Resonance Raman Spectra Of Cytochrome Research Articles

Identification of heme propionate vibrational modes in the resonance Raman spectra of cytochrome c oxidase

The propionate groups of heme a and a3 in cytochrome c oxidase (CcO) have been postulated to mediate both the electron and proton transfer within the enzyme. To establish structural markers for the propionate groups, their associated vibrational modes were identified in the resonance Raman spectra of CcO from bovine (bCcO) and Rhodobacter sphaeroides (RsCcO). The distinction between the modes from the propionates of heme a and heme a3, as well as those from the propionates on the pyrrole rings A and D in each heme, was made on the basis of H2O–D2O isotope substitution experiments combined with wavelength-selective resonance enhancement (for bCcO) or mutagenesis studies (for RsCcO).

1P-113 ブタ心筋ミトコンドリア中のチトクロムc酸化酵素の共鳴ラマンスペクトル(ヘム蛋白質,第46回日本生物物理学会年会)

1P-113 ブタ心筋ミトコンドリア中のチトクロムc酸化酵素の共鳴ラマンスペクトル(ヘム蛋白質,第46回日本生物物理学会年会)

Linearly moving low-volume spectroelectrochemical cell for microliter-scale surface-enhanced resonance Raman spectroscopy of heme proteins.

Surface-enhanced resonance Raman spectra of cytochrome c on silver electrodes coated with self-assembled monolayers of mercaptopropionic acid were recorded at different potentials using 50 microL of a micromolar solution. For this purpose, a linearly moving, low-volume, small spectroelectrochemical cell was designed and used together with a Raman microprobe. The quality of the spectra obtained is good, and the spectra show essentially the same features reported by other authors using much larger volumes. The cell described in this paper is shown to be useful for studying the spectroelectrochemistry of photosensitive compounds such as heme proteins, which are available only in very small amounts (nanomoles to picomoles).

Resonance Raman Spectra of Cytochrome c Oxidase in Whole Mitochondria

Abstract Resonance Raman spectra of cytochrome c oxidase in intact whole mitochondria were selectively observed. The Raman bands observed at 576, 517, and 369 cm-1 for the carbon monoxide-bound state were assigned to the δFe–C–O, νFe–CO, and δFe–C–O modes, respectively, of heme a3-CO complex based on the 13C18O isotopic frequency shifts. These frequencies are essentially the same as those observed for solubilized cytochrome c oxidase, which implies that the environmental structure of the dioxygen reducing site in the solubilized state is not very much altered. It is likely that the dioxygen reducing reaction studied so far for solubilized enzyme is also valid for intact whole mitochondria.

1K1115 リン脂質ベシクルに再構成したチトクロムc酸化酵素のプロトンポンプ活性と共鳴ラマンスペクトル(2.ヘム蛋白質,一般演題,日本生物物理学会第40回年会)

1K1115 リン脂質ベシクルに再構成したチトクロムc酸化酵素のプロトンポンプ活性と共鳴ラマンスペクトル(2.ヘム蛋白質,一般演題,日本生物物理学会第40回年会)

1F1130 Resonance Raman Spectra of Cytochrome c Oxidase Reconstituted in Phospholipid Vesicles

1F1130 Resonance Raman Spectra of Cytochrome c Oxidase Reconstituted in Phospholipid Vesicles

Heme-protein interactions in cytochrome c peroxidase revealed by site-directed mutagenesis and resonance Raman spectra of isotopically labeled hemes

