Laser Raman studies on cobrotoxin.

Laser Raman spectra of cobrotoxin under various conditions have been obtained. Comparison of the spectra of native cobrotoxin in lyophilized form and in aqueous solution indicates that the secondary structures of cobrotoxin are not significantly affected by the removal of the aqueous solvent. On going from the native to the partially reduced and the completely reduced, carboxy-methylated forms, characteristic peaks of the C-S-S-C and tyrosine ring in the region of 500--900 cm-1 showed definite changes in structure. The partially reduced form gave two peaks at 502 and 524 cm-1, suggesting difference in the conformation of the remaining disulfide bonds. As indicated by the present work, the conformation of the main chain of cobrotoxin in the native unperturbed state, in the partially reduced and in the completely reduced forms are the coexistence of beta-pleated sheet with random-coil structure, predominantly random coil, and predominantly random coil with the existence of an alpha-helix type structure, respectively. The effect of pH on the conformation of cobrotoxin in solution appeared to give rise to the change of the local structure of two aromatic residues common to all snake neurotoxins.     

Laser Raman spectroscopy of calf bone Gla protein

The Raman spectra of solid calf bone Gla protein in its native state, decarboxylated, with reduced disulfide bond, and as the calcium salt have been obtained. The amide I and III bands are consistent with the presence of alpha-helical, antiparallel beta-sheet, and random-coil regions in all four forms of bone Gla protein. Random coil appears to be the prevailing conformation. The protein conformation in the calcium salt exhibits an increased alpha-helix character compared to the native protein. No significant differences in the backbone conformation are observed among the native, decarboxylated, and reduced forms of bone Gla protein. The Raman band at 504 cm-1, due to the disulfide stretching vibration in native bone Gla protein, is unchanged upon decarboxylation and binding of Ca2+ to the protein, indicating the absence of any changes in the conformation around the disulfide bond in these protein species. The tryptophan and most of the tyrosine residues appear to be 'exposed' rather than 'buried' in the native protein. The environment of at least one of the phenylalanine residues changes when Ca2+ is bound to bone Gla protein. A small change also appears to take place in the environment of at least one of the tyrosine residues upon Ca2+-binding or reduction of the disulfide bond.     

Interaction of ferricytochrome c with zwitterionic phospholipid bilayers: a Raman spectroscopic study

The vibrational Raman spectra of both pure L-alpha-dipalmitoylphosphatidylcholine (DPPC) liposomes and DPPC multilayers reconstituted with ferricytochrome c under varying conditions of pH and ionic strength are reported as a function of temperature. Total integrated band intensities and relative peak height intensity ratios, two spectral scattering parameters used to determine bilayer disorder, are invariant to changes in pH and ionic strength but exhibit a sensitivity to the bilayer concentration of the ferricytochrome c. Protein concentrations were estimated by comparing the 1636 cm-1 resonance Raman line of known ferricytochrome c solutions to intensity values for the reconstituted multilayer samples. Temperature-dependent profiles of the 3100-2800 cm-1 C-H stretching, 1150-1000 cm-1 C-C stretching, 1440 cm-1 CH2 deformation, and 1295 cm-1 CH2 twisting mode regions characteristic of acyl chain vibrations reflect bilayer perturbations due to the weak interactions of ferricytochrome c. The DPPC multilamellar gel to liquid-crystalline phase transition temperature, TM, defined by either the C-H stretching mode I2935/I2880 or the C-C stretching mode I1061/I1090 peak height intensity ratios, is decreased by approximately 4 degrees C for the approximately 10(-4) M ferricytochrome c reconstituted DPPC liposomes. Other spectral features, such as the increase in the 2935 cm-1 C-H stretching mode region and the enhancement of higher frequency CH2 twisting modes, which arise in bilayers containing approximately 10(-4) M protein, are interpreted in terms of protein penetration into the hydrophobic region of the bilayer.     

