UV resonance raman spectra of ligand binding intermediates of sol-gel encapsulated hemoglobin.

We report for the first time specific conformational changes for a homogeneous population of ligand-bound adult deoxy human hemoglobin A (HbA) generated by introducing CO into a sample of deoxy-HbA with the effector, inositol hexaphosphate, encapsulated in a porous sol-gel. The preparation of ligand-bound deoxy-HbA results from the speed of ligand diffusion relative to globin conformational dynamics within the sol-gel (1). The ultraviolet resonance Raman (UVRR) difference spectra obtained reveal that E helix motion is initiated upon ligand binding, as signaled by the appearance of an alpha14beta15 Trp W3 band difference at 1559 cm(-1). The subsequent appearance of Tyr (Y8a and Y9a) and W3 (1549 cm(-1)) UVRR difference bands suggest conformational shifts for the penultimate Tyralpha140 on the F helix, the "switch" region Tyralpha42, and the "hinge" region Trpbeta37. The UVRR results expose a sequence of conformational steps leading up to the ligation-induced T to R quaternary structure transition as opposed to a single, concerted switch. More generally, this report demonstrates that sol-gel encapsulation of proteins can be used to study a sequence of specific conformational events triggered by substrate binding because the traditional limitation of substrate diffusion times is overcome.     

Near-ultraviolet magnetic circular dichroism spectroscopy of protein conformational states: correlation of tryptophan band position and intensity with hemoglobin allostery

The near-UV magnetic circular dichroism spectroscopy of the aromatic amino acid bands of hemoglobin was investigated as a potential probe of structural changes at the alpha(1)beta(2) interface during the allosteric transition. Allosteric effectors were used to direct carp and chemically modified human hemoglobins into the R (relaxed) or T (tense) state in order to determine the heme-ligation-independent spectral characteristics of the quaternary states. The tryptophan magnetic circular dichroism (MCD) peak observed at 293 nm in the R state of N-ethylsuccinimide- (NES-) des-Arg-modified human hemoglobin (Hb) was shifted to a slightly longer wavelength in the T state, consistent with the shift expected for tryptophan acting as a proton donor in a T-state hydrogen bond. Moreover, the increase observed in the T-state MCD intensity of this band relative to the R-state intensity was consistent with the effect expected for proton donation by tryptophan on the basis of the Michl perimeter model of aromatic MCD. The peak-to-trough magnitude of the R - T MCD difference spectrum is equal to 30% of the total R-state peak intensity contributed by all six tryptophans present in the human tetramer; the relative magnitude specific to the two beta37 tryptophans undergoing conformational change is estimated accordingly to be 3 times larger. The Trp-beta37 spectral shift, about 200 cm(-)(1), is in good agreement with the shifts observed in other H-bonded proton donors and provides corroborating spectral evidence for the formation in solution of a T-state Trp beta37-Asp alpha94 hydrogen bond observed in X-ray diffraction studies of deoxyHb crystals.     

Spectroscopic markers of the T<-->R quaternary transition in human hemoglobin

In this work, we use a sol-gel protocol to trap and compare the R and T quaternary states of both the deoxygenated (deoxyHb) and carbonmonoxide (HbCO) derivatives of human hemoglobin. The near infrared optical absorption band III and the infrared CO stretching band are used to detect the effect of quaternary structure on the spectral properties of deoxyHb and HbCO; comparison with myoglobin allows for an assessment of tertiary and quaternary contributions to the measured band shifts. The R<-->T transition is shown to cause a blue shift of the band III by approximately 35 cm(-1) for deoxyHb and a red shift of the CO stretching band by only approximately 0.3 cm(-1) for HbCO. This clearly shows that quaternary structure changes are transmitted to the heme pocket and that effects on deoxyHb are much larger than on HbCO, at least as far as the band energies are concerned. Experiments performed in the ample temperature interval of 300-10K show that the above quaternary structure effects are "static" and do not influence the dynamic properties of the heme pocket, at least as probed by the temperature dependence of band III and of the CO stretching band. The availability of quaternary structure sensitive spectroscopic markers and the quantitative measurement of the quaternary structure contribution to band shifts will be of considerable help in the analysis of flash-photolysis experiments on hemoglobin. Moreover, it will enable one to characterize the dynamic properties of functionally relevant hemoglobin intermediates and to study the kinetics of both the T-->R and R-->T quaternary transitions through time-resolved spectroscopy.     