Isotope labeling has been used to assign the resonance Raman spectra of cytochrome c peroxidase, expressed in Escherichia coli [CCP (MKT)], and of the D235N site mutant. 54Fe labeling establishes the coexistence of two separate bands (233 and 246 cm-1), arising from the stretching of the bond between the Fe atom and the proximal histidine ligand, His175. These are assigned to tautomers of the H-bond between the His175 imidazole NΓH proton and the Asp235 carboxylate side chain: In one tautomer the proton resides on the imidazole while in the other the proton is transferred to the carboxylate. When Asp235 is replaced by Asn, the H-bond is lost, and the Fe-His stretching frequency is markedly lowered. Two new RR bands are produced, at 205 and 185 cm-1, as a result of coupling between the shifted Fe-His vibration and a nearby porphyrin mode; the two bands share the 54Fe sensitivity expected for Fe-His stretching. C=C stretching and CβC=C bending vibrations have been separately assigned to the 2- and 4-vinyl groups of the protoheme prosthetic group via selective vinyl deuteration. In the acid form of the enzyme, the frequencies coincide for the two vinyl groups, at 1618 cm-1 for the C=C stretch, and at 406 cm-1 for the CβC=C bend. However, the 2-vinyl frequencies are elevated in the alkaline form of the enzyme, to 1628 cm-1 for C=C stretching, and to 418 cm-1 for CβC=C bending, while the 4-vinyl frequencies remain unshifted. Thus, the acid-alkaline transition involves a protein conformation change that specifically perturbs the 2-vinyl substituent. This perturbation might be a reorientation of the vinyl group, or an alteration of the porphyrin geometry that affects the porphyrin-vinyl coupling. The perturbation is attenuated when CO is bound to the enzyme; the C=C frequency is then unaffected in the alkaline form, while the CβC=C bending frequency is shifted to a smaller extent (412 cm-1). This attenuation is probably linked to inhibition of distal histidine binding to the heme Fe in the alkaline form when the CO is bound. © 1996 John Wiley & Sons, Inc.

Proton pumping activity and visible absorption and resonance Raman spectra of a cao-type cytochrome c oxidase isolated from the thermophilic bacterium Bacillus PS3

Cytochrome c oxidase having heme O in addition to heme C and heme A (cytochrome cao) [Sone, N., & Fujiwara, Y. (1991) FEBS Lett. 288, 154-158] was isolated from a thermophilic bacterium, Bacillus PS3, grown under slightly air-limited conditions. Cytochrome cao could oxidize yeast cytochrome c and N,N,N',N'-tetramethyl-p-phenylenediamine twice as fast as cytochrome caa3, which this organism yielded under normal growing conditions. Cytochrome cao also pumped protons upon cytochrome c oxidation in a way similar to cytochrome caa3. Binding of cyanide to cytochrome cao caused spin-state conversion of heme O at the binuclear center and seriously inhibited its physiological activity. A low Ki value (0.4 microM) for cyanide was found to be mainly due to a small "off" constant. Resonance Raman spectra of cytochrome cao bore close resemblance to those of cytochrome caa3 in both oxidized and reduced states, although the formyl stretching (vCH=O) band was absent. The Fe-histidine stretching (vFe-His) and Fe-CO stretching (vFe-CO) frequencies of cytochrome cao were very close to those seen for cytochrome caa3, but were distinct from those of hemoglobin and peroxidases, suggesting that the protein structure in the vicinity of heme O resembles that of the heme a3 moiety of cytochrome caa3.

Resonance Raman spectra of cytochrome a in cytochrome oxidase: Excitation in the 605‐nm absorption region

AbstractVisible excitation resonance Raman difference spectra, in resonance with the 605‐nm absorption maximum of fully reduced and mixed‐valence cyanide‐bound cytochrome oxidase, were recorded. Under these conditions, the vibrations of ferrocytochrome a are markedly enhanced owing to its dominant contribution to the 605‐nm α‐band absorption of reduced cytochrome oxidase. The effect of H/D exchange in the Raman spectra of cytochrome a was also investigated as a way to establish formyl‐ and vinyl‐peripheral substituent sensitive modes. Measurements of the depolarization ratio dependence of cytochrome a vibrational modes resulted in some isotope‐sensitive modes with characteristic depolarized behavior; however, it was observed that the majority of the visible excitation vibrations exhibit polarized values. This is interpreted as being due to a significant lowering in the molecular symmetry of the heme a porphyrin macrocycle caused by the unusual peripheral substituent pattern of heme a and, presumably, by the strong cytochrome a‐protein interactions. Both structural effects appear to result in a selective enhancement of cytochrome a Eu substituent‐sensitive modes as detected by the low‐frequency resonance Raman spectrum.

