The resonance Raman frequencies of the Fe-CO stretching and bending modes in the CO complex of cytochrome P-450cam

Resonance Raman spectra of the ferrous CO complex of cytochrome P-450cam have been observed both in its camphor-bound and free states. Upon excitation at 457.9 nm, near the absorption maximum of the Soret band, the ferrous CO complex of the camphor-bound enzyme showed an anomalously intense Raman line at 481 cm-1 besides the strong Raman lines at 1366 and 674 cm-1 for the porphyrin vibrations. The Raman line at 481 cm-1 (of the 12C16O complex) shifted to 478 cm-1 upon the substitution by 13C16O and to 473 cm-1 by 12C18O without any detectable shift in porphyrin Raman lines. This shows that the line at 481 cm-1 is assignable to Fe-CO stretching vibration. By the excitation at 457.9 nm, a weak Raman line was also observed at 558 cm-1, which was assigned to the Fe-C-O bending vibration, because it was found to shift by -14 cm-1 on 13C16O substitution while only -3 cm-1 on 12C18O substitution. These stretching and bending vibrations of the Fe-CO bond were not detected with the excitation at 413.1 nm, though the porphyrin Raman lines at 1366 and 674 cm-1 were clearly observed. When the substrate, camphor, was removed from the enzyme, the Fe-CO stretching vibration was found to shift to 464 cm-1 from 481 cm-1, while no detectable changes were found in porphyrin Raman lines. This means that the bound substrate interacts predominantly with the Fe-CO portion of the enzyme molecule.     

Resonance Raman spectra of anionic semiquinoid form of a flavoenzyme, D-amino acid oxidase

Resonance Raman (RR) spectra of the complex of anionic semiquinoid D-amino acid oxidase (DAO) with picolinate in H2O and D2O were observed in the 300-1,750 cm-1 region. RR spectra were also measured for the complex of the semiquinoid enzyme reconstituted with isotopically labeled FAD's, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]-FAD. On the basis of the isotope effects, tentative assignments of the observed bands of the anionic semiquinoid flavin were made. The spectra differ from those of oxidized, neutral semiquinoid, and anionic reduced flavins previously reported. The 1,602 cm-1 band was not shifted for any FAD labeled in ring II and/or ring III and was assigned to a ring I mode. The 1,516 cm-1 band underwent an isotopic shift upon [4a-13C]- or [4,10a-13C2]-labeling. The band was assigned to the mode containing C(4a)-C(10a) stretching. The 1,331 and 1,292 cm-1 bands shifted upon [4a-13C]- or [5-15N]-labeling and were assigned to the modes containing C(4a)-N(5) stretching. The 1,217 and 1,188 cm-1 bands were assigned to the skeletal vibrations of ring III coupled with the N(3)-H bending mode. The RR spectrum of the complex of anionic semiquinoid DAO with alpha-iminopropionate or N-methyl-alpha-iminopropionate was essentially identical with that of the complex with picolinate.     

Oxygen exchange between the Fe(IV) = O heme and bulk water for the A2 isozyme of horseradish peroxidase

Resonance Raman spectra were observed for compound II of horseradish peroxidase A2, and the Fe(IV) = O stretching Raman line was identified at 775 cm-1. This Raman line shifted to 741 cm-1 upon a change of solvent from H2(16)O to H2(18)O, indicating occurrence of the oxygen exchange between the Fe(IV) = O heme and bulk water. The oxygen exchange took place only at the acidic side of the heme-linked ionization with pKa = 6.9.     

Resonance Raman study on cytochrome c peroxidase and its intermediate. Presence of the Fe(IV) = O bond in compound ES and heme-linked ionization

