A comparison of the resonance Raman properties of the fast and slow forms of cytochrome oxidase

Resonance Raman (RR) spectra of the "rapid" and "slow" forms (Baker et al., 1987) of resting cytochrome oxidase obtained with Soret excitation at 413.1 nm are reported. There are a number of conspicuous differences between the two forms in the high-frequency region of the RR spectrum which involve changes in Raman intensity arising from a blue shift in the Soret maximum of cytochrome a3 upon conversion to the slow form. In the low-frequency region a peak present at 223 cm-1 in the rapid form shifts to 220 cm-1 in the slow form; this peak is assigned as the cytochrome a3 Fe(III)-N(His-Im) stretch. The slow form of the enzyme possesses greater intensity in RR peaks near 1620 cm-1 which have been previously attributed by others to partial photoreduction of the enzyme. We have quantitated the amount of laser-induced photoreduction in these RR spectra by comparison with the spectra of mixed-valence derivatives of the enzyme and find that these 1620-cm-1 features are unreliable indicators of photoreduction. The spectra of the fast- and slow-reacting species in H2O and D2O have been compared. The fast-reacting form exhibits a 4-cm-1 shift, from 223 to 219 cm-1, upon transferring to D2O in a peak which we assign as the cytochrome a3 Fe(III)-N(His-Im) stretch. There is a parallel shift in the feature at 1651 cm-1 due to the C = O stretch of the formyl group of cytochrome a. These deuterium shifts are not observed in the slow form.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heme pocket interactions in cytochrome c peroxidase studied by site-directed mutagenesis and resonance Raman spectroscopy

Resonance Raman spectra are reported for FeII and FeIII forms of cytochrome c peroxidase (CCP) mutants prepared by site-directed mutagenesis and cloning in Escherichia coli. These include the bacterial "wild type", CCP(MI), and mutations involving groups on the proximal (Asp-235----Asn, Trp-191----Phe) and distal (Trp-51----Phe, Arg-48----Leu and Lys) side of the heme. These spectra are used to assess the spin and ligation states of the heme, via the porphyrin marker band frequencies, especially v3, near 1500 cm-1, and, for the FeII forms, the status of the Fe-proximal histidine bond via its stretching frequency. The FeII-His frequency is elevated to approximately 240 cm-1 in CCP(MI) and in all of the distal mutants, due to hydrogen-bonding interactions between the proximal His-175 N delta and the carboxylate acceptor group on Asp-235. The FeII-His RR band has two components, at 233 and 246 cm-1, which are suggested to arise from populations having H-bonded and deprotonated imidazole; these can be viewed in terms of a double-well potential involving proton transfer coupled to protein conformation. The populations shift with changing pH, possibly reflecting structure changes associated with protonation of key histidine residues, and are influenced by the Leu-48 and Phe-191 mutations. A low-spin FeII form is seen at high pH for the Lys-48, Leu-48, Phe-191, and Phe-51 mutants; for the last three species, coordination of the distal His-52 is suggested by a approximately 200-cm-1 RR band assignable to Fe(imidazole)2 stretching.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochrome aa3 from Nitrosomonas europaea

Cytochrome c oxidase has been purified from the ammonia oxidizing chemoautotroph Nitrosomonas europaea by ion-exchange chromatography in the presence of Triton X-100. The enzyme has absorption maxima at 420 and 592 nm in the resting state and at 444 and 598 nm in the dithionite-reduced form; optical extinction coefficient (598 nm minus 640 nm) = 21.9 cm-1 nM-1. The enzyme has approximately 11 nmol of heme a and approximately 11 nmol of copper per mg of protein (Lowry procedure). There appear to be three subunits (approximate molecular weights 50,800, 38,400, and 35,500), two heme groups (a and a3), and two copper atoms per minimal unit. The EPR spectra of the resting and partially reduced enzyme are remarkably similar to the corresponding spectra of the mitochondrial cytochrome aa3-type oxidase. Although the enzyme had been previously classified as "cytochrome a1" on the basis of its ferrous alpha absorption maximum (598 nm), its metal content and EPR spectral properties clearly show that it is better classified as a cytochrome aa3. Neither the data reported here nor a review of the literature supports the existence of cytochrome a1 as an entity discrete from cytochrome aa3. The purified enzyme is reduced rapidly by ferrous horse heart cytochrome c or cytochrome c-554 from N. europaea, but not with cytochrome c-552 from N. europaea. The identity of the natural electron donor is as yet unestablished. With horse heart cytochrome c as electron donor, the purified enzyme could account for a significant portion of the terminal oxidase activity in vivo.     

