Structures of reaction intermediates of bovine cytochrome c oxidase probed by time-resolved vibrational spectroscopy

Structures of reaction intermediates of bovine cytochrome c oxidase (CcO) in the reactions of its fully reduced form with O2 and fully oxidized form with H2O2 were investigated with time-resolved resonance Raman (RR) and infrared spectroscopy. Six oxygen-associated RR bands were observed for the reaction of CcO with O2. The isotope shifts for an asymmetrically labeled dioxygen, (16)O(18)O, has established that the primary intermediate of cytochrome a3 is an end-on type dioxygen adduct and the subsequent intermediate (P) is an oxoiron species with Fe=O stretch (nu(Fe=O)) at 804/764 cm(-1) for (16)O2/(18)O2 derivatives, although it had been long postulated to be a peroxy species. The P intermediate is converted to the F intermediate with nu(Fe=O) at 785/751 cm(-1) and then to a ferric hydroxy species with nu(Fe-OH) at 450/425 cm(-1) (443/417 cm(-1) in D2O). The rate of reaction from P to F intermediates is significantly slower in D2O than in H2O. The reaction of oxidized CcO with H2O2 yields the same oxygen isotope-sensitive bands as those of P and F, indicating the identity of intermediates. Time-resolved infrared spectroscopy revealed that deprotonation of carboxylic acid side chain takes place upon deligation of a ligand from heme a3. UV RR spectrum gave a prominent band due to cis C=C stretch of phospholipids tightly bound to purified CcO.     

Resonance Raman spectroscopy of carotenoids in Photosystem I particles

Low-temperature resonance Raman (RR) spectroscopy was used for the first time to study the spectral properties, binding sites and composition of major carotenoids in spinach Photosystem I (PSI) particles. Excitation was provided by an argon ion laser at 457.9, 476.5, 488, 496.5, 502 and 514.5 nm. Raman spectra contained the four known groups of bands characteristic for carotenoids (called from nu(1) to nu4). Upon 514.5, 496.5 and 476.5 nm excitations, the nu(1)-nu(3) frequencies coincided with those established for lutein. Spectrum upon 502-nm excitation could be assigned to originate from violaxanthin, at 488 nm to 9-cis neoxanthin, and at 457.9 nm to beta-carotene and 9-cis neoxanthin. The overall configuration and composition of these bound carotenoid molecules in Photosystem I particles were compared with the composition of pigment extracts from the same PSI particles dissolved in pyridine, as well as to configuration in the main chlorophyll a/b light-harvesting protein complex of photosystem II. The absorption transitions for lutein, violaxanthin and 9-cis neoxanthin in spinach photosystem I particles are characterized, and the binding sites of lutein and neoxanthin are discussed. Resonance Raman data suggest that beta-carotene molecules are also present in all-trans and, probably, in 9-cis configurations.     

Carotenoids as a Raman-active probes of erythrocyte membrane structure.

1. Erythrocyte ghosts exhibit resonance-enhanced Raman bands at 1530 cm(-1) and 1165 cm(-1) attributable to v(-C=C-) and v(=C-C=), respectively, of the conjugated polyene chains in carotenoids. In lipid extract of ghosts, these resonance-enhanced bands lie at 1527 and 1158 cm(-1). The spectra indicate the presence of membrane-bound beta-carotene. 2. The resonance-enhanced Raman spectrum of beta-carotene in lecithin liposomes is identical to that obtained with hexane or chloroform solutions. 3. Increasing proportions of cholesterol in cholesterol-lecithin liposomes up to a cholesterol: phospholipid molar ratio of 0.8-0.9 drastically decreases the intensity of both resonance-enhanced bands. 4. In ghosts the carotenoid bands respond to membrane perturbations. Trypsinization, lysolecithin treatment and reduction of pH increase the intensities of the 1530 and 1165 cm(-1) bands. In contrast, a decrease in the intensity of both bands follows equilibration of ghosts for 15 min at approx. 50 degrees C or addition of (0.1%) sodium dodecyl sulfate. 5. We suggest that perturbants known to change lipid-protein interactions in erythrocyte membranes modify the microenvironment and/or configuration of the membrane-bound carotenoid.     