Dramatic stabilization of the native state of human carbonic anhydrase II by an engineered disulfide bond

To find a disulfide pair that could stabilize the enzyme human carbonic anhydrase II (HCA II), we grafted the disulfide bridge from the related and unusually stable carbonic anhydrase form from Neisseria gonorrhoeae (NGCA) into the human enzyme. Thus, the two Cys residues at positions 23 and 203 were engineered into a pseudo-wild-type form of HCA II (C206S), giving the mutant C206S/A23C/L203C. The disulfide bond was not formed spontaneously. The native state of the reduced form of the mutant was markedly destabilized (2.9 kcal/mol) compared to that of HCA II. Formation of a disulfide bridge was achieved by treatment by oxidized glutathione. This led to a significant stabilization of the native conformation. Compared to HCA II the unfolding midpoint for the variant was increased from 0.9 to 1.7 M guanidine HCl, corresponding to a stabilization of 3.7 kcal/mol. This makes the human enzyme almost as stable as the model protein NGCA, for which the unfolding of the native state has a midpoint at 2.1 M guanidine HCl. The stabilized protein underwent, contrary to all other investigated variants of HCA II, an apparent two-state unfolding transition, as judged from intrinsic Trp fluorescence measurements. A molten-globule intermediate is nevertheless formed but is suppressed because of the high denaturant pressure it faces upon rupture of the native state.     

Resonance Raman studies of lactoperoxidase

Resonance Raman (RR) spectra obtained at three excitation wavelengths are reported for various ferric, ferrous, and ferryl derivatives of bovine lactoperoxidase. The RR spectra of the ferric derivatives show the full complement of the vinyl stretching and scissor modes indicating that the two vinyls in the protoporphyrin IX prosthetic group are present in unmodified forms. The cysteine thiol complex exhibits a RR spectrum identical to that of the native enzyme, an observation which strongly suggests a nonheme binding site for the thiol substrates. The different ferrous complexes of lactoperoxidase which result from heme reduction at slightly alkaline and acidic pH gave identical low-frequency RR spectra. Differences are observed, however, in the high-frequency region. Reduction in the presence of cyanide, however, yields two time-resolved complexes. Changes in the ligand field during the conversion to the final form of the cyanoferrous complex are proposed based on the changes observed in the low-frequency vibrational spectrum. Comparisons are made between the low-frequency RR spectra of the limiting form of the cyanoferrous and the nitric oxide lactoperoxidase complexes. The similarity between the RR spectra of these two complexes in the 150-500 cm-1 region supports the assignment of structures for these complexes where the six-coordinate heme iron is displaced from the heme plane and away from the proximal histidine ligand.     

Iron-histidine stretching Raman line and enzymic activities of bovine and bacterial cytochrome c oxidases

Resonance Raman spectra of the reduced form of cytochrome c oxidase isolated from bovine heart and the thermophilic bacterium PS3 were investigated in relation to their H+-pumping- and cytochrome-c-oxidizing activities, which were varied by incubating the enzyme at raised temperatures or at alkaline pH at room temperature. For both the bovine and PS3 enzymes, the intensity of the iron-histidine stretching Raman line of the ferrous a3 heme (214 cm-1) exhibited an incubation-temperature-dependent change, which fell between the similar curves of the H+-pumping and cytochrome-c-oxidizing activities. The intensities of the formyl CH=O stretching Raman line of the ferrous a3 heme (1665 cm-1) as well as of other lines were insensitive to the heat treatment. The iron-histidine stretching Raman line of both enzymes showed pH-dependent intensity change which was nearly parallel with the pH dependence of cytochrome-c-oxidizing activity. Therefore, deprotonation affecting the 214 cm-1 Raman line is responsible for the decrease of activity. This limited alkaline treatment to the PS3 enzyme was reversible and the recovered enzyme exhibited Raman intensities and enzymic activities similar to the native one. However, the neutralized, bovine enzyme with a similar intensity of the 214 cm-1 line showed increased cytochrome-c-oxidizing activity and null H+-pumping activity.     