Near-UV Circular Dichroism and UV Resonance Raman Spectra of Individual Tryptophan Residues in Human Hemoglobin and Their Changes upon the Quaternary Structure Transition

The aromatic residues such as tryptophan (Trp) and tyrosine (Tyr) in human adult hemoglobin (Hb A) are known to contribute to near-UV circular dichroism (CD) and UV resonance Raman (RR) spectral changes upon the R → T quaternary structure transition. In Hb A, there are three Trp residues per αβ dimer: at α14, β15, and β37. To evaluate their individual contributions to the R → T spectral changes, we produced three mutant hemoglobins in E. coli; rHb (α14Trp→Leu), rHb (β15Trp→Leu), and rHb (β37Trp→His). Near-UV CD and UVRR spectra of these mutant Hbs were compared with those of Hb A under solvent conditions where mutant rHbs exhibited significant cooperativity in oxygen binding. Near-UV CD and UVRR spectra for individual Trp residues were extracted by the difference calculations between Hb A and the mutants. α14 and β15Trp exhibited negative CD bands in both oxy- and deoxy-Hb A, whereas β37Trp showed positive CD bands in oxy-Hb A but decreased intensity in deoxy-form. These differences in CD spectra among the three Trp residues in Hb A were ascribed to surrounding hydrophobicity by examining the spectral changes of a model compound of Trp, N-acetyl-l-Trp ethyl ester, in various solvents. Intensity enhancement of Trp UVRR bands upon the R → T transition was ascribed mostly to the hydrogen-bond formation of β37Trp in deoxy-Hb A because similar UVRR spectral changes were detected with N-acetyl-l-Trp ethyl ester upon addition of a hydrogen-bond acceptor.     

Site-specific protein dynamics in communication pathway from sensor to signaling domain of oxygen sensor protein, HemAT-Bs: Time-resolved Ultraviolet Resonance Raman Study

HemAT-Bs is a heme-based signal transducer protein responsible for aerotaxis. Time-resolved ultraviolet resonance Raman (UVRR) studies of wild-type and Y70F mutant of the full-length HemAT-Bs and the truncated sensor domain were performed to determine the site-specific protein dynamics following carbon monoxide (CO) photodissociation. The UVRR spectra indicated two phases of intensity changes for Trp, Tyr, and Phe bands of both full-length and sensor domain proteins. The W16 and W3 Raman bands of Trp, the F8a band of Phe, and the Y8a band of Tyr increased in intensity at hundreds of nanoseconds after CO photodissociation, and this was followed by recovery in ～50 μs. These changes were assigned to Trp-132 (G-helix), Tyr-70 (B-helix), and Phe-69 (B-helix) and/or Phe-137 (G-helix), suggesting that the change in the heme structure drives the displacement of B- and G-helices. The UVRR difference spectra of the sensor domain displayed a positive peak for amide I in hundreds of nanoseconds after photolysis, which was followed by recovery in ～50 μs. This difference band was absent in the spectra of the full-length protein, suggesting that the isolated sensor domain undergoes conformational changes of the protein backbone upon CO photolysis and that the changes are restrained by the signaling domain. The time-resolved difference spectrum at 200 μs exhibited a pattern similar to that of the static (reduced - CO) difference spectrum, although the peak intensities were much weaker. Thus, the rearrangements of the protein moiety toward the equilibrium ligand-free structure occur in a time range of hundreds of microseconds.     

UV resonance Raman study of streptavidin binding of biotin and 2-iminobiotin: comparison with avidin

UV resonance Raman (UVRR) spectroscopy is used to study the binding of biotin and 2-iminobiotin by streptavidin, and the results are compared to those previously obtained from the avidin-biotin complex and new data from the avidin-2-iminobiotin complex. UVRR difference spectroscopy using 244-nm excitation reveals changes to the tyrosine (Tyr) and tryptophan (Trp) residues of both proteins upon complex formation. Avidin has four Trp and only one Tyr residue, while streptavidin has eight Trp and six Tyr residues. The spectral changes observed in streptavidin upon the addition of biotin are similar to those observed for avidin. However, the intensity enhancements observed for the streptavidin Trp Raman bands are less than those observed with avidin. The changes observed in the streptavidin Tyr bands are similar to those observed for avidin and are assigned exclusively to the binding site Tyr 43 residue. The Trp and Tyr band changes are due to the exclusion of water and addition of biotin, resulting in a more hydrophobic environment for the binding site residues. The addition of 2-iminobiotin results in spectral changes to both the streptavidin and avidin Trp bands that are very similar to those observed upon the addition of biotin in each protein. The changes to the Tyr bands are very different than those observed with the addition of biotin, and similar spectral changes are observed in both streptavidin and avidin. This is attributable to hydrogen bond changes to the binding site Tyr residue in each protein, and the similar Tyr difference features in both proteins supports the exclusive assignment of the streptavidin Tyr difference features to the binding site Tyr 43.     