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 studies of lactoperoxidase

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

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).     

Resonance Raman evidence for an exchangeable protein hydrogen associated with the heme a group of cytochrome oxidase

When cytochrome-c oxidase is soaked in D2O, downshifts of the cytochrome a formyl C = O stretching mode are seen in the resonance Raman (RR) spectra (413.1 nm excitation) of both the resting and reduced forms. Other changes observed in the reduced protein RR spectra are consistent with involvement of the cytochrome a formyl group in the deuterium effect. The D2O-induced RR changes are fully developed during 3-5 days incubation, but are incomplete after 1 h. Extraction of the heme a chromophore in deuterated solvents eliminates these changes, implying that the exchangeable proton is on a protein group in the cytochrome a pocket which H-bonds to the heme formyl. The rate of the D2O exchange process is unaffected by enzyme turnover, thus reducing the likelihood that the cytochrome a formyl H-bond is directly involved in the redox-linked mechanism of proton pumping.     

Resonance Raman studies of Escherichia coli sulfite reductase hemoprotein. 2. Fe4S4 cluster vibrational modes

Resonance Raman (RR) spectra from the hemoprotein subunit of Escherichia coli sulfite reductase (SiR-HP) are examined in the low-frequency (200-500 cm-1) region where Fe-S stretching modes are expected. In spectra obtained with excitation in the siroheme Soret or Q bands, this region is dominated by siroheme modes. Modes assignable to the Fe4S4 cluster are selectively enhanced, however, with excitation at 488.0 or 457.9 nm. The assignments are confirmed by observation of the expected frequency shifts in SiR-HP extracted from E. coli grown on 34S-labeled sulfate. The mode frequencies and isotopic shifts resemble those seen in RR spectra of other Fe4S4 proteins and analogues, but the breathing mode of the cluster at 342 cm-1 is higher than that observed in the other species. Spectra of various ligand complexes of SiR-HP reveal only slight sensitivity of the cluster terminal ligand modes to the presence of exogenous heme ligands, at variance with a model of ligand binding in a bridged mode between heme and cluster. Close examination of RR spectra obtained with siroheme Soret-band excitation reveals additional 34S-sensitive features at 352 and 393 cm-1. These may be attributed to a bridging thiolate ligand.     

Raman and infrared spectra of cytochrome c peroxidase-carbon monoxide adducts in alternative conformational states

Resonance Raman (RR) spectra are reported for CO-bound cytochrome c peroxidase (CCP). At low pH, two forms are observed: form II, with nu Fe-C = 530 cm-1 and delta FeCO = 585 cm-1, and form I, with nu Fe-C = 495 cm-1 and no detectable delta FeCO. They appear to have coincident nu CO infrared bands, at 1922 cm-1. These low-pH forms, similar to those observed for horseradish peroxidase (HRP), are attributed to tilted, H-bonded CO and perpendicular CO, respectively. The frequencies differ between the two proteins, a weaker H bond to CO being indicated for CCP. As with HRP, the equilibrium between forms I and II is shifted toward the latter at increasing CO concentrations, suggesting that secondary binding of CO perturbs the distal residues. At high pH [8.4, tris(hydroxymethyl)aminomethane buffer] the form II fraction converts to another form, II', with nu FeC = 503 cm-1, delta FeCO = 575 cm-1, and nu CO = 1948 cm-1; a tilted, non-H-bonded geometry is suggested. If phosphate buffer is used, however, form II (H bonded) persists at pH 8.4. This result establishes a role for phosphate in stabilizing the H-bonded form of the enzyme; it is suggested that phosphate binds near the distal imidazole and substantially increases its pKa. The conformational state is also influenced by aging. Fresh protein contains purely high spin FeIII heme, as monitored by the high-frequency RR spectrum, and yields form II almost exclusively at elevated CO concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reversible acidic-alkaline transition of the carbon monoxide complex of cytochrome c peroxidase