Resonance Raman spectra of ferrous and ferric cytochrome c peroxidase and Compound ES and their pH dependences were investigated in resonance with Soret band. The Fe(IV) = O stretching Raman line of Compound ES was assigned to a broad band around 767 cm-1, which was shifted to 727 cm-1 upon 18O substitution. The 18O-isotopic frequency shift was recognized for Compound ES derived in H218O, but not in H216O. This clearly indicated occurrence of an oxygen exchange between the Fe(IV) = O heme and bulk water. The Fe(IV) = O stretching Raman band was definitely more intense and of higher frequency in D2O than in H2O as in Compound II of horseradish peroxidase, but in contrast with this its frequency was unaltered between pH 4 and 11. The Fe(II)-histidine stretching Raman line was assigned on the basis of the frequency shift observed for 54Fe isotopic substitution. From the intensity analysis of this band, the pKa of the heme-linked ionization of ferrocytochrome c peroxidase was determined to be 7.3. The Raman spectrum of ferricytochrome c peroxidase strongly suggested that the heme is placed under an equilibrium between the 5- and 6-coordinate high-spin structures. At neutral pH it is biased to the 5-coordinate structure, but at the acidic side of the transition of pKa = 5.5 the 6-coordinate heme becomes dominant. F- was bound to the heme iron at pH 6, but Cl- was bound only at acidic pH. Acidification by HNO3, H2SO4, CH3COOH, HBr, or HI resulted in somewhat different populations of the 5- and 6-coordinate forms when they were compared at pH 4.3. Accordingly, it is inferred that a water molecule which is suggested to occupy the sixth coordination position of the heme iron is not coordinated to the heme iron at pH 6 but that protonation of the pKa = 5.5 residue induces an appreciable structural change, allowing the coordination of the water molecule to the heme iron.     

Resonance Raman evidence for the mechanism of the allosteric control of O2-binding in a cobalt-substituted monomeric insect hemoglobin.

The substitution of iron for cobalt in the monomeric insect hemoglobin CTT (Chironomus thummi thummi) III does not alter the Bohr effect for O2-binding. The cobalt substitution in this hemoglobin allows us to identify not only the O-O and Co-O2 stretching mode but also the Co-O-O bending mode by resonance Raman spectroscopy. The assignments were made via 16O2/18O2 isotope exchange. The modes associated with the Co-O-O moiety are pH-dependent. These pH-induced changes of the resonance Raman spectra are correlated with the t = r conformation transition. At high pH (high-affinity state) two unperturbed O-O stretching modes are observed at 1,068 cm-1 (major component) and 1,093 cm-1 (minor component) for the 18O2 complex. These frequencies correspond to split modes at 1,107 cm-1 and 1,136 cm-1 and an unperturbed mode at approximately 1,153 cm-1 for the 16O2 complex. At low pH (low-affinity state) the minor component becomes the major component and vice versa. The Co-O2 stretching frequency varies for approximately 520 cm-1 (pH 5.5) to 537 cm-1 (pH 9.5) indicating a stronger (hence shorter) Co-O2 bond in the high-affinity state. On the other hand, the O-O bond is weakened upon the conversion of the low- to the high-affinity state. The Co-O-O bending mode changes from 390 cm-1 (pH 9.5) to 374 cm-1 (pH 5.5). In the deoxy form the resonance Raman spectra are essentially pH-insensitive except for a vinyl mode at 414 cm-1 (pH 5.5), which is shifted to 416 cm-1 (pH 5.5).     

Resonance Raman study of the aa3-type cytochrome oxidase of thermophilic bacterium PS3

Resonance Raman spectra of the aa3-type cytochrome oxidase of thermophilic bacterium PS3, which has a simpler subunit composition than the mitochondrial enzymes but very similar enzymatic properties, are investigated under various conditions and compared with those of mitochondrial enzymes. The intensities of the two marker lines of reduced cytochrome a3 at 1667 and 213 cm-1 had different dependences on the incubation temperatures and pH. With regard to the incubation temperature dependence, the intensity of the 1667-cm-1 line, the peripheral CH = O stretching mode of the a3 heme, behaved in nearly the same way as that of the oxidase activity whereas the intensity of the 213-cm-1 line, the Fe-histidine stretching mode of the a3 heme, exhibited a similar dependence to that of the proton pumping activity. The 213-cm-1 line disappeared upon binding of carbon monoxide, upon raising the pH above 9.2, or after incubating above 55 degrees C. The Raman line at 1611 cm-1, which was recently suggested to probe the proton pump activity [Babcock, G.T., & Callahan, P.M. (1983) Biochemistry 22, 2314-2319], remained unaltered after incubation at 60 degrees C for 20 min despite a reduction of proton pumping activity to one-third. This argues against the proposed mechanism. The frequencies of the Raman lines were the same for the intact membrane and the isolated enzyme in the reduced state. The Raman spectra of cytochrome oxidase isolated from bacterium, yeast, and bovine heart were different in the lower frequency region below 600 cm-1 but closely alike in the higher frequency region above 1200 cm-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of DNA structures by Raman spectroscopy: high-salt and low-salt forms of double helical poly(dG-dC) in H2O and D2O solutions and application to B, Z and A-DNA.

