Demonstration by ultraviolet resonance Raman spectroscopy of differences in DNA organization and interactions in filamentous viruses Pf1 and fd

Pf1, a class II filamentous virus, has been investigated by ultraviolet resonance Raman (UVRR) spectroscopy with excitation wavelengths of 257, 244, 238, and 229 nm. The 257-nm UVRR spectrum is rich in Raman bands of the packaged single-stranded DNA (ssDNA) genome, despite the low DNA mass (6%) of the virion. Conversely, the 229-nm UVRR spectrum is dominated by tyrosines (Tyr 25 and Tyr 40) of the 46-residue alpha-helical coat subunit. UVRR spectra excited at 244 and 238 nm exhibit Raman bands diagnostic of both viral DNA and coat protein tyrosines. Raman markers of packaged Pf1 DNA contrast sharply with those of the DNA packaged in the class I filamentous virus fd [Wen, Z. Q., Overman, S. A., and Thomas, G. J., Jr. (1997) Biochemistry 36, 7810-7820]. Interestingly, deoxynucleotides of Pf1 DNA exhibit sugars in the C2'-endo/anti conformation and bases that are largely unstacked, compared with C3'-endo/anti conformers and very strong base stacking in fd DNA; hydrogen-bonding interactions of thymine carbonyls are also different in Pf1 and fd. On the other hand, coat protein tyrosines of Pf1 exhibit Raman markers of ring environment identical to those of fd, including an anomalous singlet at 853 cm-1 in lieu of the canonical Fermi doublet (850/830 cm-1) found in globular proteins. The results indicate markedly different modes of organization of ssDNA in Pf1 and fd virions, despite similar environments for coat protein tyrosines, and suggest strong hydrogen-bonding interactions between DNA bases and coat subunits of Pf1 but not between those of fd. We propose that structural relationships between the protein coat and encapsidated ssDNA genome are also fundamentally different in the two assemblies.     

Tyrosine hydrogen-bonding and environmental effects in proteins probed by ultraviolet resonance Raman spectroscopy

Ultraviolet resonance Raman spectra with 229-nm excitation are reported for aqueous tyrosine and for ovomucoid third domain proteins from chicken [OMCHI3(-)] and from chachalaca [OMCHA(-)], as well as alpha 1-, alpha 2-, and beta-purothionin. At this excitation wavelength interference from phenylalanine is minimized, and it is possible to determine the frequencies of the Tyr ring modes nu 8a and nu 8b. The nu 8b frequency decreases with the degree of Tyr H-bond donation, reaching a limiting value for deprotonated tyrosine. This spectroscopic indicator of H-bond strength was calibrated by using the model compound p-cresol in H-bond acceptor solutions for which the enthalpy of H-bond formation can be obtained from the literature. With this calibration it is possible to estimate Tyr H-bond enthalpies in proteins for which Tyr is a H-bond donor; values of 13.7, 9.6, and 11.2 kcal/mol were found for OMCHA3(-) and for alpha 1- (or alpha 2-) and beta-purothionin, respectively. The intensity of the 1176-cm-1 nu 9a band of Tyr excited at 229 nm and also the intensity ratio of the Tyr 830/850-cm-1 Fermi doublet excited at 200 nm both correlate strongly with the estimated H-bond enthalpies, but large deviations are seen for the purothionins, reflecting a special environment for the Tyr residue of these proteins, which is believed to be constrained in a hydrophobic pocket. The molar intensity of the strong approximately 1000-cm-1 nu 12 band of phenylalanine in aqueous solution is about half the value observed in most proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tryptophan-lipid interactions in membrane protein folding probed by ultraviolet resonance Raman and fluorescence spectroscopy