The study of microviscosity of plasma membranes of Nitella cells during rest and excitation

Changes in the microviscosity of excitable membranes was investigated using resonance Raman spectroscopy of carotenoids. The Raman resonance spectra of carotenoids in Nitella cells were excited by 514.5 nm line of an argon ion laser. The bands at 1525 cm-1, 1160 cm-1 and 1008 cm-1 were observed and they were assigned to C=C, C-C and C-CH vibrations, respectively. The rhythmic excitation of cell reduced the intensity and increased the ratios of intensity of major carotenoid bands with no noticeable shift in the position of peaks. The Arrhenius plot of relative intensity ratios of 1525 cm-1 and 1160 cm-1 bands versus reciprocal temperature showed a change of the slope in the range of 13-18 degrees C. This indicates a membrane phase transitions in which a reorientation of carotenoids species takes place. The interpretation was supported by parallel microcalorimetric and EPR measurements. The decrease of microviscosity with increasing temperature is probably caused by changes in polyene chain conformation. It is suggested that membrane microviscosity during NH4(+)-stimulated rhythmic excitation of algal cells increases, and membrane-associated carotenoids act as microviscosity-sensitive "potential sensor" for the channel.     

Redox-triggered FTIR difference spectra of FAD in aqueous solution and bound to flavoproteins

Flavin adenine dinucleotide (FAD) and three different flavoproteins in aqueous solution were subjected to redox-triggered Fourier transform infrared difference spectroscopy. The acquired vibrational spectra show a great number of positive and negative peaks, pertaining to the oxidized and reduced state of the molecule, respectively. Density functional theory calculations on the B3LYP/6-31G(d) level were employed to assign several of the observed bands to vibrational modes of the isoalloxazine moiety of the flavin cofactor in both its oxidized and, for the first time, its reduced state. Prominent modes measured for oxidized FAD include nu(C(4)=O) and nu(C(2)=O) at 1716 and 1674 cm(-1), respectively, nu(C(4a)=N(5)) at 1580 cm(-1), and nu(C(10a)=N(1)) at 1548 cm(-1). Measured modes of the reduced form of FAD include nu(C(2)=O) at 1692 cm(-1), nu(C(4)=O) at 1634 cm(-1), and nu(C(4a)=C(10a)) at 1600 cm(-1). While the overall shape of the enzyme spectra is similar to the shape of the spectrum of free FAD, there are numerous differences in detail. In particular, the nu(C=N) modes of the flavin exhibit frequency shifts in the protein-bound form, most prominently for pyruvate oxidase where nu(C(10a)=N(1)) downshifts by 14 cm(-1) to 1534 cm(-1). The significance of this shift and a possible explanation in connection with the bent conformation of the flavin cofactor in this enzyme are discussed.     

法夫酵母 JMU-MVP14菌体中类胡萝卜素成分分析

结合薄层色谱、柱色谱、以及高效液相色谱对虾青素高产菌株-法夫酵母JMU-MVP14中的类胡萝卜素成分进行初步研究。研究结果表明,硅胶柱层析和氧化镁柱层析相结合的方法对法夫酵母JMU-MVP14菌体中的类胡萝卜素成分有很好的分离效果。经过柱层析分离纯化后,各组分中类胡萝卜素的种类单一,有利于进一步通过各种波谱技术对其进行定性。此方法弥补了单纯依靠高效液相色谱（ODS 柱）对法夫酵母 JMU-MVP14菌体中类胡萝卜素分离效果不佳,可供选择的商业化类胡萝卜素标准品少,液相保留时间漂移等因素给法夫酵母JMU-MVP14菌体中类胡萝卜素定性带来的不足。     

Resonance Raman studies indicate a unique heme active site in prostaglandin H synthase