Structural characterization of cytochrome c peroxidase by resonance Raman scattering

Resonance Raman scattering studies are reported on freshly prepared and aged ferric, ligand-free ferrous, and CO-bound ferrous cytochrome c peroxidase. The ferric form of the fresh enzyme has a heme which is penta-coordinate high spin, independent of buffer over the pH range 4.3-7, as determined by well established Raman marker lines. The aged enzyme displays a mixture of spin and coordination states, but it can be stabilized in the penta-coordinate high spin form in the presence of phosphate. These results can be accounted for by considering the size of the channel (6 A wide, 11 A long) between the distal side of the heme and the outer surface of the protein. A phosphate ion may be accommodated in this channel resulting in the stabilization of the distal heme pocket. The ferrous cytochrome c peroxidase in both the ligand-free and CO-bound states has an acidic and an alkaline form. The acidic form has the characteristic spectral features of peroxidases: a high frequency iron-histidine stretching mode (248 cm-1), a high frequency Fe-CO stretching mode (537 cm-1), and a low frequency C-O stretching mode (1922 cm-1). At alkaline pH these frequencies become similar to those of hemoglobin and myoglobin, with the corresponding modes located at 227, 510, and 1948 cm-1, respectively. We attribute the acid/alkaline transition in the ferrous forms of cytochrome c peroxidase to a rearrangement mainly of the proximal side of the heme, culminating in a change of steric interactions between the proximal histidine and the heme or of the hydrogen bonding network involving the proximal histidine. The new data presented here reconcile many inconsistencies reported in the past.     

Conformation of oxytocin studied by laser Raman spectroscopy

The peptide backbone conformation and salient structural details of oxytocin were examined by laser Raman spectroscopy. Spectra were obtained in the solid phase, water, 2H2O, and dimethyl sulfoxide solutions. A distinct Amide I band was obtained at 1663 cm-1 for aqueous and deuterated samples and 1666 cm-1 for the solid sample. A relatively high frequency Amide III band at 1260 cm-1 was obtained. It is concluded that these Amide I and III bands arise from the "beta-turn"-like conformation of oxytocin. The tyrosine side chain, according to the I850 cm-1/I830 cm-1 intensity ratio, is exposed to the solvent. The S-S stretching vibration at 512 cm-1 indicates the conformation of C-C-S-S-C-C in the disulfide bridge of oxytocin in the ring is gauche-gauche-gauche.     

Inactivation of ribonuclease inhibitor by thiol-disulfide exchange.

Porcine ribonuclease inhibitor (RI) contains 30 1/2-cystinyl residues, all of which occur in the reduced form. Reaction of the native protein with 5,5'-dithiobis (2-nitrobenzoic acid) resulted in the release of 30 mol of the product 5-mercapto-2-nitrobenzoate, and the loss of the RNase inhibitory activity. A linear relationship between the degree of modification and inactivation was observed. The rate of modification was greatly increased in the presence of 6 M guanidinium HCl. Reaction with substoichiometric amounts of 5,5'-dithiobis(2-nitrobenzoic acid) was found to yield a mixture of fully reduced active molecules, and fully oxidized inactive ones, but no partially oxidized forms were detected. This suggests that an "all-or-none" type of modification and inactivation took place. All 1/2-cystinyl residues in the inactive, monomeric inhibitor had formed disulfide bridges, judged by the absence of either free thiol groups or mixed disulfides with 5-mercapto-2-nitrobenzoate. This fully disulfide-cross-linked molecule had an open conformation compared to the native one, as shown by gel filtration and limited proteolysis. Reaction of phenylarsinoxide with vicinal dithiols yields products that are much more stable than those with monothiols. Titration of RI with this reagent yielded complete inactivation at a reagent/thiol ratio of 0.5. Taken together, these observations suggest that the thiol groups in RI have a diminished reactivity due to three-dimensional constraints. After the initial modification of a small number of thiol groups, a conformational change occurs which causes an increase in reactivity of the remaining thiols. The thiol groups are situated close enough together to permit the formation of 15 disulfide bridges in the inactive molecule.     