Aromatic amino acids of membrane proteins are enriched at the lipid-water interface. The role of tryptophan on the folding and stability of an integral membrane protein is investigated with ultraviolet resonance Raman and fluorescence spectroscopy. We investigate a model system, the β-barrel outer membrane protein A (OmpA), and focus on interfacial tryptophan residues oriented toward the lipid bilayer (trp-7, trp-170, or trp-15) or the interior of the β-barrel pore (trp-102). OmpA mutants with a single tryptophan residue at a nonnative position 170 (Trp-170) or a native position 7 (Trp-7) exhibit the greatest stability, with Gibbs free energies of unfolding in the absence of denaturant of 9.4 and 6.7 kcal/mol, respectively. These mutants are more stable than the tryptophan-free OmpA mutant, which exhibits a free energy of unfolding of 2.6 kcal/mol. Ultraviolet resonance Raman spectra of Trp-170 and Trp-7 reveal evolution of a hydrogen bond in a nonpolar environment during the folding reaction, evidenced by systematic shifts in hydrophobicity and hydrogen bond markers. These observations suggest that the hydrogen bond acceptor is the lipid acyl carbonyl group, and this interaction contributes significantly to membrane protein stabilization. Other spectral changes are observed for a tryptophan residue at position 15, and these modifications are attributed to development of a tryptophan-lipid cation-π interaction that is more stabilizing than an intraprotein hydrogen bond by ∼2 kcal/mol. As expected, there is no evidence for lipid-protein interactions for the tryptophan residue oriented toward the interior of the β-barrel pore. These results highlight the significance of lipid-protein interactions, and indicate that the bilayer provides more than a hydrophobic environment for membrane protein folding. Instead, a paradigm of lipid-assisted membrane protein folding and stabilization must be adopted.     

Protein conformation change of myoglobin upon ligand binding probed by ultraviolet resonance Raman spectroscopy

Conformational change of myoglobin (Mb) accompanied by binding of a ligand was investigated with 244 nm excited ultraviolet resonance Raman Spectroscopy (UVRR). The UVRR spectra of native sperm whale (sw) and horse (h) Mbs and W7F and W14F swMb mutants for the deoxy and CO-bound states enabled us to reveal the UVRR spectra of Trp7, Trp14, and Tyr151 residues, separately. The difference spectra between the deoxy and CO-bound states reflected the environmental or structural changes of Trp and Tyr residues upon CO binding. The W3 band of Trp7 near the N-terminus exhibited a change upon CO binding, while Trp14 did not. Tyr151 in the C-terminus also exhibited a definite change upon CO binding, but Tyr103 and Tyr146 did not. The spectral change of Tyr residues was characterized through solvent effects of a model compound. The corresponding spectral differences between CO- and n-butyl isocyanide-bound forms were much smaller than those between the deoxy and CO-bound forms, suggesting that the conformation change in the C- and N-terminal regions is induced by the proximal side of the heme through the movement of iron. Although the swinging up of His64 upon binding of a bulky ligand is noted by X-ray crystallographic analysis, UVRR spectra of His for the n-butyl isocyanide-bound form did not detect the exposure of His64 to solvent.     

Protein conformation changes of HemAT-Bs upon ligand binding probed by ultraviolet resonance Raman spectroscopy

HemAT from Bacillus subtilis (HemAT-Bs) is a heme-based O2 sensor protein that acts as a signal transducer responsible for aerotaxis. HemAT-Bs discriminates its physiological effector (O2) from other gas molecules (CO and NO), although all of them bind to a heme. To monitor the conformational changes in the protein moiety upon binding of different ligands, we have investigated ultraviolet resonance Raman (UVRR) spectra of the ligand-free and O2-, CO-, and NO-bound forms of full-length HemAT-Bs and several mutants (Y70F, H86A, T95A, and Y133F) and found that Tyr70 in the heme distal side and Tyr133 and Trp132 from the G-helix in the heme proximal side undergo environmental changes upon ligand binding. In addition, the UVRR results confirmed our previous model, which suggested that Thr95 forms a hydrogen bond with heme-bound O2, but Tyr70 does not. It is deduced from this study that hydrogen bonds between Thr95 and heme-bound O2 and between His86 and heme 6-propionate communicate the heme structural changes to the protein moiety upon O2 binding but not upon CO and NO binding. Accordingly, the present UVRR results suggest that O2 binding to heme causes displacement of the G-helix, which would be important for transduction of the conformational changes from the sensor domain to the signaling domain.     