Prostaglandin H synthase isoforms 1 and 2 (PGHS-1 and -2) catalyze the first two steps in the biosynthesis of prostaglandins. Resonance Raman spectroscopy was used to characterize the PGHS heme active site and its immediate environment. Ferric PGHS-1 has a predominant six-coordinate high-spin heme at room temperature, with water as the sixth ligand. The proximal histidine ligand (or the distal water ligand) of this hexacoordinate high-spin heme species was reversibly photolabile, leading to a pentacoordinate high-spin ferric heme iron. Ferrous PGHS-1 has a single species of five-coordinate high-spin heme, as evident from nu(2) at 1558 cm(-1) and nu(3) at 1471 cm(-1). nu(4) at 1359 cm(-1) indicates that histidine is the proximal ligand. A weak band at 226-228 cm(-1) was tentatively assigned as the Fe-His stretching vibration. Cyanoferric PGHS-1 exhibited a nu(Fe)(-)(CN) line at 446 cm(-1) and delta(Fe)(-)(C)(-)(N) at 410 cm(-1), indicating a "linear" Fe-C-N binding conformation with the proximal histidine. This linkage agrees well with the open distal heme pocket in PGHS-1. The ferrous PGHS-1 CO complex exhibited three important marker lines: nu(Fe)(-)(CO) (531 cm(-1)), delta(Fe)(-)(C)(-)(O) (567 cm(-1)), and nu(C)(-)(O) (1954 cm(-1)). No hydrogen bonding was detected for the heme-bound CO in PGHS-1. These frequencies markedly deviated from the nu(Fe)(-)(CO)/nu(C)(-)(O) correlation curve for heme proteins and porphyrins with a proximal histidine or imidazolate, suggesting an extremely weak bond between the heme iron and the proximal histidine in PGHS-1. At alkaline pH, PGHS-1 is converted to a second CO binding conformation (nu(Fe)(-)(CO): 496 cm(-1)) where disruption of the hydrogen bonding interactions to the proximal histidine may occur.     

Two-dimensional vibrational correlation spectroscopy of in vitro hydroxyapatite maturation

Two-dimensional (2-D) Raman and 2-D IR correlation spectroscopy are applied to analyze changes in the nu(4) region of the IR spectrum and in the nu(1) region of the Raman spectrum during the maturation of hydroxyapatite (HA) following the solution-mediated conversion of amorphous calcium phosphate (ACP) to HA. The nu(1) region of the Raman spectrum exhibits a frequency shift and sharpening during the maturation. Comparison of the experimental and simulated 2-D plots for this process suggests that the shift of a single peak, rather than a change in the relative intensity of two overlapped bands, is responsible for the observed spectral changes. The nu(4) mode of the PO(3-)(4) ion (T(2) symmetry in the free species) splits into a triplet with components near 563, 575, and 603 cm(-1) in HA. In addition, broad features appear at 540 and 617 cm(-1). During the latest stages of the maturation, an OH(-) librational mode develops at approximately 632 cm(-1). Changes in the relative intensities of three components of the nu(4) mode are not all correlated with each other. The synchronous 2-D plots reveal that the 563 and 603 cm(-1) pair are positively correlated while the feature at 575 cm(-1) is absent. A 587 cm(-1) mode arising from ACP is negatively correlated with the 563 and 603 cm(-1) pair and is both synchronously (positively) and asynchronously correlated with the 540 cm(-1) feature during the early stages of the maturation but is absent from 2-D plots of the later stages of the maturation. Cross correlations between the nu(4) mode and the nu(1),nu(3) contour generally confirm and extend previous assignments for the latter spectral region. Finally, the suitability of the 2-D approach for analysis of IR spectral images is examined through studies of HA crystallinity in a human iliac crest biopsy sample. Trabecular bone contains a fraction of HA that is more crystalline and mature than could be achieved in vitro during the room temperature ACP --> HA interconversion.     

IR and Raman spectroscopy in the study of carotenoids of <Emphasis Type="Italic">Cladophora rivularis</Emphasis> algae

Using Raman and infrared spectroscopy it has been found that during the normal life of algae (pH changes from 8.0 to 9.0) the content of carotenoids increases and the molecules change their conformation: the contribution of–C=C–bonds of the polyene chain of a carotenoid molecule (Raman spectroscopy) is reduced and the contribution of methyl groups (～2940 cm^–1) and aromatic C–H-plane deformation vibrations (band at 1050 cm^–1) of carotenoid molecules (infrared spectroscopy) decreases as well. It is the opinion of the authors that a change in the extracellular pH within the normal range has no influence on the content of chlorophyll a and b, but tends to increase the content and alter the conformation or structure of carotenoid molecules.     

