Resonance Raman spectra of cytochrome c oxidase. Excitation in the 600-nm region.

The resonance Raman (RR) spectra of oxidized, reduced, and oxidized cyanide-bound cytochrome c oxidase with excitation at several wavelengths in the 600-nm region are presented. No evidence is found for laser-induced photoreduction of the oxidized protein with irradiation at lambda approximately 600 nm at 195 K, in contrast to the predominance of this process upon irradiation in the Soret region at this temperature. The Raman spectra of all three protein species are very similar, and there are no Raman bands which are readily assignable to either cytochrome a or cytochrome a3 exclusively. The Raman spectra of the three protein species do, however, exhibit a number of bands not observed in the RR spectra of other hemoproteins upon exicitation in their visible absorption bands. In particular, strong Raman bands are observed in the low-frequency region of the RR spectra (less than 500 cm-1). The frequencies of these bands are similar to those of the copper-ligand vibrations observed in the RR spectra of type 1 copper proteins upon excitation in the 600-nm absorption band characteristic of these proteins. In cytochrome c oxidase, these bands do not disappear upon reduction of the protein and, therefore, cannot be attributed to copper-ligand vibrations. Thus, all the observed RR bands are associated with the two heme A moieties in the enzyme.     

Resonance Raman study of cytochrome aa3 from Sulfolobus acidocaldarius.

The single subunit terminal oxidase of Sulfolobus acidocaldarius, cytochrome aa3, was studied by resonance Raman spectroscopy. Results on the fully oxidized, the fully reduced, and the reduced carbon monoxide complex are reported and compared with those of eucaryotic cytochrome oxidase. It is shown that in both redox states the hemes a and a3 are in the six-coordinated low-spin and six-coordinated high-spin configuration, respectively. The resonance Raman spectra reveal far-reaching similarities of this archaebacterial with mammalian or plant enzymes except for the reduced form of heme a. The formyl substituent of this heme appears above 1640 cm-1, ruling out significant hydrogen bonding interactions which is in sharp contrast to beef heart cytochrome oxidase. In addition, frequency upshifts of the marker bands v4 and v2 are noted indicating differences in the electron density distribution within the molecular orbitals of the porphyrin.     

Resonance Raman evidence for low-spin Fe2+ heme a3 in energized cytochrome c oxidase: implications for the inhibition of O2 reduction

Resonance Raman (RR) spectra are reported for reduced submitochondrial particles (SMP) with excitation at 441.6 nm, where Raman bands of the cytochrome c oxidase heme a groups are selectively enhanced. Addition of ATP to energize the membranes induces the formation of a new band at 1644 cm-1 and partial loss of intensity in a band at 1567 cm-1. These changes are modeled by adding cyanide to reduced cytochrome c oxidase and are attributed to partial conversion of cytochrome (cyt) a3 from a high-spin to a low-spin state. This conversion is abolished by addition of excess oligomycin, an ATPase inhibitor, or FCCP, an uncoupler of proton translocation, and is reversed when the ATP is consumed. The observed spin-state conversion is attributed to the binding of an endogenous ligand to the cyt a3 Fe atom. This ligation is suggested to be induced by a local increase in pH and/or by a global conformation change associated with the generation of a transmembrane potential. Since O2 binding requires a vacant coordination site at cyt a3, the ligation of this site must retard O2 reduction and could thus provide a simple mechanism for energy-linked regulation of respiration. No changes in the RR spectrum were observed upon adding Ca2+ or H+ to reduced cytochrome c oxidase. The cyt a3 spin-state change associated with membrane energization is unrelated to the cyt a absorption red shift induced by adding Ca2+ or H+ to cytochrome c oxidase.     

Resonance Raman scattering from hemoproteins. Effects of ligands upon the Raman spectra of various C-type cytochromes.