Raman spectroscopy of the thermal properties of reassembled high-density lipoprotein: apolipoprotein A-I complexes of dimyristoylphosphatidylcholine

Isolated complexes of apolipoprotein A-I (apoA-I), the major apoprotein of human plasma high-density lipoproteins, and dimyristoylphosphatidylcholine (DMPC) have been prepared and studied by differential scanning calorimetry (DSC) and Raman spectroscopy. DSC studies establish that complexes having lipid to protein ratios of 200, 100, and 50 to 1 each exhibit a broad reversible thermal transition at Tc = 27 degrees C. The enthalpy of lipid melting for each of the three complexes is about 3 kcal/mol of DMPC. Raman spectroscopy indicates that the physical state of lipid molecules in the complexes is different from that in DMPC multilamellar liposomes. Analysis of the C-H stretching region (2800-3000 cm-1) of the complexes and of the pure components in water suggests that below 24 degrees C (Tc for DMPC) there is considerably less lateral order among lipid acyl chains in the complexes than in DMPC liposomes. Above 24 degrees C, these types of interactions appear to contribute equally or slightly less to the complex structure than in pure DMPC. The temperature dependence of peaks in the C-C stretching region (1000-1180 cm-1) reveals a continuous increase in the number of lipid acyl chain C-C gauche isomers over a broad range with increasing temperature. Compared to liposomes, DMPC in the complexes has more acyl chain trans isomers at temperatures above 24 degrees C; at temperatures above ca. 30 degrees C, trans isomer content is about the same for complexes and liposomes. A large change was observed in a protein vibrational band at 1340 cm-1 for pure vs. complexed apoA-I, indicating that protein hydrocarbon side chains are immobilized by lipid binding. The Raman data indicate that the reduction in melting enthalpy for complexes DMPC (approximately 3 kcal/mol) compared to that for free DMPC (approximately 6 kcal/mol) is due to reduced van der Waals interactions in the low-temperature lipid phase.     

Effects of cholesterol and adrenodoxin binding on the heme moiety of cytochrome P-450scc: a resonance Raman study

The effects of cholesterol and adrenodoxin binding on resonance Raman spectra of cytochrome P-450scc in both oxidized and CO-reduced states were examined. Upon cholesterol binding, oxidized cytochrome P-450scc showed a significant shift of spin equilibrium from low-spin to high-spin state. Addition of adrenodoxin caused a complete conversion of cholesterol-bound oxidized cytochrome P-450scc to a pure high-spin state that was considered to be in the hexacoordinated state judged by the v10 mode at 1620 cm-1 and v3 mode around 1485 cm-1. Cholesterol in substrate binding site may oppose a linear and perpendicular binding of carbon monoxide to the reduced heme iron, leading to the distorted Fe-C-O linkage. This is based on the following observations: (1) an increase of the Fe-CO stretching frequency to 483 from 477 cm-1 upon addition of cholesterol; (2) an enhanced photodissociability of bound carbon monoxide of CO complex of cytochrome P-450scc in the presence of cholesterol. As another aspect of the effect of cholesterol on the CO complex form of cytochrome P-450scc, the enhanced stability of the native form ("P-450" form) was observed. There was no additional effect of reduced adrenodoxin on the Raman spectra of the CO-reduced form of cytochrome P-450scc.     

The disulfide content of calf gamma-crystallin

The disulfide content of calf gamma-crystallin polypeptides has been investigated. The gamma-crystallin fraction of the soluble lens proteins was separated into five distinct polypeptides and characterized by isoelectric focusing, amino acid composition, and N-terminal sequence analysis to 25 residues. It has been demonstrated that 7 cysteines are present in gamma II, 4 to 5 cysteines in gamma IIIa, gamma IIIb, and gamma IV, and 6 cysteines in gamma I (beta s). Reduction of the total gamma-crystallin fraction with DTT resulted in an increase of approximately 1 to 1.5 mol of free SH per mole of protein. This increase in sulfhydryls was demonstrated to be contributed primarily by gamma II, the major polypeptide representing 50% of the total gamma-crystallin, which showed an increase of approximately 2.5 mol of sulfhydryl per mole of protein upon reduction. Insignificant disulfide content was present in gamma III and gamma IV and only a slight amount of disulfide was found in gamma I (beta s). The observed increase in sulfhydryl content upon reduction was not due to the presence of mixed disulfides of 2-mercaptoethanol, glutathione, or cysteine. The data are consistent with approximately 1 mol of intramolecular disulfide per mole of protein being present in gamma II. X-ray crystallography of gamma II has shown that the spatial location of Cys18 and Cys22 in the tertiary structure permits disulfide bond formation. Sequence analysis of the four major polypeptides of gamma-crystallin, gamma II, gamma IIIa, gamma IIIb, and gamma IV indicates that only gamma II has both Cys18 and Cys22. Cys18 is present in gamma IIIa, gamma IIIb, and gamma IV but Cys22 is replaced by His22. It is probable that the lack of disulfide in gamma IIIa, gamma IIIb, and gamma IV is due to the absence of Cys22.     