Resonant Raman quantification of zeaxanthin production from Flavobacterium multivorum

Resonant Raman scattering was used as a novel, rapid, non-destructive optical technique to measure zeaxanthin levels in Flavobacterium multivorum ATCC 55238. Culture broth, after bacterial growth for 40 h, exhibited characteristic resonance Raman vibrational modes at 1159 cm^–1 (C-C stretch) and 1525 cm^–1 (C=C stretch) upon excitation at 488 nm. A striking correlation was observed between the carotenoid level as estimated by HPLC and by resonance Raman spectroscopy.     

Measurement of hemoglobin oxygen saturation using Raman microspectroscopy and 532-nm excitation.

The resonant Raman enhancement of hemoglobin (Hb) in the Q band region allows simultaneous identification of oxy- and deoxy-Hb. The heme vibrational bands are well known at 532 nm, but the technique has never been used to determine microvascular Hb oxygen saturation (So(2)) in vivo. We implemented a system for in vivo noninvasive measurements of So(2). A laser light was focused onto areas of 15-30 microm in diameter. Using a microscope coupled to a spectrometer and a cooled detector, Raman spectra were obtained in backscattering geometry. Calibration was performed in vitro using blood at several Hb concentrations, equilibrated at various oxygen tensions. So(2) was estimated by measuring the intensity of Raman signals (peaks) in the 1,355- to 1,380-cm(-1) range (oxidation state marker band nu(4)), as well as from the nu(19) and nu(10) bands (1,500- to 1,650-cm(-1) range). In vivo observations were made in microvessels of anesthetized rats. Glass capillary path length and Hb concentration did not affect So(2) estimations from Raman spectra. The Hb Raman peaks observed in blood were consistent with earlier Raman studies using Hb solutions and isolated cells. The correlation between Raman-based So(2) estimations and So(2) measured by CO-oximetry was highly significant for nu(4), nu(10), and nu(19) bands. The method allowed So(2) determinations in all microvessel types, while diameter and erythrocyte velocity could be measured in the same vessels. Raman microspectroscopy has advantages over other techniques by providing noninvasive and reliable in vivo So(2) determinations in thin tissues, as well as in solid organs and tissues in which transillumination is not possible.     

Dark adaptation and conformations of carotenoids in the cells of <Emphasis Type="Italic">Cladophora aegagropila</Emphasis> (L). Rabenh.

Intact cells of freshwater algae Cladophora aegagropila (L). Rabenh. (synonymous to Aegagropila linnaei Kutz.) were investigated by resonance Raman spectroscopy. It was found that incubation in the dark (up to 24 h) leads to changes in the Raman spectroscopy spectrum of this species, namely to changes in the ratio of amplitudes of the I₁₅₂₃/I₁₁₅₅ and I₉₆₀/I₁₀₀₄ bands and in the half width of band in the region of 1523 cm–1. We suggested that the adaptation of algae to the dark alters the conformation of the molecule of the carotenoid by delocalization of π-electrons in the polyene chain of the molecule and changes the orientation of the ring. Moreover, the composition of carotenoids, as well as their location in the cell and microenvironment in the pigment–protein complexes can change: in the absence of illumination, the distribution of carotenoids in algal cells is more uniform. These changes are probably caused either by changes in the location of cell organelles or by carotenoid redistribution between photosynthetic membranes, plastoglobules, and lipophilic formations in the cytoplasm.     

Soret-excited Raman spectroscopy of the spinach cytochrome b6f complex. Structures of the b- and c-type hemes, chlorophyll a, and beta-carotene