Resonance Raman spectra were measured for various C-type cytochromes (mammalian cytochrome c, bacterial cytochrome c3, algal photosynthetic cytochrome f, and alkylated cytochrome c) and a B-type cytochrome (cytochrome b5) in their reduced and oxidized states. (1) For ferrous alkylated cytochrome c, a Raman line sensitive to the replacement of an axial ligand of the heme iron uas found around 1540 cm=1. This ligand-sensitive Raman line indicated the transition from acidic (1545 cm-1) to alkaline (1533 cm-1) forms with pK 7.9. The pH dependence of the Raman spectrum corresponded well to that of the optical absorption spectra. (2) For ferrous cytochrome f, the ligand-sensitive Raman line was found at the same frequency as cytochrome c (1545 cm-1). Accordingly two axial ligands are likely to be histidine and methionine as in cytochrome c. (3) For ferrous cytochrome c3, the frequency of the ligand-sensitive Raman line was between those of cytochrome c and cytochrome b5. Since two axial ligands of the heme iron in cytochrome c3 might be histidines. However, a combination of histidine and methionine as a possible set of two axial ligands was not completely excluded for one or two of the four hemes. (4) In ferrous cytochrome b5, two weak Raman lines appeared at 1302 and 1338 cm-1 instead of the strongest band at 1313 cm-1 of C-type ferrous cytochromes. This suggests the practical use of these bands for the identification of types of cytochromes. The difference in frequency and intensity between B- and C-types of hemes implies that the low effective symmetry of the heme in ferrous cytochrome c is due to vibrational coupling of ring modes with peripheral substituents rather than geometrical disortion of heme.     

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.     

Conformational changes in cytochrome c and cytochrome oxidase upon complex formation: a resonance Raman study

The fully oxidized complex of cytochrome c and cytochrome oxidase formed at low ionic strength was studied by resonance Raman spectroscopy. The spectra of the complex and of the individual components were compared over a wide frequency range using Soret band excitation. In both partners of the complex, structural changes occur in the heme groups and in their immediate protein environment. The spectra of the complex in the 1600-1700 cm-1 frequency range were dominated by bands from the cytochrome oxidase component, whereas those in the 300-500 cm-1 range were dominated by bands from the cytochrome c component, hence allowing separation of the contributions from the two individual species. For cytochrome c, spectral changes were observed which correspond to the induction of the conformational state I and the six-coordinated low-spin configuration of state II on binding to cytochrome oxidase. While in state I the structure of cytochrome c is essentially the same as in solution, state II is characterized by a structural rearrangement of the heme pocket, leading to a weakening of the axial iron-methionine bond and an opening of the heme crevice which is situated in the center of the binding domain for cytochrome oxidase. The relative contributions of the two cytochrome c states were estimated to be approximately in the ratio 1:1 in the complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Continuous flow-resonance Raman spectroscopy of an intermediate redox state of cytochrome C.

An intermediate redox state of cytochrome c at alkaline pH, generated upon rapid reduction by sodium dithionite, has been observed by resonance Raman (RR) spectroscopy in combination with the continuous flow technique. The RR spectrum of the intermediate state is reported for excitation both in the (alpha, beta) and the Soret optical absorption band. The spectra of the intermediate state are more like those of the stable reduced form than those of the stable oxidized form. For excitation of 514.5 nm, the most prominent indication of an intermediate state is the wave-number shift of one RR band from 1,562 cm-1 in the stable oxidized state through 1,535 cm-1 in the intermediate state to 1,544 cm-1 in the stable reduced state. For excitation at 413.1 nm, a band, present at 1,542 cm-1 in the stable reduced state but not present in the stable oxidized state, is absent in the intermediate state. We interpret the intermediate species as the state where the heme iron is reduced but the protein remains in the conformation of the oxidized state, with methionine-80 displaced as sixth ligand to the heme iron, before relaxing to the conformation of the stable reduced state, with methionine-80 returned as sixth ligand.     

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)     

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.     

Flavin and heme structures in lactate:cytochrome c oxidoreductase: a resonance Raman study

Resonance Raman spectra of Hansenula anomala L-lactate:cytochrome c oxidoreductase (or flavocytochrome b2), of its cytochrome b2 core, and of a bis(imidazole) iron-protoporphyrin complex were obtained at the Soret preresonance from the oxidized and reduced forms. Raman contributions from both the isoalloxazine ring of flavin mononucleotide (FMN) and the heme b2 were observed in the spectra of oxidized flavocytochrome b2. Raman diagrams showing frequency differences of selected FMN modes between aqueous and proteic environments were drawn for various flavoproteins. These diagrams were closely similar for flavocytochrome b2 and for flavodoxins. This showed that the FMN structure must be very similar in both types of proteins, despite their very different proteic pockets. However, the electron density at this macrocycle was found to be higher in flavocytochrome b2 than in these electron transferases. No significant difference was observed between the heme structures in flavocytochrome b2 and in cytochrome b2 core. The porphyrin center-N(pyrrole) distances in the oxidized and reduced heme b2 were estimated to be 1.990 and 2.022 A from frequencies of porphyrin skeletal modes, respectively. The frequency of the vinyl stretching mode of protoporphyrin was found to be very affected in resonance Raman spectra of flavocytochrome b2 and of cytochrome b2 core (1634-1636 cm-1) relative to those observed in the spectra of iron-protoporphyrin [bis(imidazole)] complexes (1620 cm-1). These specificities were interpreted as reflecting a near coplanarity of the vinyl groups of heme b2 with the pyrrole rings to which they are attached. The low-frequency regions of resonance Raman indicated that the iron atoms of the four hemes b2 are in the porphyrin plane whatever their oxidation state. The histidine-Fe-histidine symmetric stretching mode was located at 205 cm-1 in the spectra of flavocytochrome b2 and of cytochrome b2 core. It was insensitive to the iron oxidation state and indicated strong Fe-His bonds in both states.     

