UV resonance Raman-selective amide vibrational enhancement: quantitative methodology for determining protein secondary structure

We have directly determined the amide band resonance Raman spectra of the "average" pure alpha-helix, beta-sheet, and unordered secondary structures by exciting within the amide pi-->pi* transitions at 206.5 nm. The Raman spectra are dominated by the amide bands of the peptide backbone. We have empirically determined the average pure alpha-helix, beta-sheet, and unordered resonance Raman spectra from the amide resonance Raman spectra of 13 proteins with well-known X-ray crystal structures. We demonstrate that we can simultaneously utilize the amide I, II, and III bands and the Calpha-H amide bending vibrations of these average secondary structure spectra to directly determine protein secondary structure. The UV Raman method appears to be complementary, and in some cases superior, to the existing methods, such as CD, VCD, and absorption spectroscopy. In addition, the spectra are immune to the light-scattering artifacts that plague CD, VCD, and IR absorption measurements. Thus, it will be possible to examine proteins in micelles and other scattering media.     

Conformation of phosphatidylserine in bilayers as studied by Fourier transform infrared spectroscopy

The 13C labeled lipid 1[1'-13C]DPPS-NH4+ and its metal salts were used to unambiguously assign all carbonyl vibrations in the infrared spectrum of phosphatidylserines. It is shown that the C=O stretching band at 1741 cm-1 of phosphatidylserines previously assigned to the sn-1 C = O vibration contains contributions from both the sn-1 and the sn-2 carbonyls. The C=O stretching band at frequencies between 1715 and 1730 cm-1 previously assigned to the sn-2 C=O vibration also contains contributions from both carbonyl groups. The frequency dependence observed with the ester carbonyls primarily reflects hydrogen bonding and the polarity of the immediate vicinity. Conformational changes are accounted for in terms of frequency shifts if the conformational change involves the disposition of the C=O groups and in turn the hydrogen bonding properties. The infrared spectra of phospholipids dispersed in aqueous medium in the liquid crystalline state are inconsistent with a simple phospholipid conformation, e.g., with a conformation as found in the single-crystal structure of 1,2-dimyristoyl-sn-phosphatidylcholine and 1,2-dilauroyl-rac-phosphatidylethanolamine. The spectra support the hypothesis proposed earlier (Hauser et al., Biochemistry, 1988) on the basis of existing single-crystal phospholipid structures and NMR evidence. The hypothesis states that several conformations are present in liquid crystalline phospholipid dispersions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Laser Raman investigation of the conformation of human immunoglobulin G

Laser Raman spectra of human immunoglobulin G in neutral solution, as well as in the lyophilized and alkaline-denatured states are presented. In the spectrum of the native protein, the amide III band appears at 1240 cm-1 and is assigned to the presence of beta-sheet structure. From its intensity, using a procedure described in this paper, we evaluate the beta-structure content to 37 +/- 4%. This result is supported by the strong amide I' band at 1667 cm-1 and by the presence in the spectra of two bands at 991 and 1078 cm-1, respectively assigned to the C-C and C-N skeletal stretching modes. The differences between the spectrum of the lyophilized powder and that of the solution show that the lyophilization process induces conformational changes that perturb the local environment of some of the tryptophan residues and alter the secondary structure of immunoglobulin G. The beta-structure appears to be more uniform and more abundant in solution. When the protein is denatured at pH 11, the amide III and amide I'bands, which become weaker and broader, shift in frequency from 1240 to 1248 cm-1 and from 1667 to 1656 cm-1 respectively. These changes indicate a decrease in the amount of beta-structure and a transition toward a much more disordered conformation. During the denaturation, the intensities of many bands of the aromatic chromophores change, notably the tryptophan peaks at 879, 1359 and 1573 cm-1.     