Soret-excited resonance Raman (RR) spectra of the spinach cytochrome b6f complex (cyt b6f) are reported for the oxidized, native, ascorbate-reduced, and dithionite-reduced forms. Using excitations at 441.6, 413.1, and 406.7 nm, RR contributions of chlorophyll a, beta-carotene, the c-type heme of cytochrome f, and the b-type hemes of cytochrome b6 of the b6f complex were identified and the data compared to those previously obtained for the Rhodospirillum rubrum bc1 complex [Le Moigne, C., Schoepp, B., Othman, S., Verméglio, A., and Desbois, A. (1999) Biochemistry 38, 1066-1076]. RR bands arising from the b(6)f-associated chlorophyll a and beta-carotene pigments were found to be particularly intense in the spectra excited at 441.6 nm. The frequencies of the phorbin skeleton of chlorophyll a at 1606, 1552, and 1525 cm(-1) are typical of a Mg atom with a single axial ligand. Strong RR bands corresponding to stretching or deformation modes of beta-carotene were detected at 1137, 1157, 1191, 1216, and 1531 cm(-1) in the different forms of cyt b6f. This set of frequencies is assigned to an all-trans configuration of the polyene chain. The redox titrations of the b(6)f complex allow the characterization of RR bands of the three hemes. The nu10, nu2, nu3, and nu8 modes of reduced cyt f are detected at 1619, 1591, 1492, and 356 cm(-1), respectively. From this set of frequencies, one can conclude that the particular histidine/amine heme coordination found in the truncated soluble domain of cyt f is a specific feature of the entire cyt f included in the b6f complex. The frequencies of the nu2, nu8, and nu10 marker modes are consistent with different conformations for the two b-type hemes of cyt b6f. One of these hemes is strongly distorted (nu2, nu8, and nu10 at 1581, 351, and 1610 cm(-1), respectively), while the other one is planar (1586, 345, and 1618 cm(-1), respectively). Largely different structures for the b-type hemes appear to be a common property for the bc1/b6f complexes.     

Redox infrared markers of the heme and axial ligands in microperoxidase: bases for the analysis of c-type cytochromes

Structural changes accompanying the change in the redox state of microperoxidase-8 (MP8), the heme-octapeptide obtained from cytochrome c, and its complexes with (methyl)imidazole ligands were studied by electrochemically induced Fourier transform IR (FTIR) difference spectroscopy. To correlate with confidence IR modes with a specific electronic state of the iron, we used UV-vis and electron paramagnetic resonance spectroscopy to define precisely the heme spin state in the samples at the millimolar concentration of MP8 required for FTIR difference spectroscopy. We identified four intense redox-sensitive IR heme markers, nu38 at 1,569 cm(-1) (ox)/1,554 cm(-1) (red), nu42 at 1,264 cm(-1) (ox)/1,242 cm(-1) (red), nu43 at 1,146 cm(-1) (ox), and nu44 at 1,124-1,128 cm(-1) (ox). The intensity of nu42 and nu43 was clearly enhanced for low-spin imidazole-MP8 complexes, while that of nu44 increased for high-spin MP8. These modes can thus be used as IR markers of the iron spin state in MP8 and related c-type cytochromes. Moreover, one redox-sensitive band at 1,044 cm(-1) (red) is attributed to an IR marker specific of c-type hemes, possibly the delta(CbH3)(2,4) heme mode. Other redox-sensitive IR bands were assigned to the MP8 peptide backbone and to the fifth and sixth axial heme ligands. The distinct IR frequencies for imidazole (1,075 cm(-1)) and histidine (1,105 cm(-1)) side chains in the imidazole-MP8 complex allowed us to provide the first direct determination of their pKa at pH 9 and 12, respectively.     

Resonance Raman characterization of the P intermediate in the reaction of bovine cytochrome c oxidase

Reduced cytochrome c oxidase binds molecular oxygen, yielding an oxygenated intermediate first (Oxy) and then converts it to water via the reaction intermediates of P, F, and O in the order of appearance. We have determined the iron-oxygen stretching frequencies for all the intermediates by using time-resolved resonance Raman spectroscopy. The bound dioxygen in Oxy does not form a bridged structure with Cu(B) and the rate of the reaction from Oxy to P (P(R)) is slower at higher pH in the pH range between 6.8 and 8.0. It was established that the P intermediate has an oxo-heme and definitely not the Fe(a(3))-O-O-Cu(B) peroxy bridged structure. The Fe(a(3))=O stretching (nu(Fe=O)) frequency of the P(R) intermediate, 804/764 cm(-1) for (16)O/(18)O, is distinctly higher than that of F intermediate, 785/750 cm(-1). The rate of reaction from P to F in D(2)O solution is evidently slower than that in H(2)O solution, implicating the coupling of the electron transfer with vector proton transfer in this process. The P intermediate (607-nm form) generated in the reaction of oxidized enzyme with H(2)O(2) gave the nu(Fe=O) band at 803/769 cm(-1) for H(2)(16)O(2)/H(2)(18)O(2) and the simultaneously measured absorption spectrum exhibited the difference peak at 607 nm. Reaction of the mixed valence CO adduct with O(2) provided the P intermediate (P(M)) giving rise to an absorption peak at 607 nm and the nu(Fe=O) bands at 804/768 cm(-1). Thus, three kinds of P intermediates are considered to have the same oxo-heme a(3) structure. The nu(4) and nu(2) modes of heme a(3) of the P intermediate were identified at 1377 and 1591 cm(-1), respectively. The Raman excitation profiles of the nu(Fe=O) bands were different between P and F. These observations may mean the formation of a pi cation radical of porphyrin macrocycle in P.     