Polyanion binding to cytochrome c probed by resonance Raman spectroscopy

The interaction of ferricytochrome c with negatively charged heteropolytungstates was studied by resonance Raman spectroscopy. In analogy to previous findings on ferricytochrome c bound to other types of charged interface (Hildebrandt, P. and Stockburger, M. (1989) Biochemistry 28, 6710-6721, 6722-6728), it was shown that in these complexes the conformational states I and II are stabilized. While in state I, the structure is the same as is in the uncomplexed heme protein, in state II three different coordination configurations coexist, i.e., a six-coordinated low-spin, a five-coordinated high-spin and a six-coordinated high-spin form. These configurations constitute thermal coordination equilibria whose thermodynamic properties were determined. The detailed analysis of the low-frequency resonance Raman spectra reveals that in state II the heme pocket assumes an open structure leading to a significantly higher flexibility of the heme group compared to the native ferricytochrome c. It is concluded that these structural changes are the result of Coulombic attractions between the polyanions and the lysine residues around the exposed heme edge which destabilize the heme crevice. Modifications of these interactions upon variation of the ionic strength, the pH or the type of the polytungstate are sensitively reflected by changes of the coordination equilibria in state II as well as of the conformational equilibrium of state I and state II. The conformational changes in state II significantly differ from those associated with the alkaline transition of ferricytochrome c. However, there are some structural similarities between the acid form of the heme protein stable below pH 2.5 in aqueous solution and the six-coordinated high-spin configuration of the bound ferricytochrome c at neutral pH (state II). This suggests that electrostatic interactions with the heteropolytungstates perturb the ionic equilibria of those amino acid side chains which are involved in the acid-induced transition leading to a significant upshift of the apparent pKa.     

Tyrosine and tryptophan modification monitored by ultraviolet resonance Raman spectroscopy

Nitration of tyrosine with tetranitromethane shifts the tyrosine absorption spectrum and abolishes its 200 nm-excited resonance Raman spectrum. There is no detectable resonance Raman contribution from either reactants or products. Likewise, modification of tryptophan with 2-hydroxy-5-nitrobenzyl bromide (HNBB) shifts its absorption spectrum and abolishes its 218 nm-excited resonance Raman spectrum. In this case resonance Raman bands due to HNBB are seen, but are readily distinguishable from the tryptophan spectrum, can be computer-subtracted. When stellacyanin was treated with tetranitromethane the UV resonance Raman spectrum was greatly attenuated; quantitation of the 850 cm-1 tyrosine band intensity gave a value of 4.3 tyrosines modified out of the seven present in stellacyanin, in good agreement with an estimate of 4.7 from the absorption spectrum. For cytochrome c, the resonance Raman spectrum indicates that two out of the four tyrosines are modified by tetranitromethane treatment, consistent with the crystal structure, which shows two buried tyrosines and two at the protein surface. Treatment of stellacyanin with HNBB gave a reduction in the tryptophan spectrum, excited at 218 nm, consistent with one of the three tryptophans being modified. These modification procedures should be useful in distinguishing spectra of buried tyrosine and tryptophan residues from those at the surface.     

Study of ATP binding in the active site of Na,K-ATPase as probed by ultraviolet resonance Raman spectroscopy

The ultraviolet resonance Raman (UV RR) spectra of functional ATP/membrane-bound Na⁺K⁺-ATPase complexes have been obtained. The substrate binding in the enzyme active site has been shown to be accompanied with significant changes in the electronic vibrational structure of the adenine ring. From the spectral analysis of ATP, 8-Br-ATP and 6-NHMe-adenine at various pH values the conclusion was made that N1 and the NH₂, group and, probably, N7 of the substrate adenine part, interact with the protein surroundings via hydrogen bonds.     

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.     

Interactions of soluble guanylate cyclase with diatomics as probed by resonance Raman spectroscopy

Soluble guanylate cyclase (sGC, EC 4.6.1.2) acts as a sensor for nitric oxide (NO), but is also activated by carbon monoxide in the presence of an allosteric modulator. Resonance Raman studies on the structure-function relations of sGC are reviewed with a focus on the CO-adduct in the presence and absence of allosteric modulator, YC-1, and substrate analogues. It is demonstrated that the sGC isolated from bovine lung contains one species with a five-coordinate (5c) ferrous high-spin heme with the Fe-His stretching mode at 204 cm(-1), but its CO adduct yields two species with different conformations about the heme pocket with the Fe-CO stretching (nuFe-CO) mode at 473 and 489 cm(-1), both of which are His- and CO-coordinated 6c ferrous adducts. Addition of YC-1 to it changes their population and further addition of GTP yields one kind of 6c (nuFe-CO=489 cm(-1)) in addition to 5c CO-adduct (nuFe-CO=521 cm(-1)). Under this condition the enzymatic activity becomes nearly the same level as that of NO adduct. Addition of gamma-S-GTP yields the same effect as GTP does but cGMP and GDP gives much less effects. Unexpectedly, ATP cancels the effects of GTP. The structural meaning of these spectroscopic observations is discussed in detail.     