Cysteine conformation and sulfhydryl interactions in proteins and viruses. 3. Quantitative measurement of the Raman S-H band intensity and frequency.

The bond stretching vibration of the cysteine sulfhydryl (SH) group in a typical protein generates a Raman band in the spectral interval 2500-2600 cm-1, a region devoid of interference from any other fundamental mode of vibration of the protein. The relatively high Raman cross section associated with the S-H stretching vibration, the sensitivity of the vibrational frequency to hydrogen bonding interactions and side chain configurations, and the dependence of the Raman intensity on thiol-thiolate equilibria, combine to make the Raman SH band a potentially valuable marker of protein sulfhydryl interactions and a unique indicator of sulfhydryl participation in thiol-disulfide oxidoreductase activity. In order to exploit Raman spectroscopy for these purposes, accurate and precise measurements of Raman SH band profiles are required. We show here that the required precision and accuracy can be achieved by use of the Raman band corresponding to the stretching vibration of in situ nitrogen gas as a quantitative intensity and frequency standard. The Raman Q-branch center of the N2 band occurs at 2330.7 cm-1.     

Molecular conformations of cerebrosides in bilayers determined by Raman spectroscopy.

Vibrational Raman spectra of the solid and gel phases of bovine brain cerebrosides and the component fractions, kerasin and phrenosin, provide conformational information for these glycosphingolipids in bilayer systems. The carbon-carbon stretching mode profiles (1,150-1,000 cm-1) indicate that at 22 degrees C the alkyl chains assume an almost all-trans arrangement. These spectral data, combined with those from the C-H stretching region (3,050-2,800 cm-1), show that phrenosin forms the most highly ordered polycrystalline solid and kerasin the most ordered gel phase. The conformation of the unsaturated, 24-carbon acyl chains is monitored independently by a skeletal stretching mode at 1,112 cm-1. The alkyl chains in the kerasin and phrenosin gels are sufficiently extended to allow interdigitation of the 24-carbon acyl chains across the midplane of the bilayer. The amide I vibrational mode occurs at a lower frequency in solid phrenosin than kerasin, a shift consistent with stronger hydrogen bounding. This band is broadened and shifted to higher frequencies, however, in the phrenosin gel phase. In both the solid and gel phases natural cerebroside exhibits a composite amide I mode. The disruptive effects on cerebroside chain packing and headgroup orientation arising from mixing with dimyristoyl phosphatidylcholine are examined. Vibrational data for cerebroside are also compared to those for ceramide, sphingosine, and distearoyl phosphatidylcholine structures. Spectral interpretations are discussed in terms of calorimetric and X-ray structural data.     

Conformational differences in protein disulfide linkages between normal hair and hair from subjects with trichothiodystrophy: a quantitative analysis by Raman microspectroscopy

Raman spectra of normal hair shafts and hair shafts from patients exhibiting trichothiodystrophy (TTD) were obtained using line focus laser illumination. Because hair from TTD patients has a significant decrease in the content of the sulfur-containing amino acids in comparison to normal hair, the 550-500 cm(-1) disulfide stretching mode region of the Raman spectrum was examined in detail. A quantitative spectral analysis demonstrates significant increases in the two energetically less favored gauche-gauche-trans (g-g-t) and trans-gauche-trans (t-g-t) forms. These observations suggest that the increased amounts of these less stable disulfide conformers are contributing factors to or associated with the hair brittleness observed for this congenital disorder. Structure-spectra correlations for the three dominant disulfide conformers are confirmed by quantum chemical calculations using modern density functional theory (DFT).     