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.     

Electronic state of heme in cytochrome oxidase. I. Magnetic circular dichroism of the isolated enzyme and its derivatives.

Magnetic circular dichroism (MCD) spectra have been recorded for beef heart cytochrome oxidase and a number of its inhibitor complexes. The resting enzyme exhibits a derivate shape Faraday C term in the Soret region, characteristic of low spin ferric heme, which accounts for 50% of the total oxidase heme a. The remaining heme a (50%) is assigned to the high spin state. MCD temperature studies, comparison with the MCD spectra of heme a-imidazole model compounds, and ligand binding (cyanide, formate) studies are consistent with these spin state assignments in the oxidized enzyme. Furthermore, the ligand binding properties and correlations between optical and MCD parameters indicate that in the resting enzyme the low spin heme a is due solely to cytochrome a3+ and the high spin heme a to cytochrome a33+. The Soret MCD of the reduced protein is interpreted as th sum of two MCD curves: an intense, asymmetric MCD band very similar to that exhibited by deoxymyoglobin which we assign to paramagnetic high spin cytochrome a3(2+) and a weaker, more symmetric MCD contribution, which is attributed to diamagnetic low spin cytochrome a2+. Temperature studies of the Soret MCD intensity support this proposed spin state heterogeneity. Ligand binding (CO, CN-) to the reduced protein eliminates the intense MCD associated with high spin cytochrome a3(2+); however, the band associated with cytochrome a2+ is observed under these conditions as well as in a number of inhibitor complexes (cyanide, formate, sulfide, azide) of the partially reduced protein. The MCD spectra of oxidized, reduced, and inhibitor-complexed cytochrome oxidase show no evidence for heme-heme interaction via spectral parameters. This conclusion is used in conjunction with the fact that ferric, high spin heme exhibits weak MCD intensity to calculate the MCD spectra for the individual cytochromes of the oxidase as well as the spectra for some inhibitor complexes of cytochrome a3. The results are most simply interpreted using the model we have recently proposed to account for the electronic and magnetic properties of cytochrome (Palmer, G., Babcock, F.T., and Vcikery, L.E. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 2206-2210).     

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.     

Nitrosyl cytochrome c oxidase. Formation and properties of mixed valence enzyme

We report the first resonance Raman scattering studies of NO-bound cytochrome c oxidase. Resonance Raman scattering and optical absorption spectra have been obtained on the fully reduced enzyme (a2+, a2+(3) NO) and the mixed valence enzyme (a3+, a2+(3) NO). Clear vibrational frequency shifts are detected in the lines associated with cytochrome a in comparing the two redox states. With 441.6 nm excitation the fully reduced preparation yields a spectrum similar to that of carbon monoxide-bound cytochrome c oxidase and is dominated by the spectrum of reduced cytochrome a. In contrast, in the mixed valence preparation no contributions from reduced cytochrome a are evident in the spectrum, verifying that this heme is no longer in the Fe2+ state. In the mixed valence NO-bound samples, a line appears at approximately 545 cm-1, a frequency similar to that found in NO-bound hemoglobin and myoglobin and assigned as an Fe-N-O-bending mode in those proteins. We do not detect this line in the spectrum of the fully reduced NO-bound enzyme. The carbonyl line of the cytochrome a3 heme formyl group in the fully reduced NO-bound enzyme appears at approximately equal to 1666 cm-1 in the resonance Raman spectrum. In the mixed valence NO-bound preparation the frequency of the carbonyl line increases by 1.2 cm-1 to approximately equal to 1667 cm-1. Thus, modes in cytochrome a2+(3) NO are sensitive to the redox state of the cytochrome a and/or CuA centers. We propose that the redox sensitivity of the formyl mode and the Fe-N-O mode results from an interaction between cytochrome a2+(3) (NO) and the cytochrome a-CuA pair, and is linked to the cytochrome a3 (NO) by the coupling between CuB and the NO-bound cytochrome a3 heme.     