Characteristics in tyrosine coordinations of four hemoglobins M probed by resonance Raman spectroscopy

Resonance Raman spectra of four hemoglobins (Hbs) M with tyrosinate ligand, that is, Hb M Saskatoon (beta distal His----Tyr), Hb M Hyde Park (beta proximal His----Tyr), Hb M Boston (alpha distal His----Tyr), and Hb M Iwate (alpha proximal His----Tyr), were investigated in order to elucidate structural origins for distinctly facile reducibility of the abnormal subunit of Hb M Saskatoon in comparison with other Hbs M. All of the Hbs M exhibited the fingerprint bands for the Fe-tyrosinate proteins around 1600, 1500, and 1270 cm-1. However, Hb M Saskatoon had the lowest Fe-tyrosinate stretching frequency and was the only one to display the Raman spectral pattern of a six-coordinate heme for the abnormal beta subunit; the others displayed the patterns of a five-coordinate heme. The absorption intensity of Hb M Saskatoon at 600 nm indicated a transition with a midpoint pH at 5.2, whereas that of Hb M Boston was independent of pH from 7.2 to 4.8. The fingerprint bands for the tyrosinate coordination as well as the Fe-tyrosinate stretching band disappeared for Hb M Saskatoon at pH 5.0, and the resultant Raman spectrum resembled that of metHb A, while those bands were clearly observed for Hb M Boston at pH 5.0 and for two Hbs M at pH 10.0. These observations suggest that the unusual characteristics of the heme in the abnormal beta chain of Hb M Saskatoon result from the weak Fe-tyrosinate bond, which allows weak coordination of the proximal histidine, giving rise to the six-coordinate high-spin state at pH 7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lignin radicals in the plant cell wall probed by Kerr-gated resonance Raman spectroscopy

Lignin radicals are crucial intermediates for lignin biosynthesis in the cell wall of vascular plants. In this work they were for the first time, to our knowledge, selectively observed in wood cell walls by laser-based Kerr-gated resonance Raman spectroscopy, and the observations were supported by density functional theory prediction of their vibrational properties. For dry wood cells a lignin radical Raman band is observed at 1,570 cm(-1) irrespective of species. For wet beech cells they were generated in situ and observed at 1,606 cm(-1). DFT/B3LYP/6-31+G(d) modeling results support that in beech they are formed from syringyl (S) phenolic moieties and in spruce from guaiacyl (G) phenolic moieties. The observed lignin radical band is predicted as G is approximately 1,597 cm(-1) and S is approximately 1,599 cm(-1), respectively, and is assigned the (Wilson notation) nu(8a) phenyl ring mode. The RR band probes lignin radical properties, e.g., spin density distribution, and these respond to charge polarization or hydrogen bonding to proximate water molecules. These observations can be crucial for an understanding of the factors that control cell wall structure during biosynthesis of vascular plants and demonstrate the unique potential of RR spectroscopy of lignin radicals.     

Structure of the filamentous bacteriophage,Pf3, by X-ray fiber diffraction

Fiber diffraction data have been obtained for the filamentous bacteriophage Pf3. The virus crystallizes on a hexagonal net with lattice constants a = b = 5.61 nm and c = 7.50 nm at 0% relative humidity and a = 6 = 6.20 nm and c = 7.99 nm at 98% relative humidity. The X-ray diffraction pattern resembles those of two other bacteriophages, Pf 1 and Xf, and therefore belongs to class II. The data are consistent with a 27₅ helix symmetry with an axial rise of 0.277 (dry) to 0.296 (wet) nm.     

Early events in apomyoglobin unfolding probed by laser T-jump/UV resonance Raman spectroscopy