Raman spectra of poly(dG-dC) . poly(dG-dC) in D2O solutions of high (4.0M NaCl) and low-salt (0.1M NaCl) exhibit differences due to different nucleotide conformations and secondary structures of Z and B-DNA. Characteristic carbonyl modes in the 1600-1700 cm-1 region also reflect differences in base pair hydrogen bonding of the respective GC complexes. Comparison with A-DNA confirms the uniqueness of C = O stretching frequencies in each of the three DNA secondary structures. Most useful for qualitative identification of B, Z and A-DNA structures are the intense Raman lines of the phosphodiester backbone in the 750-850 cm-1 region. A conformation-sensitive guanine mode, which yields Raman lines near 682, 668, or 625 cm-1 in B (C2'-endo, anti), A (C3'-endo, anti) or Z (C3'-endo, syn) structures, respectively, is the most useful for quantitative analysis. In D2O, the guanine line of Z-DNA is shifted to 615 cm-1, permitting its detection even in the presence of proteins.     

On the structures of flavoprotein D-amino acid oxidase purple intermediates. A resonance Raman study

Resonance Raman (RR) spectra were obtained in H2O or D2O solution for the purple intermediates of D-amino acid oxidase (DAO) with isotopically labeled substrates, i.e., [1-13C]-, [2-13C]-, [3-13C]-, [15N]-, and [3,3,3-D3]alanine; [carboxyl-13C]- and [15N]proline. RR spectra were also measured for the intermediates of DAO reconstituted with isotopically labeled FAD's, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]FAD in D2O. The isotopic shift of the 1692 cm-1 band upon [15N]- or [2-13C]-substitution of alanine indicates that the band is due to the C = N stretching mode of an imino acid derived from D-alanine, i.e., alpha-iminopropionate. The 1658 cm-1 band with D-proline was also assigned to the C = N stretching mode of an imino acid derived from D-proline, i.e., delta 1-pyrrolidine-2-carboxylate, since the band shifts to 1633 cm-1 upon [15N]-substitution and its stretching frequency is generally found in this frequency region. Since the band shifts to low frequency in D2O, the imino acid should have a protonated imino group such as the C = N+1H form. The intense band at 1363 cm-1 with D-alanine was assigned to a mixing of the CO2- symmetric stretching and CH3 symmetric deformation modes in alpha-iminopropionate, based on the isotope effects. The 1359 cm-1 band with D-proline has probably contributions of CO2- symmetric stretching and CH2 wagging, considering the isotope effects with [carboxyl-13C]proline. The 1359 cm-1 band with D-proline was split into 1371 cm-1 and 1334 cm-1 bands in D2O. As this splitting of the 1359 cm-1 band with D-proline in D2O can not be interpreted only by the replacement of the C = N+1-H proton by deuterium, the carboxylate of the imino acid probably interacts with the enzyme through some proton(s) exchangeable by deuterium(s) in D2O. The bands around 1605 cm-1 which shift upon [4a-13C]- and [4,10a-13C2]-labeling of FAD are derived from a fully reduced flavin, because the isotopic shifts of the band are very different from those of the bands of oxidized or semiquinoid flavin observed near 1605 cm-1.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Resonance Raman study on photoreduction of cytochrome c oxidase: distinction of cytochromes a and a3 in the intermediate oxidation states

Occurrence of photoreduction of bovine cytochrome c oxidase was confirmed with the difference absorption spectra and oxygen consumption measurements for the enzyme irradiated with laser light at 406.7, 441.6, and 590 nm. The resonance Raman spectra were obtained under the same experimental conditions as those adopted for the measurements of oxygen consumption and difference absorption spectra. The photoreduction was more effective upon irradiation at shorter wavelengths and was irreversible under anaerobic conditions. However, upon aeration into the cell, the original oxidized form was restored. It was found that aerobic laser irradiation produces a photo steady state of the catalytic dioxygen reduction and that the Raman scattering from this photo steady state probes cytochrome a2+ and cytochrome a3(3)+ separately upon excitations at 441.6 and 406.7 nm, respectively. The enzyme was apparently protected from the photoreduction in the spinning cell with the spinning speed between 1 and 1500 rpm. These results were explained satisfactorily with the reported rate constant for the electron transfer from cytochrome a to cytochrome a3 (0.58 s-1) and a comparable photoreduction rate of cytochrome a. The anaerobic photoreduction did give Raman lines at 1666 and 214 cm-1, which are characteristic of the ferrous high-spin cytochrome a3(2)+, but they were absent under aerobic photoreduction. The formyl CH = O stretching mode of the a3 heme was observed at 1671 cm-1 for a2+a3(2)+CO but at 1664 cm-1 for a2+a3(2)+CN-, indicating that the CH = O stretching frequency reflects the pi back-donation to the axial ligand similar to the oxidation state marker line (v4).     