The Soret absorption band of the ferrous carbon monoxide (CO) complex of cytochrome c peroxidase exhibited a blue shift from 423.7 to 420 nm upon an increase in pH from 6.5 to 8.5. The spectral change was reversible with an isosbestic point at 422 nm. The pH dependence of this spectral change gave a sigmoidal curve fitted well to a theoretical curve of a cooperative release of two protons with a pK value of 7.5, indicating the existence of the acidic and alkaline forms of the ferrous CO enzyme. Upon irradiation of light flash (100 J of power and 30-microseconds), the heme-bound CO was readily dissociated in both acidic and alkaline forms with a quantum yield of approximately unity. On the other hand, the rate of recombination of the dissociated CO with the heme iron was significantly different between these two forms; the recombination rate constants were 1.1 X 10(3) and 3.0 X 10(4) M-1 S-1 at 25 degrees C for the acidic and alkaline forms, respectively. At intermediate pH values, kinetics of recombination were biphasic, consisting of the slow and fast processes with the appropriate rate constants mentioned above. When the fraction of the fast process was plotted against pH, the pH profile coincided with the spectrophotometric pH titration curve described above. Thus, it was concluded that the acidic and alkaline forms of the enzyme were responsible for the slow and fast processes, respectively. In infrared spectroscopy, the acidic form showed a narrow CO stretching band at 1922 cm-1 with a half-band width of 12.5 cm-1, while the alkaline form exhibited a broad CO-stretching band at 1948 cm-1 with a half-band width of 33 cm-1. Significance of these results are discussed in relation to the structure of the heme vicinity on the CO complex of cytochrome c peroxidase.     

Unusual CO bonding geometry in abnormal subunits of hemoglobin M Boston and hemoglobin M Saskatoon

To clarify the role of the proximal histidine (F8-His), distal His (E7-His), and E11 valine (E11-Val) in ligand binding of hemoglobin (Hb), we have investigated the resonance Raman (RR) spectra of the carbon monoxide adduct of Hbs M (COHb M) in which one of these residues was genetically replaced by another amino acid in either the alpha or beta subunit. In the fully reduced state, all Hbs M gave v3 at approximately 1472 cm-1 and vFe-His at 214-218 cm-1, indicating that they have a pentacoordinate heme and the heme iron is bound to either E7-His or F8-His. The porphyrin skeletal vibrations of the COHb M were essentially unaltered by replacements of E7- or F8-His with tyrosine (Tyr) and of E11-Val by glutamic acid (Glu). The vCO, vFe-CO, and delta Fe-C-O frequencies of COHb M Iwate (alpha F8-His----Tyr), COHb M Hyde Park (beta F8-His----Tyr), and COHb M Milwaukee (beta E11-Val----Glu) were nearly identical with those of COHb A. In contrast, the RR spectra of COHb M Boston (alpha E7-His----Tyr) and COHb M Saskatoon (beta E7-His----Tyr) gave two new Raman bands derived from the abnormal subunits, vFe-CO at 490 cm-1 and vCO at 1972 cm-1, in addition to those from the normal subunits at 505 cm-1 (vFe-CO) and 1952 cm-1 (vCO). The CO adduct of the abnormal subunits exhibited apparently no photodissociation upon illumination of CW laser with a stationary cell under which the normal subunit exhibited complete photodissociation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Raman spectra of heme a, cytochrome oxidase-ligand complexes, and alkaline denatured oxidase.

We report 441.6 nm excitation resonance Raman spectra of oxidized and reduced monomeric heme a-imidazole, cytochrome oxidase-exogenous ligand complexes in various redox states, and alkaline denatured oxidase. These data show that, in reduced oxidase, the cytochrome a3 Raman spectrum has bands at 215, 364, 1230, and 1670 cm-1 not observed in the cytochrome a spectrum. The appearance of these bands in the reduced cytochrome a3 spectrum is due to interactions between the heme a of cytochrome a3 and its protein environment and not to intrinsic properties of heme a. These interactions are pH sensitive and strongly influence the vibrational spectra of both heme a groups. We assign the 1670-cm-1 band to the heme a formyl substituent and propose that the intensity of the 1670 cm-1 is high for reduced cytochrome a3 because the C==O lies in the porphyrin plane and is very weak for oxidized and reduced cytochrome a, oxidized cytochrome a3, and oxidized and reduced heme a-imidazole because the C==O lies out of the plane. We suggest that movement of the C==O in and out of the plane explains the ligand induced spectral shift in the optical absorption spectrum of reduced cytochrome a3. Finally, we confirm the observation of Adar & Yonetani (private communication) that, under laser illumination, resting oxidase is photoreactive.     