Thermal and pH stabilities of alkaline phosphatase from bovine intestinal mucosa: a FTIR study

The inactivation of alkaline phosphatase (AP) from bovine intestinal mucosa caused by lowering the p2H from 10.4 to 5.4 or by increasing the temperature from 25 degrees C to 70 degrees C were not followed by significant FTIR changes, indicating that the native conformation of AP was preserved under these conditions. Further decrease of p2H from 5.4 to 3.4 leaded to small infrared spectral changes of AP in the amide I' and amide II regions that were similar to the infrared spectral changes of AP induced by raising the temperature from 70 degrees C to 80 degrees C. The increase of temperature from 70 degrees C to 80 degrees C promoted the formation of intermolecular beta-sheets at the expense of some alpha-helix structures as evidenced by the appearance of the 1684 cm-1 and 1620 cm-1 component bands and the disappearance of the 1651-1657 cm-1 component band. This conformational change was followed by a sharp increase of the 2H/H exchange rate. CD spectra confirmed the FTIR results and were very sensitive to the variation of alpha-helix content while FTIR spectra were more receptive to the changes of beta-sheet structures.     

AMPA receptor-mediated excitotoxicity in human NT2-N neurons results from loss of intracellular Ca2+ homeostasis following marked elevation of intracellular Na+

We studied the gas phase spectrum of the deuterated ethynyl radical C2D in the region between 3196 and 3243 cm-1 using a Faraday LMR spectrometer in combination with a CO overtone laser. The C2D radicals were generated in a dc glow discharge containing helium, deuterium, and acetylene. We observed a hot band between two vibronic 2Pi states with an origin at 3225 cm-1. The lower level is assigned to the first excited bending level of the electronic X ground state. The upper level corresponds to the first excited electronic state A at 3513 cm-1, which was observed previously [J. Mol. Struct. 190, 41-60 (1988)]. This region is subject to strong vibronic interaction, caused by mixing of the electronic X ground state with the A state at 3513 cm-1. From the analysis of the spectra we could determine the orbital g factor of the upper level, which gave important information about the mixing ratios. In addition we were able to derive a precise term value for the first excited bending level of the electronic X ground state. The experimentally derived molecular parameters are compared with theoretically calculated values, obtained by ab initio calculations. Copyright 1998 Academic Press.     

Hot Bands of Water up to 6nu2-5nu2 in the 933-2500 cm-1 Region

Emission spectra have been recorded for hot water at temperatures up to 1550 degreesC. Separate spectra have been recorded in the 800-1900 and 1800-2500 cm-1 range. Assignments are made using a linelist generated from high accuracy, variational nuclear motion calculations, and energy differences. The spectra contain many hot bending transitions of the form (0n0)-(0n-10), where states up to n = 6 have been assigned. Detailed analysis shows that the spectra contain lines from 34 separate vibrational bands including other hot bending transitions and the difference bands (030)-(100), (110)-(020), (011)-(020), (100)-(010), (040)-(110), (040)-(011), (120)-(030), (012)-(030), (011)-(100), (110)-(001), and (101)-(110), all of which have not been observed previously. From a total of 8959 lines recorded, 6810 have been assigned; 4556 of these lines are new. These spectra represent the first detection of the (060) vibrational band, for which a band origin of 8870.54 +/- 0.05 cm-1 is determined. The (050) band origin is confirmed as 7542.40 +/- 0.03 cm-1. The assignments extend the range of J and Ka values observed for the bending states, particularly for (050) and (060), where 63 and 27 different rotational levels, respectively, have now been observed; 53 frequencies given in HITRAN are corrected. Copyright 1999 Academic Press.     