Tissue-specific accumulation of carotenoids in carrot roots

Raman spectroscopy can be used for sensitive detection of carotenoids in living tissue and Raman mapping provides further information about their spatial distribution in the measured plant sample. In this work, the relative content and distribution of the main carrot (Daucus carota L.) root carotenoids, α-, β-carotene, lutein and lycopene were assessed using near-infrared Fourier transform Raman spectroscopy. The pigments were measured simultaneously in situ in root sections without any preliminary sample preparation. The Raman spectra obtained from carrots of different origin and root colour had intensive bands of carotenoids that could be assigned to β-carotene (1,520 cm⁻¹), lycopene (1,510 cm⁻¹) and α-carotene/lutein (1,527 cm⁻¹). The Raman mapping technique revealed detailed information regarding the relative content and distribution of these carotenoids. The level of β-carotene was heterogeneous across root sections of orange, yellow, red and purple roots, and in the secondary phloem increased gradually from periderm towards the core, but declined fast in cells close to the vascular cambium. α-carotene/lutein were deposited in younger cells with a higher rate than β-carotene while lycopene in red carrots accumulated throughout the whole secondary phloem at the same level. The results indicate developmental regulation of carotenoid genes in carrot root and that Raman spectroscopy can supply essential information on carotenogenesis useful for molecular investigations on gene expression and regulation.     

Substrate-specific interactions with the heme-bound oxygen molecule of nitric-oxide synthase

We report the characterization by resonance Raman spectroscopy of the oxygenated complex (Fe(II)O(2)) of nitric-oxide synthases of Staphylococcus aureus (saNOS) and Bacillus subtilis (bsNOS) saturated with N(omega)-hydroxy-l-arginine. The frequencies of the nu(Fe-O) and nu(O-O) modes were 530 and 1135 cm(-), respectively, in both the presence and absence of tetrahydrobiopterin. On the basis of a comparison of these frequencies with those of saNOS and bsNOS saturated with l-arginine (nu(Fe-O) at 517 cm(-1) and nu(O-O) at 1123 cm(-1)) and those of substrate-free saNOS (nu(Fe-O) at 517 and nu(O-O) at 1135 cm(-1)) (Chartier, F. J. M., Blais, S. P., and Couture, M. (2006) J. Biol. Chem. 281, 9953-9962), we propose two models that account for the frequency shift of nu(Fe-O) (but not nu(O-O)) upon N(omega)-hydroxy-l-arginine binding as well as the frequency shift of nu(O-O) (but not nu(Fe-O)) upon l-arginine binding. The implications of these substrate-specific interactions with respect to catalysis by NOSs are discussed.     

Resonance Raman and ligand binding studies of the oxygen-sensing signal transducer protein HemAT from Bacillus subtilis