Observation of the FeIV=O stretching Raman band for a thiolate-ligated heme protein. Compound I of chloroperoxidase.

The FeIV=O stretching vibration has never been identified for a cysteine-coordinated heme enzyme. In this study, resonance Raman and visible absorption spectra were observed simultaneously for transient species in the catalytic reaction of chloroperoxidase with hydrogen peroxide by using our original apparatus for mixed-flow and Raman/absorption simultaneous measurements. For the first intermediate, the FeIV=O stretching Raman band was observed at 790 cm-1, which shifted to 756 cm-1 with the 18O derivative, but the v4 band was too weak to be identified. This suggested the formation of an oxoferryl porphyrin pi cation radical. The second intermediate gave an intense v4 band at 1,372 cm-1 but no oxygen isotope-sensitive Raman band, suggesting oxygen exchange with bulk water.     

Resonance Raman spectra of carbon-13- and nitrogen-15-labeled riboflavin bound to egg-white flavoprotein.

The resonance Raman spectra of [2-13C]-, [4a-13C]-, [4-13C]-8 [10a-13C]-, [2,4,4a, 10a-13C]-, [5-15N]-, [1,3-15N]-, and [1,3,5-15N]riboflavin bound to egg-white proteins were observed for N(3)-H and N(3)-D forms with spontaneous Raman technique by using the 488.0-nm excitation line of an argon ion laser. The fluorescence of riboflavin was quenched by forming a complex with egg-white riboflavin binding protein. The in-plane displacements of the C(2), C(4a), N(1), N(3), and N(5) atoms during each Raman active vibration were calculated from the observed isotopic frequency shifts. The 1252-cm-1 mode of the N(3)-H form was found to involve large vibrational displacements of the C(2) and N(3) atoms and to be strongly coupled with the N(3)-H bending mode. This line can be used as an indicator for state of N(3)-H...protein interaction. The 1584-cm-1 mode, which is known to be resonance-enhanced upon excitation near the 370-nm absorption band, was accompanied by the displacement of the N(5) atom in particular. The 1355-cm-1 mode was most strongly resonance-enhanced by the 450-nm absorption band and involved the displacements of all carbon atoms of ring III. Both lines can be used as structure probes for elucidating the structure of electronically excited states of isoalloxazine.     

Appearance of the v(FeIV = O) vibration from a ferryl-oxo intermediate in the cytochrome oxidase/dioxygen reaction

Time-resolved resonance Raman spectra have been recorded during the reaction of fully reduced (a2+a3(2+)) cytochrome oxidase with dioxygen at room temperature. In the spectrum recorded at 800 microseconds subsequent to carbon monoxide photolysis, a mode is observed at 790 cm-1 that shifts to 755 cm-1 when the experiment is repeated with 18O2. The frequency of this vibration and the magnitude of the 18O2 isotopic frequency shift lead us to assign the 790-cm-1 mode to the FeIV = O stretching vibration of a ferryl-oxo cytochrome a3 intermediate that occurs in the reaction of fully reduced cytochrome oxidase with dioxygen. The appearance and vibrational frequency of this mode were not affected when D2O was used as a solvent. This result suggests that the ferryl-oxo intermediate is not hydrogen bonded. We have also recorded Raman spectra in the high-frequency (1000-1700 cm-1) region during the oxidase/O2 reaction that show that the oxidation of cytochrome a2+ is biphasic. The faster phase is complete within 100 microseconds and is followed by a plateau region in which no further oxidation of cytochrome a occurs. The plateau persists to approximately 500 microseconds and is followed by the second phase of oxidation. These results on the kinetics of the redox activity of cytochrome a are consistent with the branched pathway discussed by Hill et al. [Hill, B., Greenwood, C., & Nichols, P. (1986) Biochim. Biophys. Acta 853, 91-113] for the oxidation of reduced cytochrome oxidase by O2 at room temperature.     

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.     

