G) and O6-methylguanosine (O6MeG) that are soluble in non-aqueous, low dielectric solvents at appreciable concentrations. Previous experiments on this system have revealed different rotation dynamics for the amino bonds within the CG base pair, an observation that could be explained by the presence of rare tautomers (P.O. Lowdin, Reviews of Modern Physics 35,724 (1963)), or by mutual polarization of the base pairs (L.D. Williams, N.G. Williams and B.R. Shaw,J.Am.Chem.Soc. 112,829 (1990)). The IR spectra in the OH and NH stretching region indicate formation of hydrogen-bonded CG base pairs and self associates in 1,2-dichlorobenzene over a temperature range from 10 to 290K. Changes in the lineshapes and intensities of the IR bands with temperature correlate with phase transitions of the solvent, but no evidence is seen for an OH stretching band that would indicate the formation of hydroxyl tautomers within base pairs. Similarly, the relative intensities of the C = O stretching bands of CG in cyclohexane solution remain constant over this same temperature range, confirming that within the base pair, the tautomeric states of the bases remain essentially unperturbed in the 2-amino/6-keto form of G and the 2-keto/4-amino form of C. The spectra of O6-MeG aid in the band assignments, since this molecule is frozen in an equivalent of the 2-amino/6-hydroxyl tautomer, but without the OH group and its associated stretching band. We conclude that the probability of tautomerism does not appear to be sufficient to explain the different rotation dynamics for the two amino bonds of the CG base pair. Rather it is argued that mutual polarization within the base pair, which would increase the bond order of the amino bond of C within the base pair, can explain the results without the formation of unconventional tautomers.     

Cryoprotection of lipid membranes for high-resolution solid-state NMR studies of membrane peptides and proteins at low temperature

Solid-state NMR spectra of membrane proteins often show significant line broadening at cryogenic temperatures. Here we investigate the effects of several cryoprotectants to preserve the spectral resolution of lipid membranes and membrane peptides at temperatures down to ~200 K. Trehalose, glycerol, dimethylsulfoxide (DMSO), dimethylformamide (DMF), and polyethylene glycol (PEG), were chosen. These compounds are commonly used in protein crystallography and cryobiology. ¹³C and ¹H magic-angle-spinning spectra of several types of lipid membranes show that DMSO provides the best resolution enhancement over unprotected membranes and also best retards ice formation at low temperature. DMF and PEG-400 show slightly weaker cryoprotection, while glycerol and trehalose neither prevent membrane line broadening nor prevent ice formation under the conditions of our study. Neutral saturated-chain phospholipids are the most amenable to cryoprotection, whereas negatively charged and unsaturated lipids attenuate cryoprotection. ¹³C–¹H dipolar couplings and ³¹P chemical shift anisotropies indicate that high spectral resolution at low temperature is correlated with stronger immobilization of the lipids at high temperature, indicating that line narrowing results from reduction of the conformational space sampled by the lipid molecules at high temperature. DMSO selectively narrowed the linewidths of the most disordered residues in the influenza M2 transmembrane peptide, while residues that exhibit narrow linewidths in the unprotected membrane are less impacted. A relatively rigid β-hairpin antimicrobial peptide, PG-1, showed a linewidth increase of ~0.5 ppm over a ~70 K temperature drop both with and without cryoprotection. Finally, a short-chain saturated lipid, DLPE, exhibits excellent linewidths, suggesting that it may be a good medium for membrane protein structure determination. The three best cryoprotectants found in this work—DMSO, PEG, and DMF—should be useful for low-temperature membrane-protein structural studies by SSNMR without compromising spectral resolution.     

Optical and IR absorption as probe of dynamics of heme proteins

The spectroscopy of horseradish peroxidase with and without the substrate analogue benzohydroxamic acid (BHA) was monitored in different solvents as a function of the temperature in the interval from 10 to 300 K. Thermal broadening of the Q(0,0) optical absorption band arises mainly from interaction of the electronic pi --> pi transition with the heme vibrations. In contrast, the width of the IR absorption band of CO bound to heme is controlled by the coupling of the CO transition moment to the electric field of the protein matrix. The IR bandwidth of the substrate free enzyme in the glycerol/H2O solvent hardly changes in the glassy matrix and strongly increases upon heating above the glass transition. Heating of the same enzyme in the trehalose/H2O glass considerably broadens the band. The binding of the substrate strongly diminishes the temperature broadening of the CO band. This result is consistent with the view that the BHA strongly reduces the amplitude of vibrations of the heme pocket environment. Unusually strong thermal broadening of the CO band above the glass transition is interpreted to be caused by thermal population of a very flexible excited conformational substate. The thermal broadening of the same band in the trehalose glass is caused by an increase of the protein vibrational amplitude in each of the conformational substates, their population being independent of the temperature in the glassy matrix.     

Site-selected fluorescence spectra of porphyrin derivatives of heme proteins

The emission spectra of the porphyrin in metal-free and Zn cytochrome c and in metal-free mesoporphyrin derivatives of horseradish peroxidases A and C, leghemoglobin, and myoglobin were examined as a function of temperature and excitation wavelength. At room temperature, the emission spectra were unresolved and were independent of excitation wavelength. At low temperature (4.2 K), the spectra depended upon excitation wavelength: using narrow-band excitation into the high-energy side of the 0-1 and 0-0 bands gave unresolved emission spectra whereas excitation into the low-energy side produced quasi-line spectra. The resolved spectra were different for the five proteins and further varied with pH, indicating chromophore-protein interactions. The spectra are interpreted in terms of site selection and phonon interactions.     

Studies on the reaction coordinates of the water oxidase in PS II membrane fragments from spinach.

The temperature dependence of the rate constants of the univalent redox steps YzoxSi----YzSi + 1 (i = 0,1,2) and YzoxS3----(YzS4)----YzSo + O2 in the water oxidase was investigated by measuring time resolved absorption changes at 355 nm induced by a laser flash train in dark adapted PS II membrane fragments from spinach. Activation energies of 5.0, 12.0 and 36.0 kJ/mol were obtained for the reactions YzoxSi----YzSi + 1 with i = 0,1 and 2, respectively. The reaction YzoxS3----(YzS4)----YzS0 + O2 exhibits a temperature dependence with a characteristic break point at 279 K with activation energies of 20 kJ/mol (T greater than 279 K) and 46 kJ/mol (T less than 279 K). Evaluation of the data within the framework of the classical Marcus theory of nonadiabatic electron transfer [(1985) Biochim. Biophys. Acta 811, 265-322] leads to the conclusion that the S2 oxidation to S3 is coupled with significant structural changes. Furthermore, the water oxidase in S3 is inferred to attain two different conformational states with populations that markedly change at a characteristic transition temperature.