Resonance Raman characterization of the 7-ns photoproduct of (carbonmonoxy)hemoglobin: implications for hemoglobin dynamics

Resonance Raman spectra are reported for deoxyhemoglobin (deoxyHb) and the (carbonmonoxy)hemoglobin (HbCO) photoproduct Hb by use of 7-ns YAG laser pulses at wavelengths of 416 and 532 nm, where enhancement is observed for totally symmetric and nontotally symmetric modes, respectively. The frequencies of the porphyrin skeletal modes v10, v2, v19, v11, and v3 have been determined to be 1602, 1559, 1553, 1542, and 1466 cm-1 in Hb. These frequencies are 2-3 cm-1 lower than the corresponding frequencies for deoxyHb. The v19 and v11 frequencies are at the expected values for a Ct-N distance of 2.057 A, the known core size for a 6-coordinate high-spin FeII-porphyrin complex. The remaining frequencies, however, deviate from the core size correlations for these modes in the same direction as do those of deoxyHb, suggesting that the porphyrin ring is domed in both species. Thus, the heme structure is similar for deoxyHb and Hb but is slightly expanded in the latter. The expanded heme in Hb implies a restraint on the full out-of-plane displacement of the Fe atom, by an estimated approximately 0.1 A relative to deoxyHb. This could result from a residual interaction with the CO molecule if the latter remains held by the protein against the Fe atom, in a high-spin 6-coordinate complex. The available spectroscopic evidence suggests that such a complex may be stabilized at 4 K but is unlikely to persist at room temperature beyond the electronic relaxation (0.35 ps) of the electronically excited heme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resonance Raman investigations of Escherichia coli-expressed Pseudomonas putida cytochrome P450 and P420.

High-resolution resonance Raman spectra of the ferric, ferrous, and carbonmonoxy (CO)-bound forms of wild-type Escherichia coli-expressed Pseudomonas putida cytochrome P450cam and its P420 form are reported. The ferric and ferrous species of P450 and P420 have been studied in both the presence and absence of excess camphor substrate. In ferric, camphor-bound, P450 (mos), the E. coli-expressed P450 is found to be spectroscopically indistinguishable from the native material. Although substrate binding to P450 is known to displace water molecules from the heme pocket, altering the coordination and spin state of the heme iron, the presence of camphor substrate in P420 samples is found to have essentially no effect on the Raman spectra of the heme in either the oxidized or reduced state. A detailed study of the Raman and absorption spectra of P450 and P420 reveals that the P420 heme is in equilibrium between a high-spin, five-coordinate (HS,5C) form and low-spin six-coordinate (LS,6C) form in both the ferric and ferrous oxidation states. In the ferric P420 state, H2O evidently remains as a heme ligand, while alterations of the protein tertiary structure lead to a significant reduction in affinity for Cys(357) thiolate binding to the heme iron. Ferrous P420 also consists of an equilibrium between HS,5C and LS,6C states, with the spectroscopic evidence indicating that H2O and histidine are the most likely axial ligands. The spectral characteristics of the CO complex of P420 are found to be almost identical to those of a low pH of Mb. Moreover, we find that the 10-ns transient Raman spectrum of the photolyzed P420 CO complex possesses a band at 220 cm-1, which is strong evidence in favor of histidine ligation in the CO-bound state. The equilibrium structure of ferrous P420 does not show this band, indicating that Fe-His bond formation is favored when the iron becomes more acidic upon CO binding. Raman spectra of stationary samples of the CO complex of P450 reveal VFe-CO peaks corresponding to both substrate-bound and substrate-free species and demonstrate that substrate dissociation is coupled to CO photolysis. Analysis of the relative band intensities as a function of photolysis indicates that the CO photolysis and rebinding rates are faster than camphor rebinding and that CO binds to the heme faster when camphor is not in the distal pocket.     

Electron structure of the heme of reduced cytochrome P450 and P420 from the data of low-temperature magnetic circular dichroism

Magnetic circular dichroism spectra (MCD) of reduced cytochromes P450 and P420 from rabbit liver microsomes have been recorded and analyzed for the 350-600 nm spectral region in the temperature interval from 2 to 290 K. The shape, intensity and temperature dependence of the MCD of reduced P450 in the Soret region are quite different from that of other high-spin ferrous hemoproteins, whose heme iron is coordinated to the imidazole of histidine (deoxymyoglobin, deoxyhemoglobin, reduced peroxidase and cytochrome c oxidase). Assuming that in the reduced P450 as well as in its CO-complex the protein-derived ligand is mercaptide (RS-) the differences can be explained by the existence of two electronic transitions in the Soret region: the common for hemoproteins pi----pi porphyrin transition and sulfur to porphyrin charge-transfer transition, p+(Sp)----eg (pi). The unusual spectral characteristics of the CO-complex of P450 have been ascribed earlier to strong configurational interaction of these two transitions. From the similarities of the Soret MCD and their temperature dependences for the reduced P420 and for other high-spin ferrous hemoproteins one can conclude that heme iron of the reduced P420 is high-spin and is coordinated to the imidazole of histidine. The zero-field splitting parameter D of the spin Hamiltonian has been estimated from the MCD temperature dependences. The obtained splitting of approximately 30 cm-1 for P450 and of approximately 10 cm-1 for P420 exceeds that for myoglobin and hemoglobin (approximately 5 cm-1).     