Direct detection of a dioxygen adduct of cytochrome a3 in the mixed valence cytochrome oxidase/dioxygen reaction

Time-resolved resonance Raman spectra have been recorded during the reaction of mixed valence (a3+ a2+(3)) cytochrome oxidase with dioxygen at room temperature. In the spectrum recorded at 10 microseconds subsequent to carbon monoxide photolysis, a mode is observed at 572 cm-1 that shifts to 548 cm-1 when the experiment is repeated with 18O2. The appearance of this mode is dependent upon the laser intensity used and disappears at higher incident energies. The high frequency data in conjunction with the mid-frequency data allow us to assign the 572 cm-1 mode to the Fe-O stretching vibration of the low-spin O2 adduct that forms in the mixed valence cytochrome oxidase/dioxygen reaction. The 572 cm-1 v(Fe2(+)-O2) frequency in the mixed valence enzyme/O2 adduct is essentially identical to the 571 cm-1 frequency we measured for this mode during the reduction of O2 by the fully reduced enzyme (Varotsis, C., Woodruff, W. H., and Babcock, G. T. (1989) J. Am. Chem. Soc. 111, 6439-6440; Varotsis, C., Woodruff, W. H., and Babcock, G. T. (1990) J. Am. Chem. Soc. 112, 1297), which indicates that the O2-bound cytochrome a3 site is independent of the redox state of the cytochrome a/CuA pair. The photolabile oxy intermediate is replaced by photostable low- or intermediate-spin cytochrome a3+(3), with t1/2 congruent to 200 microseconds.     

A resonance Raman study on a reaction intermediate of Pseudomonas L-phenylalanine oxidase (deaminating and decarboxylating).

Resonance Raman (RR) spectra of purple intermediates of L-phenylalanine oxidase (PAO) with non-labeled and isotopically labeled phenylalanines as substrates, i.e., [1-13C], [2-13C], [ring-U-13C6], and [15N]phenylalanines, were measured with excitation at 632.8 nm within the broad absorption band around 540 nm. The spectra obtained resemble those of purple intermediates of D-amino acid oxidase (DAO). The isotope effects on the 1,665 cm-1 band with [15N] or [2-13C]phenylalanine indicate that the band is due to the C = N stretching mode of an imino acid derived from phenylalanine, i.e., alpha-imino-beta-phenylpropionate. The intense band at 1,389 cm-1 is contributed to by the CO2- symmetric stretching and C-CO2- stretching modes of alpha-imino-beta-phenylpropionate. The 1,602 cm-1 band, which does not shift upon isotopic substitution of phenylalanine, corresponds to the 1,605 cm-1 band of DAO purple intermediates and was assigned to a vibrational mode associated with the C(10a) = C(4a) - C(4) = O moiety of reduced flavin. These results confirm that PAO purple intermediates consist of the reduced enzyme and an imino acid derived from a substrate, and suggest that the plane defined by C(10a) = C(4a) - C(4) = O of reduced flavin and the plane containing H2+N = C - CO2- of an imino acid are arranged in close contact to each other, generating a charge-transfer interaction.     

Optical and resonance Raman spectroscopy of the heme groups of the quinol-oxidizing cytochrome aa3 of Bacillus subtilis.

The cytochrome aa3-type terminal quinol oxidase of Bacillus subtilis catalyzes the four-electron reduction of dioxygen to water. It resembles the aa3-type cytochrome-c oxidase in using heme A as its active-site chromophores but lacks the CuA center and the cytochrome-c oxidizing activity of the mitochondrial enzyme. We have used optical and resonance Raman spectroscopies to study the B. subtilis oxidase in detail. The alpha-band absorption maximum of the reduced minus oxidized enzyme is shifted by 5-7 nm to the blue relative to most other aa3-type oxidases, and accordingly, we designate the Bacillus enzyme as cytochrome aa3-600. The shifted optical spectrum cannot be ascribed to an alteration in the strength of the hydrogen bond between the formyl group of the low-spin heme and its environment, as the Raman line assigned to this mode in aa3-600 has the same frequency and degree of resonance enhancement as the low-spin heme a formyl mode in most other aa3-type oxidases. Raman modes arise at 194 and 214 cm-1 in aa3-600, whereas a single band at about 214 cm-1 is assigned to the iron-histidine stretch for the other aa3-type oxidases. Possible explanations for the occurrence of these two modes are discussed. Comparison of formyl and vinyl modes and heme skeletal vibrational modes in different oxidation states of aa3-600 and of beef heart cytochrome-c oxidase shows a strong similarity, which suggests conservation of essential features of the heme environments in these oxidases.     