Raman, infrared, and circular dichroism spectroscopic studies on metallothionein: a predominantly "turn"-containing protein

Raman and IR spectra of rabbit liver metallothionein 1 (MT-1) containing 7 mol of either cadmium or zinc ions reveal high-lying amide III bands between 1290 and 1330 cm-1, indicative of beta-turns. A comparison of the splitting pattern in the amide III region below 1290 cm-1 and in the amide I band between 1600 and 1700 cm-1, with the normal-mode calculations of Lagant et al. [Lagant, P., Vergoten, G., Fleury, G., & Loucheux-Lefebvre, M. (1984a) Eur. J. Biochem. 139, 137-148; Lagant, P., Vergoten, G., Fleury, G., & Loucheux-Lefebvre, M. (1984b) Eur. J. Biochem. 139, 149-154; Lagant, P., Vergoten, G., Fleury, G., & Loucheux-Lefebvre, M. (1984c) J. Raman Spectrosc. 15, 421-423] and Krimm and Bandekar [Krimm, S., & Bandekar, J. (1980) Biopolymers 19, 1-29], suggests that metal-bound (holo) MT-1 consists largely of beta-turns of type II. In contrast, the metal-free (apo) protein displays a predominantly unordered conformation. The Raman spectra of the holoproteins below 1000 cm-1 are characterized by several unusual skeletal stretching and bending modes. The spectral pattern between 760 and 800 cm-1 in conjunction with the splitting of the amide I band agrees closely with the normal-mode calculations of Lagant et al. (1984b) on model peptides and is indicative of the presence of type III beta-turns (or 3(10)-helical segments) in MT-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resonance Raman spectra of bovine liver catalase: enhancement of proximal tyrosinate vibrations

Resonance Raman spectra of native bovine liver ferri-catalase have been obtained in the 200-1800 cm-1 region. Excitation at a series of wavelengths ranging from 406.7 to 514.5 nm has been used and gives rise to distinct sets of resonance Raman bands. Excitation within the Soret and Q-bands of the heme group produces the expected set of polarized and nonpolarized porphyrin modes, respectively. The frequencies of the porphyrin skeletal stretching bands in the 1450-1700 cm-1 region indicate that catalase contains only five-coordinate, high-spin heme groups. In addition to the porphyrin modes, bovine liver catalase exhibits bands near 1612 and 1520 cm-1 that are attributable to ring vibrations of the proximal tyrosinate that are enhanced via resonance with a proximal tyrosinate----Fe(III) change transfer transition centered near 490 nm. Similar bands have been observed in mutant hemoglobins that have tyrosinate axial ligands and in other Fe(III)-tyrosinate proteins. No resonance Raman bands have been observed that can be attributed to degraded hemes. The spectra are relatively insensitive to pH over the range of 5-10, and the same spectra are observed for catalase samples that do and do not contain tightly bound NADPH. Resonance Raman spectra of the fluoride complex exhibit porphyrin skeletal stretching modes that show it to be six coordinate, high spin, while the cyanide complex is six coordinate, low spin. Both the azide and thiocyanate complexes, however, are spin-state mixtures with the high-spin form predominant.     

Observation of the Fe4+ = O stretching Raman band for cytochrome oxidase compound B at ambient temperature

Resonance Raman and visible absorption spectra were simultaneously observed for cytochrome oxidase reaction intermediates at 5 degrees C by using the artificial cardiovascular system (Ogura, T., Yoshikawa, S., and Kitagawa, T. (1989) Biochemistry 28, 8022-8027) and a device for Raman/absorption simultaneous measurements (Ogura, T., and Kitagawa, T. (1988) Rev. Sci. Instrum. 59, 1316-1320). The Fe4+ = O stretching (nu FeO) Raman band was observed at 788 cm-1 for compound B for the first time. This band showed the 16O/18O isotopic frequency shift (delta nu FeO) by 40 cm-1, in agreement with that for horseradish peroxidase compound II (nu FeO = 787 cm-1 and delta nu FeO = 34 cm-1). In the time region when the FeII-O2 stretching band for compound A and the nu FeO band for compound B were coexistent, a Raman band assignable to the Fe3+-O-O-Cu2+ linkage was not recognized.     