Structure and architecture of the bacterial virus fd. An infrared linear dichroism study

Oriented gels of intact bacterial virus fd have been invetigated by infrared linear dichroism. Infrared absorption band maxima and dichroism indicate an alpha-helix content of the major coat protein of 95-100%. The alpha-helical rods of the coat protein are aligned parallel to the long axis of the virion with an inclination roughly estimated to approximately 37 degree. The presence of DNA infrared bands at 968, 885, 830 and 799 cm-1, the absence of a band at 860 cm-1 and the perpendicular polarization of the symmetric PO-2 stretching vibration at 1085 cm-1 are all indicative of a B-type backbone conformation in the single-stranded DNA. We find no evidence for specific interaction between aromatic side groups (phenylalanine, tyrosine) and the DNA bases. Our results independently confirm most features of the model of Marvin and co-workers [2,15 ] based on low-resolution X-ray diffraction studies. However, our findings contradict their suggestion of an A-type DNA in the bacterial virus fd. Two results are consistent with rigid and stable order in the virus. First, over a 4-day period, 65% of the peptide hydrogens remain unexchanged with deuterium. Second, changes in the relative humidity of the sample do not result in any shifts in the DNA spectrum that are characteristic of free DNA.     

Correction of complete transposition of great vessels by the Mustard technic (26 cases operated)

Laser Raman spectra of neurotoxins of Pelamis platurus (yellow-bellied sea snake) and Laticauda semifasciata (broad-banded blue sea snake) were investigated. The amide I band appeared at 1672 cm-1 for both toxins, which presents an indication of anti-parallel beta structure. Since this agrees well with the result from the CD-ORD studies of snake neurotoxin, it was concluded that snake neurotoxins mainly consist of beta structure. The amide III band appeared at 1245 cm-1 for P. platurus toxin and 1248 cm-1 for L. semifasciata toxin. The four disulfide bonds present in the toxin have a very similar geometry. After vigorous heat treatment, the backbone configuration of the toxin molecule basically remained the same although it was partially denatured. The major peak at 512 cm-1 was not altered by the heat treatment but a new shoulder appeared at 546 cm-1. This suggests that a new type of S-S stretching vibration (trans-gauche-trans) was produced as a result of heat treatment. However, the majority of the S-S vibrations remained in the gauche-gauche-gauche orientation. A substantial change in the interactions between a tyrosine aromatic ring and neighboring residues was apparently the alteration caused by the heat treatment.     

ATP-Induced phosphorylation of the sarcoplasmic reticulum Ca2+ ATPase: molecular interpretation of infrared difference spectra

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.     

Determination of secondary structure of normal fibrin from human peripheral blood

The secondary structure of human fibrin from normal donors and from bovine and suilline plasma was studied by Fourier transform ir spectroscopy and a quantitative analysis of its secondary structure was suggested. For this purpose, a previously experimented spectrum deconvolution procedure based on the use of the Conjugate Gradient Minimisation Algorithm with the addition of suitable constraints was applied to the analysis of conformation-sensitive amide bands. This procedure was applied to amide I and III analysis of bovine and suilline fibrin, obtained industrially, and to amide III analysis of human fibrin clots. The analysis of both amide I and III in the first case was useful in order to test the reliability of the method. We found bovine, suilline, and human fibrin to contain about 30% alpha-helix (amide I and III components at 1653 cm-1, and 1312 and 1284 cm-1, respectively), 40% beta-sheets (amide I and III components at 1625 and 1231 cm-1, respectively) and 30% turns (amide I and III components at 1696, 1680, 1675 cm-1, and 1249 cm-1, respectively). The precision of the quantitative determination depends on the amount of these structures in the protein. Particularly, the coefficient of variation is < 10% for percentage values of amide I and III components > 15 and 5%, respectively. The good agreement of our quantitative data, obtained separately by amide I and amide III analysis, and consistent with a previous fibrinogen (from commercial sources) study that reports only information about fibrin beta-sheet content obtained by factor analysis, leads us to believe that the amounts of secondary structures found (alpha-helix, beta-sheets, and turns) are accurate.     