Effects of temperature and glycerol on the resonance Raman spectra of cytochrome c peroxidase and selected mutants

The high-frequency resonance Raman spectra of FeIII yeast native cytochrome c peroxidase (CCP) and five of its mutants [CCP(MI), Phe-51, Leu-48, Lys-48, Asn-235, and Phe-191] were recorded in phosphate buffer, pH 7.0, and in glycerol/phosphate mixtures at 295 and 10 K. Glycerol induces heme coordination changes in some of the CCP mutants at room temperature. It apparently weakens the binding of the Fe atom to ligands in the distal heme cavity and drives the heme toward the 5-coordinate, high-spin state. At 10 K, native CCP and all the mutants (except Phe-51 which remains 6-coordinate, high-spin) show various distributions of spin and coordination states which differ from those observed at 295 K. Upon cooling in phosphate buffer, pH 7, and to a much lesser extent in 66% glycerol/phosphate, an internal strong-field ligand is coordinated to the Fe. A likely candidate is H2O-595, which could become a strong-field ligand on H-bonding and/or proton transfer to H2O-648, and/or the distal His-52. However, distal His-52 itself cannot be ruled out as the coordinating ligand considering that the Phe-51 mutant, which binds H2O-595 at room temperature, does not show a large 6-coordinate, low-spin component at 10 K like the other mutants. These results clearly indicate that the Fe coordination in CCP and its mutants is sensitive to both temperature and solvent composition.

Cytochrome c oxidase: evidence for interaction of water molecules with cytochrome a

The resonance Raman spectra of cytochrome c oxidase in protonated buffer compared to that in deuterated buffer indicate that water molecules are near the heme of cytochrome a. Differences in widths of the heme line at 1610 cm-1, after short exposure to D2O, and, additionally, of the heme line at 1625 cm-1, after long exposure, can be accounted for by changes in resonance vibrational energy transfer between modes of cytochrome a2+ and the bending mode of water molecules in the heme pocket. On the basis of the assignment of these modes, we place one water molecule near the vinyl group and one water molecule near the formyl group of the cytochrome a heme. These water molecules may play several possible functional roles.

Ultraviolet resonance Raman spectra of cytochrome c conformational states.

Ultraviolet resonance Raman (UV RR) spectra are reported for ferricytochrome c from tuna and horse heart at pH 1.6, 7, 10, and 13, representing distinct conformational states of the protein (states II, III, IV, and V, respectively). The spectra were obtained with pulsed laser excitation at 200 and 218 nm, via H2 Raman shifting the fourth harmonic output of a pulsed YAG laser. At these deep UV wavelengths, strong enhancement is observed for vibrational modes associated with tryptophan, tyrosine, and phenylalanine side chains and with the amide groups of the polypeptide backbone. The amide I peak frequency is consistent with a dominant contribution from alpha-helical regions, although a broad high-frequency tail reflects a variety of unordered conformations. The peak frequency is 12 cm-1 higher for cytochrome c from tuna than from horse, suggesting a less tightly wound structure, which is consistent with the lower denaturation temperature previously reported for the tuna protein. The amide I peak broadens when native protein (state III) is converted to the low- or high-pH forms (states II and IV), reflecting some disordering of the polypeptide chain, but the peak frequencies are unshifted, establishing that the alpha-helical segments are not completely unfolded in these states. Raising the pH to 13 (state V), however, does produce a frequency upshift, reflecting helix unfolding. The amide II and III frequencies are likewise consistent with a dominant alpha-helix contribution in the native proteins; they gain intensity, and amide III is shifted to a lower frequency, in states II and IV, consistent with partial disordering.(ABSTRACT TRUNCATED AT 250 WORDS)

Raman characterization of axial ligands for penta- and hexacoordinate ferric high- and intermediate-spin (octaethylporphyrinato)iron(III) complexes. Elucidation of unusual resonance Raman spectra of cytochrome c'