Heme structure of hemoglobin M Iwate [alpha 87(F8)His-->Tyr]: a UV and visible resonance Raman study

Heme structures of a natural mutant hemoglobin (Hb), Hb M Iwate [alpha87(F8)His-->Tyr], and protonation of its F8-Tyr were examined with the 244-nm excited UV resonance Raman (UVRR) and the 406.7- and 441.6-nm excited visible resonance Raman (RR) spectroscopy. It was clarified from the UVRR bands at 1605 and 1166 cm(-)(1) characteristic of tyrosinate that the tyrosine (F8) of the abnormal subunit in Hb M Iwate adopts a deprotonated form. UV Raman bands of other Tyr residues indicated that the protein takes the T-quaternary structure even in the met form. Although both hemes of alpha and beta subunits in metHb A take a six-coordinate (6c) high-spin structure, the 406.7-nm excited RR spectrum of metHb M Iwate indicated that the abnormal alpha subunit adopts a 5c high-spin structure. The present results and our previous observation of the nu(Fe)(-)(O(tyrosine)) Raman band [Nagai et al. (1989) Biochemistry 28, 2418-2422] have proved that F8-tyrosinate is covalently bound to Fe(III) heme in the alpha subunit of Hb M Iwate. As a result, peripheral groups of porphyrin ring, especially the vinyl and the propionate side chains, were so strongly influenced that the RR spectrum in the low-frequency region excited at 406.7 nm is distinctly changed from the normal pattern. When Hb M Iwate was fully reduced, the characteristic UVRR bands of tyrosinate disappeared and the Raman bands of tyrosine at 1620 (Y8a), 1207 (Y7a), and 1177 cm(-)(1) (Y9a) increased in intensity. Coordination of distal His(E7) to the Fe(II) heme in the reduced alpha subunit of Hb M Iwate was proved by the observation of the nu(Fe)(-)(His) RR band in the 441.6-nm excited RR spectrum at the same frequency as that of its isolated alpha chain. The effects of the distal-His coordination on the heme appeared as a distortion of the peripheral groups of heme. A possible mechanism for the formation of a Fe(III)-tyrosinate bond in Hb M Iwate is discussed.     

UV resonance Raman study of beta93-modified hemoglobin A: chemical modifier-specific effects and added influences of attached poly(ethylene glycol) chains.

The reactive sulfhydryl group on Cys beta93 in human adult hemoglobin (HbA) has been the focus of many studies because of its importance both as a site for synthetic manipulation and as a possible binding site for nitric oxide (NO) in vivo. Despite the interest in this site and the known functional alterations associated with manipulation of this site, there is still considerable uncertainty as to the conformational basis for these effects. UV resonance Raman (UVRR) spectroscopy is used in this study to evaluate the conformational consequences of chemically modifying the Cys beta93 sulfhydryl group of both the deoxy and CO-saturated derivatives of HbA using different maleimide and mixed disulfide reagents. Included among the maleimide reagents are NEM (n-ethylmaleimide) and several poly(ethylene glycol) (PEG)-linked maleimides. The PEG-based reagents include both different sizes of PEG chains (PEG2000, -5000, and -20000) and different linkers between the PEG and the maleimide. Thus, the effect on the conformation of both linker chemistry and PEG size is evaluated. The spectroscopic results reveal minimal perturbation of the global structure of deoxyHbA for the mixed disulfide modification. In contrast, maleimide-based modifications of HbA perturb the deoxy T state of HbA by "loosening" the contacts associated with the switch region of the T state alpha(1)beta(2) interface but do not modify the hinge region of this interface. When the NEM-modified HbA is also subjected to enzymatic treatment to remove the C-terminal Arg alpha141 (yielding NESdes-ArgHb), the resulting deoxy derivative exhibits the spectroscopic features associated with a deoxy R state species. All of the CO-saturated derivatives exhibit spectra that are characteristic of the fully liganded R structure. The deoxy and CO derivatives of HbA that have been decorated on the surface with large PEG chains linked to the maleimide-modified sulfhydryl through a short linker group all show a general intensity enhancement of the tyrosine and tryptophan bands in the UVRR spectrum. It is proposed that this effect arises from the osmotic impact of a large, close PEG molecule enveloping the surface of the protein.     