Hydrogen-bond interactions of the primary donor of the photosynthetic purple sulfur bacterium Chromatium tepidum

We have used near-infrared Fourier transform (pre)resonance Raman spectroscopy to determine the protein interactions with the bacteriochlorophyll (BChl) dimer constituting the primary electron donor, P, in the reaction center (RC) from the thermophilic purple sulfur bacterium Chromatium tepidum. In addition, we report the alignment of partial sequences of the L and M protein subunits of C. tepidum RCs in the vicinity of the primary donor with those of Rhodobacter sphaeroides and Rhodopseudomonas viridis. Taken together, these results enable us to propose the hydrogen-bonding pattern and the H-bond donors to the conjugated carbonyl groups of P. Selective excitation (1064-nm laser radiation) of the FT (pre)-resonance Raman spectra of P in its neutral (P degree) and oxidized (P degree +) states were obtained via their electronic absorption bands at 876 and 1240 nm, respectively. The P degree spectrum exhibits vibrational frequencies at 1608, 1616, 1633, and 1697 cm-1 which bleach upon P oxidation. The P degree + spectrum exhibits new bands at 1600, 1639, and 1719 cm-1. The 1608-cm-1 band, which downshifts to 1600 cm-1 upon oxidation, is assigned to a CaCm methine bridge stretching mode of the P dimer, indicating that each BChl molecule possesses a single axial ligand (His L181 and His M201, from the sequence alignment). The 1616- and 1633-cm-1 bands correspond to two H-bonded pi-conjugated acetyl carbonyl groups of each BChl molecule. with different H-bond strengths: the 1616-cm-1 band is assigned to the PL C2 acetyl group which is H-bonded to a histidine residue (His L176), while the 1633-cm-1 band is assigned to the PM C2 acetyl carbonyl, H-bonded to a tyrosine residue (Tyr M196). Both PL and PM C9 keto carbonyls are free from interactions and vibrate at the same frequency (1697 cm-1). Thus, the H-bond pattern of the primary donor of C. tepidum differs from that of Rb. sphaeroides in the extra H-bond to the PM C2 acetyl carbonyl group; that of PL is H-bonded to a histidine residue in both primary donors (His L168 in Rb. sphaeroides and His L176 in C. tepidum). The P degree/P degree + redox midpoint potentials were measured to be +497 and +526 mV for isolated C. tepidum RCs with and without the associated tetraheme cytochrome c subunit, respectively, and +502 mV for intracytoplasmic membranes. The positive charge localization was estimated to be 69% in favor of PL, indicating a more delocalized situation over the primary donor of C. tepidum than that of Rb. sphaeroides (estimated to be 80% on PL). These differences in physicochemical properties are discussed with respect to the proposed structural model for the microenvironment of the primary donor of C. tepidum.     

Effect of ethanol on the protein secondary structure of the human gastric mucosa, in vitro

The effect of ethanol on the secondary conformational structure of proteins of the human gastric mucosa was investigated by attenuated total reflection/Fourier transform infrared (ATR/FT-IR) spectroscopy. The IR peak intensity and position of each structural component of gastric mucosa was found to change significantly with the ethanol concentration and length of exposure. The peak intensity due to the beta-sheet and/or beta-turn conformational structure in amide I and II bands of gastric mucosa clearly increased after treatment with ethanol. Moreover, the peak at 1635 cm-1 shifted to 1630 cm-1 after treatment with 40% ethanol for 3 h, or 80% ethanol for 1 h, and a distinct shoulder also appeared at 1643 cm-1. This shift occurred more rapidly and was more pronounced after exposure of mucosa to 80% ethanol, compared with the effect of 40% ethanol, but the alpha-helical structure at the amide I and II bands was not influenced by either concentration of ethanol. Ethanol treatment might also transform the secondary structure of amide III in gastric mucosa from an alpha-helix to a mainly random coil with extensive unfolding. The absorption between 1180 and 980 cm-1, which is assigned to glycoprotein structure, was also reduced after treatment with ethanol. This strongly indicates that ethanol influences the conformation of the lipids and proteins of human gastric mucosa, leading to their deformation.     