Raman spectroscopy of cytoplasmic muscle fiber proteins. Orientational order.

The polarized Raman spectra of glycerinated and intact single muscle fibers of the giant barnacle were obtained. These spectra show that the conformation-sensitive amide I, amide III, and C-C stretching vibrations give Raman bands that are stronger when the electric field of both the incident and scattered radiation is parallel to the fiber axis (Izz). The detailed analysis of the amide I band by curve fitting shows that approximately 50% of the alpha-helical segments of the contractile proteins are oriented along the fiber axis, which is in good agreement with the conformation and composition of muscle fiber proteins. Difference Raman spectroscopy was also used to highlight the Raman bands attributed to the oriented segments of the alpha-helical proteins. The difference spectrum, which is very similar to the spectrum of tropomyosin, displays amide I and amide III bands at 1,645 and 1,310 cm-1, respectively, the bandwidth of the amide I line being characteristic of a highly alpha-helical biopolymer with a small dispersion of dihedral angles. A small dichroic effect was also observed for the band due to the CH2 bending mode at 1,450 cm-1 and on the 1,340 cm-1 band. In the C-C stretching mode region, two bands were detected at 902 and 938 cm-1 and are both assigned to the alpha-helical conformation.     

Direct detection of Fe(IV)[double bond]O intermediates in the cytochrome aa3 oxidase from Paracoccus denitrificans/H2O2 reaction

We report the first evidence for the formation of the "607- and 580-nm forms" in the cytochrome oxidase aa3/H2O2 reaction without the involvement of tyrosine 280. The pKa of the 607-580-nm transition is 7.5. The 607-nm form is also formed in the mixed valence cytochrome oxidase/O2 reaction in the absence of tyrosine 280. Steady-state resonance Raman characterization of the reaction products of both the wild-type and Y280H cytochrome aa3 from Paracoccus denitrificans indicate the formation of six-coordinate low spin species, and do not support, in contrast to previous reports, the formation of a porphyrin pi-cation radical. We observe three oxygen isotope-sensitive Raman bands in the oxidized wild-type aa3/H2O2 reaction at 804, 790, and 358 cm-1. The former two are assigned to the Fe(IV)[double bond]O stretching mode of the 607- and 580-nm forms, respectively. The 14 cm-1 frequency difference between the oxoferryl species is attributed to variations in the basicity of the proximal to heme a3 His-411, induced by the oxoferryl conformations of the heme a3-CuB pocket during the 607-580-nm transition. We suggest that the 804-790 cm-1 oxoferryl transition triggers distal conformational changes that are subsequently communicated to the proximal His-411 heme a3 site. The 358 cm-1 mode has been found for the first time to accumulate with the 804 cm-1 mode in the peroxide reaction. These results indicate that the mechanism of oxygen reduction must be reexamined.     

Resonance Raman investigation of CO-ligated monomeric insect hemoglobins. Direct evidence for reciprocal changes in iron-axial ligand bonds induced by allosteric transitions

The resonance Raman spectra of the two affinity states of the CO-ligated monomeric insect hemoglobins, Chironomus thummi thummi (CTT) III ad IV, have been investigated. We have identified (via 54Fe/57Fe and 13C18O/12C16O isotope exchange) the Fe-N epsilon(His) stretching mode at approximately 317 cm-1. This stretching mode changes from 329 (pH 5.5) to 317 cm-1 (pH 9.5) reflecting the pH-induced t in equilibrium with r conformational transition. The Fe-CO stretching mode is also pH-sensitive changing from 483 (pH 5.2) to 485 cm-1 (pH 9.2) in 57Fe CTT III . 13C18O complex. However the C-O stretching mode is pH-insensitive. The nonallosteric monomeric insect hemoglobin CTT I does not exhibit a pH-dependence of these vibrational modes. pH-Induced effects were also observed for a vinyl bending mode at 379 cm-1 (pH 9.5) in CTT III deuterated at the beta-carbons of the vinyls in position 2 and 4. It shifts to 390 cm-1 at pH 5.5. The other vinyl vibration at 573 cm-1 exhibits intensity enhancement via through-space coupling with the Fe-C-O bending mode. Our resonance Raman data provide the first direct evidence that the trans-effect is operative as a trigger mechanism for ligand-binding in monomeric allosteric insect hemoglobins. In going from the low-affinity to the high-affinity state, the Fe-N epsilon(His) bond becomes weaker, whereas the Fe-CO bond becomes stronger.