Ultraviolet-resonance raman spectroscopy of the filamentous virus Pf3: interactions of Trp 38 specific to the assembled virion subunit

The class II filamentous virus Pf3 packages a circular single-stranded DNA genome of approximately 5833 [corrected] nucleotides within a cylindrical capsid constructed from approximately 2500 [corrected] copies of a 44 residue alpha-helical subunit. The single tryptophan residue (Trp 38) of the capsid subunit is located within a basic C-terminal sequence (.R(+)WIK(+)AQFF). The local environment of Trp 38 in the native Pf3 assembly has been investigated using 229 nm excited ultraviolet-resonance Raman (UVRR) spectroscopy and fluorescence spectroscopy. Trp 38 exhibits an anomalous UVRR signature in Pf3, including structure-diagnostic Raman bands (763, 1228, 1370, and 1773 cm(-)(1)) that are greatly displaced from corresponding Raman markers observed in either detergent-disassembled Pf3, class I filamentous viruses, most globular proteins, or aqueous L-TRP. An unusual and highly quenched fluorescence spectrum is also observed for Trp 38. These distinctive UVRR and fluorescence signatures together reflect interactions of the Trp 38 side chain that are specific to the native PF3 assembly. The experimental results on PF3 and supporting spectroscopic data from other proteins of known three-dimensional structure favor a model in which pi electrons of the Trp 38 indolyl ring interact specifically with a basic side chain of the subunit C-terminal sequence. Residues Arg 37 AND Lys 40 are plausible candidates for the proposed cation-pi interaction of Trp 38. The present study suggests that raman spectroscopy may be a generally useful probe of interactions between the indolyl pi-electron system of tryptophan and electropositive groups in proteins and their assemblies.     

Changes in hydrogen bonding and environment of tryptophan residues on helix F of bacteriorhodopsin during the photocycle: a time-resolved ultraviolet resonance Raman study

Protein structural changes during the photocycle of bacteriorhodopsin were examined by time-resolved ultraviolet resonance Raman (UVRR) spectroscopy. Most of the 244-nm UVRR difference signals of Trp were assigned to either Trp182 or Trp189 using the Trp182 --> Phe and Trp189 --> Phe mutants. The W17 mode of Trp182 shows a wavenumber downshift in the M(1) --> M(2) transition, indicating an increase in hydrogen bonding strength at the indole nitrogen. On the other hand, Trp189 shows Raman intensity increases of the W16 and W18 modes ascribable to an increased hydrophobic interaction. These observations suggest that the tilt of helix F, which ensures that reprotonation of the Schiff base is from the cytoplasmic side, occurs in the M(1) --> M(2) transition. In the M(2) --> N transition, the environment of Trp189 returns to the initial state, whereas the hydrophobic interaction of Trp182 decreases drastically. The decrease in hydrophobic interaction of Trp182 in the N state suggests an invasion of water molecules that promote the proton transfer from Asp96 to the Schiff base. Structural reorganization of the protein after the tilt of helix F may be important for efficient reprotonation of the Schiff base.     

Picosecond structural dynamics of myoglobin following photodissociation of carbon monoxide as revealed by ultraviolet time-resolved resonance Raman spectroscopy

Picosecond protein dynamics of myoglobin in response to structural changes in heme upon CO dissociation were observed in a site-specific fashion for the first time using time-resolved UV resonance Raman spectroscopy. Transient UV resonance Raman spectra showed several phases of intensity changes in both tryptophan and tyrosine Raman bands. Five picoseconds after dissociation, the W18, W16, and W3 bands of tryptophan residues and the Y8a band of tyrosine residues decreased in intensity, followed by recovery of the Y8a band intensity in hundreds of picoseconds and recovery of the tryptophan bands in nanoseconds. These spectral changes suggest that the change in heme structure impulsively drives concerted movement of the EF helical section and that rearrangements toward a deoxy structure occur in the heme vicinity and in the A helix within a time frame of sub-nanoseconds to nanoseconds.     