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 pyridoxal Schiff bases

Resonance Raman (RR) spectra are reported for amino acid and amine adducts of pyridoxal 5'-phosphate (PLP) and 5'-deoxypyridoxal (5'-dPL) in aqueous solution. For the valine adducts, a detailed study has been carried out on solutions at pH and pD 5, 9, and 13, values at which the pyridine and imine protons are successively ionized, and on the adducts formed from 15N-valine, alpha-deuterovaline, and N-methyl-PLP. Good quality spectra were obtained, despite the strong fluorescence of pyridoxal Schiff bases, by adding KI as a quencher, and by exciting the molecules on the blue side of their absorption bands: 406.7 nm (cw Kr+ laser) for the pH 5 and 9 species (lambda max = 409 and 414 nm), and 354.7 nm (pulsed YAG laser, third harmonic) for the pH 13 species (lambda max = 360 nm). A prominent band at 1646 cm-1 is assigned to the imine C=N stretch via its 13 cm-1 15N shift. A 12 cm-1 down-shift of the band in D2O confirms that the Schiff base linkage is protonated at pH 9. Deprotonation at pH 13 shifts VC = N from 1646 to 1629 cm-1, values typical of conjugated Schiff bases. The strongest band in the spectrum, at 1338 cm-1, shifts to 1347 cm-1 upon pyridine protonation at pH 5, and is assigned to a ring mode with a large component of phenolate C-O stretch. A shoulder on its low-frequency side is assigned to the C4-C4' stretch. Large enhancements of these modes can be understood qualitatively in terms of the dominant resonance structures contributing to the ground and resonant excited states. A number of weaker bands are observed, and assigned to pyridine ring modes. These modes gain significantly in intensity, while the exocyclic modes diminish, when the spectra are excited at 266 nm (YAG laser, fourth harmonic) in resonance with ring-localized electronic transitions.     

Resonance Raman investigation of the interaction of thromboxane synthase with substrate analogues

Thromboxane synthase is a hemethiolate enzyme that catalyzes the isomerization of prostaglandin H2 to thromboxane A2. We report the first resonance Raman (RR) spectra of recombinant human thromboxane synthase (TXAS) in both the presence and the absence of substrate analogues U44069 and U46619. The resting enzyme and its U44069 complex are found to have a 6-coordinate, low spin (6c/ls) heme, in agreement with earlier experiments. The U46619-bound enzyme is detected as a 6c/ls heme too, which is in contradiction with a previous conclusion based on absorption difference spectroscopy. Two new vibrations at 368 and 424 cm(-1) are observed upon binding of the substrate analogues in the heme pocket and are assigned to the second propionate and vinyl bending modes, respectively. We interpret the changes in these vibrational modes as the disruption of the protein environment and the hydrogen-bonding network of one of the propionate groups when the substrate analogues enter the heme pocket. We use carbocyclic thromboxane A2 (CTA2) to convert the TXAS heme cofactor to its 5-coordinate, high spin (5c/hs) form, as is confirmed by optical and RR spectroscopy. In this 5c/hs state of the enzyme, the Fe-S stretching frequency is determined at 350 cm(-1) with excitation at 356.4 nm. This assignment is supported by comparison to the spectrum of resting enzyme excited at 356.4 nm and by exciting at different wavelengths. Implications of our findings for substrate binding and the catalytic mechanism of TXAS will be discussed.     

Spectroscopic studies of an insect hemoglobin from the backswimmer Buenoa margaritacea (Hemiptera:Notonectidae).

Hemoglobin (Hb) isolated from the backswimmer Buenoa margaritacea has been analyzed spectroscopically. The met form at pH less than 6 shows a 30nm red shift in the Qv and Qo bands and a 5nm red shift in the Soret band compared to mammalian Hb, while only minor differences are seen in the spectra of the CO and O2 adducts of Hb from Buenoa and mammals. EPR spectra of the metHb show a superposition of signals; at low pH they are mainly of axial high-spin character, while at high pH a low-spin signal predominates with an O-type g-tensor (2.54, 2.61, 1.85) comparable to that of hydroxy myoglobin. Infrared spectra of Hb12C-16O at pH 8.2 reveal two major absorption bands at 1934 cm-1 and 1967 cm-1, which shift to 1892 cm-1 and 1923 cm-1, respectively, for Hb12C-18O. As isolated the Buenoa Hb consists of several isozymes, all of which have a histidine as the proximal ligand of the heme iron.     

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.