Conformational changes in bacteriorhodopsin associated with protein-protein interactions: a functional alpha I-alpha II helix switch?

Fourier transform infrared spectra of bacteriorhodopsin samples were obtained in conditions in which the aggregation state of the protein (i.e., monomeric or trimeric) was modified by different treatments. Two approaches were followed: (1) renaturation of bacteriorhodopsin starting from bacterioopsin dissolved in SDS and (2) reconstitution of bacterioopsin in Halobacterium lipid liposomes at two different lipid/protein ratios. Concomitant with the gradual recovery of the native interactions between transmembrane helices, we observed clear and gradual changes in the infrared absorption spectra in the amide I band and also in the band at 1741 cm-1. These processes were found to be compatible with the two-state oligomerization model. The whole set of experiments shows that the band at 1665 cm-1 in the deconvoluted spectra appears only when monomers interact forming trimers, even when the lattice is not present. This implies that the trimeric organization of bacteriorhodopsin is responsible for the unique features described in the amide I of purple membrane. The spectroscopic changes detected can be attributed to changes in secondary structure compatible with the interconversion of alpha I and alpha II helices. However, the exact nature and functional relevance of these changes is still unknown.     

Unusual helix-containing greek keys in development-specific Ca(2+)-binding protein S. 1H, 15N, and 13C assignments and secondary structure determined with the use of multidimensional double and triple resonance heteronuclear NMR spectroscopy

Multidimensional heteronuclear NMR spectroscopy has been used to determine almost complete backbone and side-chain 1H, 15N, and 13C resonance assignments of calcium loaded Myxococcus xanthus protein S (173 residues). Of the range of constant-time triple resonance experiments recorded, HNCACB and CBCA(CO)NH, which correlate C alpha and C beta with backbone amide resonances of the same and the succeeding residue respectively, proved particularly useful in resolving assignment ambiguities created by the 4-fold internal homology of the protein S amino acid sequence. Extensive side-chain 1H and 13C assignments have been obtained by analysis of HCCH-TOCSY and 15N-edited TOCSY-HMQC spectra. A combination of NOE, backbone amide proton exchange, 3JNH alpha coupling constant, and chemical shift data has been used to show that each of the protein S repeat units consists of four beta-strands in a Greek key arrangement. Two of the Greek keys contain a regular alpha-helix between the third and fourth strands, resulting in an unusual and possibly unique variation on this common folding motif. Despite similarity between two nine-residue stretches in the first and third domains of protein S and one of the Ca(2+)-binding sequences in bovine brain calmodulin [Inouye, S., Franceschini, T., & Inouye, M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6829-6833], the protein S topology in these regions is incompatible with an EF-hand calmodulin-type Ca(2+)-binding site.     

Measurements of HDO between 4719 and 5843 cm-1

Fourier transform spectrometer data of heavy water vapor were obtained at resolutions of 0.01 and 0.02 cm-1. The spectra were analyzed to obtain line center frequencies and line strengths of HD16O from 4719 to 5843 cm-1. The analysis included 1700 assigned lines of the (011)-(000), (200)-(000), (120)-(000), and (021)-(010) bands. This is the first report of assignments in the weak (120)-(000) band which borrows much of its strength from the (200)-(000) band. The measurements provided in this work are a marked improvement over the HDO listing presently available on the HITRAN database in the spectral region. Copyright 1997 Academic Press. Copyright 1997Academic Press     

Mechanism for the recognition and activation of substrate in medium-chain acyl-CoA dehydrogenase