HemAT-Bs is a heme-containing signal transducer protein responsible for aerotaxis of Bacillus subtilis. The recombinant HemAT-Bs expressed in Escherichia coli was purified as the oxy form in which oxygen was bound to the ferrous heme. Oxygen binding and dissociation rate constants were determined to be k(on) = 32 microm(-1) s(-1) and k(off) = 23 s(-1), respectively, revealing that HemAT-Bs has a moderate oxygen affinity similar to that of sperm whale myoglobin (Mb). The rate constant for autoxidation at 37 degrees C was 0.06 h(-1), which is also close to that of Mb. Although the electronic absorption spectra of HemAT-Bs were similar to those of Mb, HemAT-Bs showed some unique characteristics in its resonance Raman spectra. Oxygen-bound HemAT-Bs gave the nu(Fe-O(2)) band at a noticeably low frequency (560 cm(-1)), which suggests a unique hydrogen bonding between a distal amino acid residue and the proximal atom of the bound oxygen molecule. Deoxy HemAT-Bs gave the nu(Fe-His) band at a higher frequency (225 cm(-1)) than those of ordinary His-coordinated deoxy heme proteins. CO-bound HemAT-Bs gave the nu(Fe-CO) and nu(C-O) bands at 494 and 1964 cm(-1), respectively, which fall on the same nu(C-O) versus nu(Fe-CO) correlation line as that of Mb. Based on these results, the structural and functional properties of HemAT-Bs are discussed.     

Heme structures of five variants of hemoglobin M probed by resonance Raman spectroscopy

The alpha-abnormal hemoglobin (Hb) M variants show physiological properties different from the beta-abnormal Hb M variants, that is, extremely low oxygen affinity of the normal subunit and extraordinary resistance to both enzymatic and chemical reduction of the abnormal met-subunit. To get insight into the contribution of heme structures to these differences among Hb M's, we examined the 406.7-nm excited resonance Raman (RR) spectra of five Hb M's in the frequency region from 1700 to 200 cm(-1). In the high-frequency region, profound differences between met-alpha and met-beta abnormal subunits were observed for the in-plane skeletal modes (the nu(C=C), nu(37), nu(2), nu(11), and nu(38) bands), probably reflecting different distortions of heme structure caused by the out-of-plane displacement of the heme iron due to tyrosine coordination. Below 900 cm(-1), Hb M Iwate [alpha(F8)His --> Tyr] exhibited a distinct spectral pattern for nu(15), gamma(11), delta(C(beta)C(a)C(b))(2,4), and delta(C(beta)C(c)C(d))(6,7) compared to that of Hb M Boston [alpha(E7)His --> Tyr], although both heme irons are coordinated by Tyr. The beta-abnormal Hb M variants, namely, Hb M Hyde Park [beta(F8)His --> Tyr], Hb M Saskatoon [beta(E7)His --> Tyr], and Hb M Milwaukee [beta(E11)Val --> Glu], displayed RR band patterns similar to that of metHb A, but with some minor individual differences. The RR bands characteristic of the met-subunits of Hb M's totally disappeared by chemical reduction, and the ferrous heme of abnormal subunits was no longer bonded with Tyr or Glu. They were bonded to the distal (E7) or proximal (F8) His, and this was confirmed by the presence of the nu(Fe-His) mode at 215 cm(-1) in the 441.6-nm excited RR spectra. A possible involvement of heme distortion in differences of reducibility of abnormal subunits and oxygen affinity of normal subunits is discussed.     

Micro-Raman characterisation of the R to T state transition of haemoglobin within a single living erythrocyte

We present the first recorded Raman spectra of haemoglobin in both the R and T states from within a single living erythrocyte using 632.8 nm excitation. Bands characteristic of low spin haems are observed in oxygenated and carboxylated erythrocytes at approx. 1636 (nu(10)), 1562-1565 (nu(2)), 1250-1245 cm(-1) (nu(13)) and 1226-1224 cm(-1) (nu(5)+nu(8)). The spectra of deoxygenated and methaemoglobin erythrocytes have characteristic high spin bands at approx. 1610-1606 cm(-1) (nu(10)), 1582-1580 (nu(37)), 1547-1544 (nu(11)), 1230-1220 cm(-1) (nu(13)) and 1215-1210 cm(-1) (nu(5)+nu(8)). Bands at 1172 (nu(30)), 976 (nu(45)) and 672 (nu(7)) cm(-1) appear to be enhanced at 632.8 nm in low spin haems. The oxidation state marker band (nu(4)) at 1364-1366 cm(-1) appeared invariant within this domain in all single cells and conditions investigated contrary to other resonance Raman studies on haem isolates. The information gained by in vivo single erythrocyte molecular analysis has important ramifications to the understanding of fundamental physiological processes and may have applications in the diagnosis and treatment of red blood cell disorders.