Early events in the unfolding of apomyoglobin are studied with time-resolved ultraviolet resonance Raman (UVRR) spectroscopy coupled to a laser-induced temperature jump (T-jump). The UVRR spectra provide simultaneous probes of the aromatic side-chain environment and the amide backbone conformation. The amide bands reveal helix melting, with relaxation times of 70 and 16 micros at pH 5.5 and 4, respectively, in reasonable agreement with previously reported amide I' FTIR/T-jump relaxations (132 and 14 micros at pD 5.5 and 3). The acceleration at pH 4 is consistent with destabilization of the hydrophobic AGH core of the protein via protonation of a pair of buried histidines. The same relaxation times are found for intensity loss by the phenylalanine F12 band, signaling solvent exposure of the phenyl rings. There are seven Phe residues, distributed throughout the protein; they produce a global response, parallel to helix melting. Relaxation of the tryptophan W16 intensity also parallels helix melting at pH 5.5 but is twice as fast, 7 micros, at pH 4. The pH 5.5 signal arises from Trp 7, which is partially solvent-exposed, while the pH 4 signal arises from the buried Trp 14. Thus, Trp 14 is exposed to the solvent prior to helix melting of the AGH core, suggesting initial displacement of the A helix, upon which Trp 14 resides. All of the UVRR signals show a prompt response, within the instrument resolution (approximately 60 ns), which accounts for half of the total relaxation amplitude. This response is attributed to solvent penetration into the protein, possibly convoluted with melting of hydrated helix segments.     

Investigation of higher order structures of proteins by ultraviolet resonance Raman spectroscopy.

Progress in laser technology and light detection devices have enabled us to explore protein structures and their dynamics by using time-resolved resonance Raman spectroscopy. It is in the last decade that Raman spectra of proteins excited at 200-240 nm have brought about rich structural information. The technological developments in deep UV resonance Raman spectroscopy are reviewed first, and the unique information on proteins obtainable from such spectra are summarized. As an application of this technique to investigations of the higher order structures of proteins, studies on the quaternary structure transition of haemoglobin are described.     

Nucleic acid derivatives studied by preresonance and resonance Raman spectroscopy in the ultraviolet region

Raman spectra of AMP, UTP, GMP, and CMP, and of their bromo-derivatives (8 Br-ATP, 8-bromo-adenosine, 8-bromo-guanosine, 5-bromo-deoxyuridine, 5-bromo-cytidine), are reported. They are obtained using excitation wavelengths of 457.9 nm (ionized continuous argon laser) and of 300 nm (tunable pulsed dye laser). Comparison of spectra leads to the following observations: (1) preresonances Raman effects on nucleotides spectra at 300 nm; (2) resonance Raman effects on bromoderivatives spectra at 300 nm; (3) in dilute solution (10^?4M), shifts and enhancements of Raman lines of the bromo-derivatives with respect to the corresponding lines of nucleoctides. On the basis of these comparisons, the assignments of the Raman lines are discussed, This provide the necessary background for the understanding of the properties of selected groups in DNA in dilute solution. The new experimental set-up for measurements of Raman spectra using excitation in the uv regions is described. It is specially designed to incorporate the pulsed feature of the excitation laser and for correcting of the instabilities of the sourse.     

Substrate polarization by residues in delta 5-3-ketosteroid isomerase probed by site-directed mutagenesis and UV resonance Raman spectroscopy.

delta 5-3-Ketosteroid isomerase (KSI: EC 5.3.3.1) of Pseudomonas testosteroni catalyzes the isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by the stereospecific transfer of the steroid 4 beta-proton to the 6 beta-position, using Tyr-14 as a general acid and Asp-38 as a base. Ultraviolet resonance Raman (UVRR) spectra have been obtained for the catalytically active double mutant Y55F + Y88F, which retains Tyr-14 as the only tyrosine residue (referred to as the Y14(0) mutant), and the Y14F mutant, which has 50,000-fold lower activity. The UVRR results establish that binding of the product analog and competitive inhibitors 19-nortestosterone or 4-fluoro-19-nortestosterone to the Y14(0) mutant does not result in the formation of deprotonated Tyr-14. The UVRR spectra of the steroid inhibitors show large decreases in the vinyl and carbonyl stretching frequencies on binding to the Y14(0) enzyme but not on binding to the Y14F enzyme. These changes cannot be mimicked by protonation of the steroids. For 19-nortestosterone, the vinyl and carbonyl stretching frequencies shift down (with respect to the values in aqueous solution) by 18 and 27 cm-1, respectively, on binding to Y14(0) KSI. It is proposed that the changes in the steroid resonance Raman spectrum arise from polarization of the enone moiety via the close proximity of the charged Asp-38 side chain to the vinyl group and the directional hydrogen bond between Tyr-14 and the 3-carbonyl oxygen of the steroid enone. The 230-nm-excited UVRR spectra do not, however, show changes that are characteristic of strong hydrogen bonding from the tyrosine hydrogen. It is proposed that this hydrogen bonding is compensated by a second hydrogen bond to the Tyr-14 oxygen from another protein residue. UVRR spectra of the Y14(0) enzyme obtained using 200 nm excitation show enhancement of the amide II and S Raman bands. The secondary structure of KSI was estimated from the amide II and S intensities and was found to be low in alpha-helical structure. The alpha-helix content was estimated to be in the range of 0-25% (i.e., 10 +/- 15%).     