The mechanism underlying the recognition and activation of the substrate for medium-chain acyl-CoA dehydrogenase (MCAD) was spectroscopically investigated using 3-thiaacyl-CoAs as substrate analogs. The complex of MCAD with 3-thiaoctanoyl-CoA (3-thia-C8-CoA) exhibited a charge-transfer (CT) band with a molar extinction coefficient of epsilon808 = 9.1 mM-1.cm-1. With increasing 3-thiaacyl-chain length, the CT-band intensity of the complex decreased concomitantly with changes in the FAD absorption at 416 and 482 nm, and no CT band was detected in complexes with chain-lengths longer than C15. Detailed analysis of the absorption spectra suggested that the complexed states represent a two-state equilibrium between the CT-inducing form and the CT-non-inducing form. 13C-NMR measurements with 13C-labeled ligand clarified that 3-thia-C8-CoA is complexed to MCAD in an anionic form with signals detected at 163.7 and 101.2 ppm for 13C(1) and 13C(2), respectively. In the MCAD complex with 13C(1)-labeled 3-thia-C12-CoA, two signals for the bound ligand were observed at 163.7 and 198.3 ppm, and assigned to the anionic and neutral forms, respectively. Only the neutral form signal was measured at 200.6 ppm in the complex with 13C(1)-labeled 3-thia-C17-CoA. These results indicate that the CT band can be explained in terms of an internal equilibrium between anionic (CT-inducing) and neutral (CT-non-inducing) forms of the bound ligand. Resonance Raman spectra of the MCAD.3-thia-C8-CoA complex, with excitation at the CT band, showed enhanced bands, among which the 854- and 1,368-cm-1 bands were assigned to the S-C(2) stretching mode of the ligand and to flavin band VII, respectively. Since the enhanced bands were observed at the same wave numbers in complexes with C8, C12, and C14-ligands, it appears that the CT-inducing form shares a common alignment relative to oxidized flavin irrespective of differences in the acyl-chain length. However, with longer ligands, the degree of resonance enhancement of the Raman bands decreased in parallel with the CT-band intensity; this is compatible with the increase in the CT-non-inducing form in complexes with longer ligands. Furthermore, the pH dependence of the CT band gave an apparent pKa = 5.6-5.7 for ligands with chain-lengths of C8-C12. The NMR measurements revealed that, like chain-length dependence, the pH dependence can be explained by a two-state equilibrium derived from the protonation/deprotonation of the CT-inducing form of the bound ligand. On the basis of these results we have established a novel model to explain the mechanism of recognition and activation of the substrates/ligands by MCAD.     

Multiple thermotropic state transitions in erythrocyte membranes. A laser-Raman study of the CH-stretching and acoustical regions

1. We have examined the Raman scattering from erythrocyte ghosts at 2700 cm-1 -3000 cm-1 (CH-stretching region), that at low frequencies due to acoustical vibrations, as well as that due to v (-C=C-) and v (=C-C=) of beta-carotene, in the temperature range of -30 degrees C to +30 degrees C. For comparison we have also evaluated the Rama spectra of liposomes composed of lecithins+/-cholesterol. 2. Plots vs. temperature of the intensities (I) of the approx. 2880 cm-1 and 2932 cm-1 bands relative to the intensity of the thermally stable 2850 cm-1 band, i.e. the (I2880/I2850) and (I2932/I2850) ratios, revealed discontinuities in both erythrocyte membranes and liposomes. 3. In erythrocyte membranes, plots of (I approximately 2880/I2850) and (I approximately 2932/I 2850) reveal several discontinuities. Those reported by (I approximately 2880/I 2850) occur at +17 degrees C and approx. -4 degrees C. The ratio (I approximately 2932/I 2850) reveals a transition between -20 degrees C and +10 degrees C, 1/2 maximal at -8 degrees C. The ratio (I 1527/I 1158), representing the relative intensity of v (-C=C-) vs. v (=C-C=) of membrane-bound beta-carotene, changes discontinuously with the 17 degrees C transition, but is stable over the rest of the temperature change studied. 4. The data indicate that a major membrane-state transition takes place below 0 degrees C but that an additional transition occurs at approx. 17 degrees C.     