Biochemical and biophysical studies on cytochrome c oxidase XVI. Circular dichroic study of cytochrome c oxidase and its ligand complexes

1. CD spectra of cytochrome c oxidase have been determined both in the absence and presence of the extrinsic ligands CO, NO, cyanide and azide.2. CO and NO affect the CD spectrum of cytochrome c oxidase in a similar way.3. Cyanide and azide also affect the CD spectrum of cytochrome c oxidase in a similar way, but distinctly different from CO and NO.4. From the CD spectra of the oxidized and reduced enzyme, in the presence and absence of extrinsic ligands, CD difference spectra (reduced minus oxidized) are calculated for the so-called cytochrome a and cytochrome a₃ moieties of the enzyme.5. These spectra are largely dependent on the extrinsic ligand used. It is therefore concluded that these spectra do not represent independent cytochrome a and cytochrome a₃ difference spectra, but that heme-heme interactions occur within the cytochrome c oxidase molecule, in such a way that binding of a ligand to one of the heme a groups of cytochrome c oxidase affects the spectral properties of the other heme a group.6. As a consequence, ligand-binding studies cannot give information as to the pre-existence of separate cytochrome a and cytochrome a₃ moieties in the absence of extrinsic ligands.     

Infrared spectra of carbon monoxide bound to mitochondria from diverse species and tissues reveal structurally similar cytochrome c oxidase dioxygen reaction sites

Infrared bands for CO bound to mitochondria from bovine and porcine hearts, bovine brain, rat kidney, and blowfly flight muscle and to intact blowfly flight muscle have been measured in the carbon-oxygen stretch region. Each spectrum contains a narrow band near 1963 cm-1 similar to the major band found earlier for the carbonyl cytochrome c oxidase purified from bovine heart. A second band near 1959 cm-1 ascribed to a less stable conformer of the purified oxidase carbonyl is also detected in mitochondria. These spectra support very similar CO (and O2) binding sites among all the oxidases examined whether the enzyme is purified or is still within mitochondria or intact tissue and therefore suggest that the reduced heme A ligand binding site has been highly conserved during evolution.     

细胞色素C与细胞色素氧化酶之间相互作用的共振RAMAN光谱研究

分别于５１４．５ｎｍ及６０４ｕｍ波长激发下,对游离的细胞色素Ｃ,细胞色素氧化酶以及细胞色素Ｃ和细胞色素氧化酶的复合体的共振拉曼光谱进行了分析比较,在形成复合体时,双方蛋白的共振拉曼谱均有所变化,一个共同的特征性变化是Ａ２ｇｖ２２１１３０ｃｍ－１,ｖ２１１３１２ｃｍ－１,ｖ２０１４００ｃｍ－２,和ｖ１９１５８４ｃｍ－１强度都有增强,其中变化最明显的是Ａ２ｇｖ１９１５８４ｃｍ－１峰,在游离态中,Ｉ１５４０／ｉ１５８２＞１,在结合态中Ｉ１５５０／Ｉ１５８２＜１。     

Resonance Raman spectroscopy of cytochrome oxidase using Soret excitation: selective enhancement, indicator bands, and structural significance for cytochromes a and a3

Resonance Raman studies of oxidized and reduced cytochrome oxidase and liganded derivatives of the oxidized enzyme have been performed by using direct-Soret excitation at 413.1 and 406.7 nm, as well as near-Soret excitation (457.9 nm) and alpha-band excitation (604.6 nm). The Soret results clearly show selective enhancement of Raman modes of the hemes of cytochromes a and a3, depending upon the excitation wavelength chosen. For the preparations employed in this study, photoreduction of cytochrome oxidase in the laser beam was not a significant problem. Resonance Raman frequencies sensitive to oxidation state and spin state or core expansion of the a and a3 hemes are identified and correlated with those previously identified for other heme proteins. An unusual low-frequency (less than 500 cm(-1)) spectrum is observed for oxidized high-spin cytochrome a3, which may be due to axial nonheme structures in this cytochrome.     

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.