Coordination structures and reactivities of compound II in iron and manganese horseradish peroxidases. A resonance Raman study

Resonance Raman investigations on compound II of native, diacetyldeuteroheme-, and manganese-substituted horseradish peroxidase (isozyme C) revealed that the metal-oxygen linkage in the compound differed from one another in its bond strength and/or structure. Fe(IV) = O stretching frequency for compound II of native enzyme was pH sensitive, giving the Raman line at 772 and 789 cm-1 at pH 7 and 10, respectively. The results confirmed the presence of a hydrogen bond between the oxo-ligand and a nearby amino acid residue (Sitter, A. J., Reczek, C. M., and Terner, J. (1985) J. Biol. Chem. 260, 7515-7522). The Fe(IV) = O stretch for compound II of diacetylheme-enzyme was located at 781 cm-1 at pH 7 which was 9 cm-1 higher than that of native enzyme compound II. At pH 10, however, the Fe(IV) = O stretch was found at 790 cm-1, essentially the same frequency as that of native enzyme compound II. The pK value for the pH transition, 8.5, was also the same as that of native compound II. Unlike in native enzyme, D2O-H2O exchange did not cause a shift of the Fe(IV) = O frequency of diacetylheme-enzyme. Thus, the metal-oxygen bond at pH 7 was stronger in diacetylheme-enzyme due to a weaker hydrogen bonding to the oxo-ligand, while the Fe(IV) = O bond strength became essentially the same between both enzymes at alkaline pH upon disruption of the hydrogen bond. A much lower reactivity of the diacetylheme-enzyme compound II was accounted to be due to the weaker hydrogen bond. Compound II of manganese-substituted enzyme exhibited Mn(IV)-oxygen stretch about 630 cm-1, which was pH insensitive but down-shifted by 18 cm-1 upon the D2O-H2O exchange. The finding indicates that its structure is in Mn(IV)-OH, where the proton is exchangeable with a water proton. These results establish that the structure of native enzyme compound II is Fe(IV) = O but not Fe(IV)-OH.     

Inter- and intramolecular disulfide bond formation and related structural changes in the lens proteins. A Raman spectroscopic study in vivo of lens aging

Raman spectra have been measured for intact rat lens nuclei at various stages of aging in an attempt to gain further insight into age-related structural changes in the lens proteins, especially changes concerning protein sulfhydryl groups. Two Raman bands at 2579 and 2561 cm-1 were observed to be assignable to SH stretching modes of the cysteine residues. These bands have been attributed to "exposed" and "buried" sulfhydryl groups of the lens proteins, respectively, on the basis of a model compound study. The relative intensities of both SH stretching modes decreased with lens aging, and concurrently the intensity of a S-S stretching mode at 509 cm-1 due to disulfide bridges increased, suggesting that not only exposed but also buried protein sulfhydryl groups are converted to disulfide groups as a result of aging. The rate of the intensity decrease in the 2561 cm-1 band was similar to that in the 2579 cm-1 band. Therefore, it seems likely that the sulfhydryl groups in the two distinct environments are nearly equally subjected to the oxidation. Cysteine and cystine residues of the lens proteins gave their C-S stretching modes at 708 cm-1, indicating that they predominantly assume PC and/or PN conformers. The intensity ratio of a tyrosine doublet near 840 cm-1 (I832/I855) changed from approximately 0.86 to approximately 0.81 with the aging of the rat lens. This result implies that some tyrosine residues undergo a change in their hydrogen bonding environments during the course of aging. Of particular importance is that the relative intensity change of the tyrosine doublet with normal aging and that with cataract formation are in opposite directions.