Proton uptake by carboxylic acid groups upon photoreduction of the secondary quinone (QB) in bacterial reaction centers from Rhodobacter sphaeroides: FTIR studies on the effects of replacing Glu H173

In the photosynthetic reaction center (RC) from Rhodobacter sphaeroides, Glu H173, located approximately 7 A from the center of the secondary quinone acceptor QB, is expected to contribute to proton uptake upon QB- formation in response to the movement of an electron in its vicinity. Steady-state FTIR difference spectroscopy provides a method to monitor proton uptake by carboxylic acids upon photochemical changes. The FTIR spectra corresponding to the photoreduction of QB were obtained at pH 7 for RCs containing Glu (native), Gln (EQ H173), or Asp (ED H173) at the H173 site. No new bands were observed in the carboxylic acid region (1770-1700 cm-1) in any of the mutant RCs compared to native RCs. In addition, the positive band at 1728 cm-1, previously assigned to Glu L212 [Nabedryk, E., Breton, J., Hienerwadel, R., Fogel, C., M?ntele, W., Paddock, M. L., and Okamura, M. Y. (1995) Biochemistry 34, 14722-14732], remained present in all of the mutant RCs. This result shows that Glu H173 is not a major contributor to proton uptake upon QB- formation and further strengthens the assignment of the 1728 cm-1 band to Glu L212. An increase in the 1728 cm-1 band was observed in the EQ H173 RCs compared to that of either the ED H173 or native RCs. These changes are consistent with Glu and Asp at H173 remaining ionized in the QB and QB- states. Changes in the absorption regions of the semiquinone and amide or side chain groups in the spectra of the mutant RCs suggest slight changes in the protein structure compared to those of native RCs, which could contribute to the altered kinetics observed in the mutant RCs.     

Involvement of glutamic acid 278 in the redox reaction of the cytochrome c oxidase from Paracoccus denitrificans investigated by FTIR spectroscopy

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 M?ntele, 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.     

Redox dependent changes at the heme propionates in cytochrome c oxidase from Paracoccus denitrificans: direct evidence from FTIR difference spectroscopy in combination with heme propionate 13C labeling

Specific isotope labeling at the carboxyl groups of the four heme propionates of cytochrome c oxidase from Paracoccus denitrificans was used in order to assign signals observed in electrochemically induced redox Fourier transform infrared (FTIR) difference spectra of this enzyme. For this purpose, the hemA gene of the P. denitrificans strain PD1222, coding for 5-aminolevulinate synthase, was deleted by partial replacement with a kanamycin resistance cartridge, resulting in a stable 5-aminolevulinic acid (ALA) auxotrophy. Normal growth of this deficient strain and cytochrome c oxidase yield comparable to that of P. dentrificans wild-type strain PD1222 could be obtained by supplementation with 0.1 mM ALA in the growth medium. Visible spectra and reduced-minus-oxidized FTIR spectra showed that the purified cytochrome c oxidase had spectral characteristics identical to those of the wild-type enzyme. The decrease of a negative signal at 1676 cm-1 in the reduced-minus-oxidized FTIR difference spectra of the 13C-labeled cytochrome c oxidase in comparison to those of the unlabeled protein allowed the assignment of this signal to a COOH vibration mode of at least one of the four heme propionates. Moreover, a negative band at approximately 1570 cm-1 shifted to smaller wavenumbers in the spectra of the 13C-labeled enzyme in comparison to the spectra of the unlabeled enzyme and was thus assigned to contributions from an antisymmetric COO- mode of one or more of the four heme propionates. Additionally, a positive signal at 1538 cm-1 shifted to approximately 1500 cm-1 in the spectra of the isotopically labeled protein and was therefore assigned to at least one antisymmetric COO- mode of the heme propionates. A negative signal at 1390 cm-1, which has been shifted to 1360 cm-1 in the spectra of the 13C-labeled enzyme, is due to a symmetric COO- mode from at least one heme propionate. These results suggest that at least two of the four heme propionates in cytochrome c oxidase undergo significant vibrational changes upon reduction of the enzyme, either by protonation/deprotonation or by environmental changes.