Distinct heme-substrate interactions of lactoperoxidase probed by resonance Raman spectroscopy: difference between animal and plant peroxidases

Resonance Raman scattering from cow milk lactoperoxidase (LPO) and its complexes with various electron donors and inhibitors was investigated. The Raman spectrum of LPO is strikingly close to that of hog intestinal peroxidase but distinctly dissimilar to that of horseradish peroxidase (HRP). The v10 frequency suggested the six-coordinate high-spin structure of heme for native LPO in contrast with the five-coordinate high-spin structure for HRP. For the v10 band, benzohydroxamic acid caused a frequency shift with HRP but not with LPO. Guaiacol, o-toluidine, and histidine brought about a frequency shift of the v4 mode for LPO but not for HRP. The frequency shift was restored upon removal of the substrate or inhibitor by dialysis. The down shift of the v4 frequency is considered to represent an appreciable donation of electrons from the substrate or inhibitor to the porphyrin LUMO and thus their direct interaction with the heme group. From the relative intensity of the shifted and unshifted v4 lines, the dissociation constant was determined to be Kd = 52 mM for guaiacol and Kd = 87 mM for histidine at pH 7.4. The binding of histidine was relatively retarded in the presence of sulfate anion (Kd = 150 mM for 0.53 M sulfate present), and imidazole alone yielded no frequency shift, indicating the binding of the carboxyl group of histidine to the protein cationic site on one hand and a weak charge-transfer interaction between the imidazole group and the heme group on the other.     

Differences in flexibility of active sites of cytochromes P450 probed by resonance Raman and UV-Vis absorption spectroscopy.

Spectroscopic methods reveal differences in flexibility and stability of P450 forms. Among microsomal P450s, the most flexible active site has been found in the CYP3A4 enzyme as it is compressible and the heme vinyl side chains may adopt two different conformations. On the other hand, active site of this enzyme denatures quite easily upon hydrostatic pressure. The most rigid active site able to withstand the effect of high pressure has CYP1A2. The bacterial CYP102 (BM3) flavocytochrome has also a rather stable, but flexible active site. The differences between CYP3A4 and CYP1A2 active sites apparently reflect their ability to bind various substrates: whereas the CYP3A4 binds a vast variety of structures, the CYP1A2 preferentially binds planar, aromatic structures and its substrate specificity is relatively narrow.     

Differences in coordination states of substituted tyrosine residues and quaternary structures among hemoglobin M probed by resonance Raman spectroscopy

Among the four types of hemoglobin (Hb) M with a substitution of a tyrosine (Tyr) for either the proximal (F8) or distal (E7) histidine in the α or β subunits, only Hb M Saskatoon (βE7Tyr) assumes a hexacoordinate structure and its abnormal subunits can be reduced readily by methemoglobin (metHb) reductase. This is distinct from the other three M Hbs. To gain new insight into the cause of the difference, we examined the ionization states of E7 and F8 Tyrs by UV resonance Raman (RR) spectroscopy and Fe–O(Tyr) bonding by visible RR spectroscopy. Hb M Iwate (αF8Tyr), Hb M Boston (αE7Tyr), and Hb M Hyde Park (βF8Tyr) exhibited two extra UV RR bands at 1,603 cm⁻¹ (Y8a′) and 1,167 cm⁻¹ (Y9a′) arising from deprotonated (ionized) Tyr, but Hb M Saskatoon displayed the UV RR bands of protonated (unionized) Tyr at 1,620 and 1,175 cm⁻¹ in addition to those of deprotonated Tyr. Evidence for the bonding of both ionization states of Tyr to the heme in Hb M Saskatoon was provided by visible RR spectroscopy. These results indicate that βE7Tyr of Hb M Saskatoon is in equilibrium between protonated and deprotonated forms, which is responsible for facile reducibility. Comparison of the UV RR spectral features of metHb M with that of metHb A has revealed that metHb M Saskatoon and metHb M Hyde Park are in the R (relaxed) structure, similar to that of metHb A, whereas metHb M Iwate, metHb M Boston and metHb M Milwaukee are in the T (tense) quaternary structure.