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTRaman characterization of axial ligands for penta- and hexacoordinate ferric high- and intermediate-spin (octaethylporphyrinato)iron(III) complexes. Elucidation of unusual resonance Raman spectra of cytochrome c'Junji Teraoka and Teizo KitagawaCite this: J. Phys. Chem. 1980, 84, 15, 1928–1935Publication Date (Print):July 1, 1980Publication History Published online1 May 2002Published inissue 1 July 1980https://doi.org/10.1021/j100452a013RIGHTS & PERMISSIONSArticle Views148Altmetric-Citations70LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts

Photoreductive titration of the resonance Raman spectra of cytochrome oxidase in whole mitochondria

A photoreductive titration of the resonance Raman (RR) spectra of cytochrome c oxidase in whole mitochondria was recorded by exploiting the preferential enhancement of the Raman signals of reduced cytochrome oxidase excited at 441.6 nm. When the sample was cooled to about--10 degrees C, it was possible to slow down the photoreductive effect of the laser and to record RR spectra at various states of reduction. Compared to the earliest recorded scan (most oxidized), the dithionite-reduced sample shows the appearance of new bands at 216, 363, 560, and 1665 cm-1. At intermediate stages of photoreduction, the 216- and 560-cm-1 bands appear before the 363- and 1665-cm-1 bands; photoreduction induces full intensity in the former bands, whereas the latter bands are photoreduced to 50% of the dithionite-reduced intensity. The relative intensities of a doublet at 1609--1623 cm-1 are affected by reduction: the band at 1609 cm-1 is weaker in the earlier scans; in later scans this band has grown to equal intensity with the 1623-cm-1 band. We conclude that this reductive titration of the RR spectrum of cytochrome c oxidase reflects three states in its reduction. The behavior of the doublet at 1609--1623 cm-1 suggests that the two hemes are nonequivalent but interacting. The band at 216 cm-1 may be indicative of an iron-copper interaction that is affected by the presence of external ligands.

Resonance Raman spectra of cytochrome C and oxyhemoglobin using pulsed laser excitation and optical multichannel detection

The resonance Raman spectra of dilute cytochrome c and oxyhemoglobin solutions obtained with high peak power (>1 MW) laser excitation at 532.0 nm and optical multichannel analysis (OMA) are presented. The frequencies and intensities of bands in resonance Raman spectra obtained under these conditions are directly comparable to spectra derived from 0.15 W peak power excitation using cw lasers. No anomalous spectral features are observed. These pulsed-laser/OMA spectra are used to comment on the applicability of such techniques for time-resolved resonance Raman spectroscopy of biopolymers.

Fast Raman spectroscopy of cytochrome c using intracavity resonance Raman amplification

The resonance Raman spectrum of cytochrome c (5 × 10 −4 M) was obtained using the method of resonance Raman amplification (RRA) in a dye laser resonator in 30 ns. The second harmonic of a Q-switched Nd glass laser was used as an excitation source and the spectra were recorded using a photographic plate.

Determination of oxidation and spin states of heme iron. Resonance raman spectroscopy of cytochrome c, microperoxidase, and horseradish per oxidase

Resonance Raman spectra of cytochrome c in solution are compared with those of lyophilized cytochrome c, a heme undecapeptide from cytochrome c, and horseradish peroxidase. Spectral lines sensitive to iron oxidation state and spin state are identified. The spectra are not noticeably influenced by the protein environment of the heme chromophore.

Resonance Raman spectroscopy of cytochrome [formula omitted] oxidase and electron transport particles with excitation near the Soret band

We report the resonance Raman spectra of cytochrome c oxidase, both solubilized and in electron transport particles using laser excitation near the Soret band. As in the spectra of other hemoproteins, such as cytochrome c , the shape and intensity of a number of bands change when the oxidation state is varied. However, one of the hemes of solubilized cytochrome c oxidase shows redox behavior which is anomalous. Spectra of electron transport particles are dominated by cytochrome c oxidase. There are, however, definite differences between spectra of solubilized cytochrome c oxidase and electron transport particles in the oxidized states.