Hemoglobin site-mutants reveal dynamical role of interhelical H-bonds in the allosteric pathway: time-resolved UV resonance Raman evidence for intra-dimer coupling

The dynamical effect of eliminating specific tertiary H-bonds in the hemoglobin (Hb) tetramer has been investigated by site-directed mutagenesis and time-resolved absorption and ultraviolet resonance Raman (UVRR) spectroscopy. The Trp alpha 14...Thr alpha 67 and Trp beta 15...Ser beta 72 H-bonds connect the A and E helices in the alpha and beta chains, and are proposed to break in the earliest protein intermediate (Rdeoxy) following photo-deligation of HbCO, along with a second pair of H-bonds involving tyrosine residues. Mutation of the acceptor residues Thr alpha 67 and Ser beta 72 to Val and Ala eliminates the A-E H-bonds, but has been shown to have no significant effect on ligand-binding affinity or cooperativity, or on spectroscopic markers of the T-state quaternary interactions. However, the mutations have profound and unexpected effects on the character of the Rdeoxy intermediate, and on the dynamics of the subsequent steps leading to the T state. Formation of the initial quaternary contact (RT intermediate) is accelerated, by an order of magnitude, but the locking-in of the T state is delayed by a factor of 2. These rate effects are essentially the same for either mutation, or for the double mutation, suggesting that the alpha beta dimer behaves as a mechanically coupled dynamical unit. Further evidence for intra-dimer coupling is provided by the Rdeoxy UVRR spectrum, in which either or both mutations eliminate the tyrosine difference intensity, although only tryptophan H-bonds are directly affected. A possible mechanism for mechanical coupling is outlined, involving transmission of forces through the alpha(1)beta(1) (and alpha(2)beta(2)) interface. The present observations establish that quaternary motions can occur on the approximately 100 ns time-scale. They show also that a full complement of interhelical H-bonds actually slows the initial quaternary motion in Hb, but accelerates the locking in of the T-contacts.     

Protonation and hydrogen-bonding state of the distal histidine in the CO complex of horseradish peroxidase as studied by ultraviolet resonance Raman spectroscopy

Ultraviolet resonance Raman (UVRR) spectroscopy has been used to characterize the structure and hydrogen bonding state of the distal histidine (His42) in horseradish peroxidase (HRP) complexed with carbon monoxide (HRP-CO). The HRP-CO - HRP UVRR difference spectrum in D(2)O solution at pD 7.0 shows two positive peaks at 1408 and 1388 cm(-)(1), which are ascribable to medium-to-weak and strong hydrogen bonding states, respectively, of the protonated imidazolium side chain of His42 in HRP-CO. Both His42 peaks decrease in intensity with increase of pD with a midpoint of transition at pD 8.8, indicating that the pK(a) of His42 in HRP-CO is 8.8. The CO ligand exhibits two C-O stretching Raman peaks at 1932 and 1902 cm(-)(1), the latter of which diminishes at alkaline pD and is assignable to a strong hydrogen-bonded state. It is most probable that the imidazolium side chain of His42 forms a strong hydrogen bond with CO, giving a His42 peak at 1388 cm(-)(1) and a CO peak at 1902 cm(-)(1), in one conformer. The other hydrogen bonding state of His42, giving the 1408 cm(-)(1) peak, is ascribed to another conformer forming a medium-to-weak hydrogen bond with a water molecule in the distal cavity. The present finding that His42 can act as a strong proton donor to CO and decrease the CO bond order is consistent with the role of His42 as a general acid to cleave the O-O bond of hydrogen peroxide, a specific oxidizing agent, in the catalytic cycle of HRP.     

UV Raman studies of peptide conformation demonstrate that betanova does not cooperatively unfold.

We used UV resonance Raman spectroscopy (UVRR) excited within the peptide bond pi --> pi* electronic transitions and within the aromatic amino acid pi --> pi* electronic transitions to examine the temperature dependence of the solution conformation of betanova, a 20-residue beta-sheet polypeptide [Kortemme, T., Ramirez-Alvarado, M., and Serrano, L. (1998) Science 281, 253-256]. The 206.5 nm excited UVRR enhances the amide vibrations and demonstrates that betanova has a predominantly beta-sheet structure between 5 and 82 degrees C. The 229 nm excited UVRR, which probes the tyrosine and tryptophan side chain vibrations, shows an increase in the solvent exposure of the tryptophan side chains as the temperature is increased. Our results are consistent with the existence of an intermediate state similar to that calculated by Bursulaya and Brooks [Bursulaya, B. D., and Brooks, C. L. (1999) J. Am. Chem. Soc. 121, 9947-9951] and exclude the previously proposed two-state cooperative folding mechanism. Betanova's structure appears to be molten globule over the 3-82 degrees C temperature range of our study.     

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.     

Vibrational and electronic couplings in ultraviolet resonance Raman spectra of cyclic peptides

Ultraviolet resonance Raman (UVRR) spectra are reported for a series of cyclic amides. 2-Azacyclotridecone, which has a 13-membered ring, shows a classic trans-amide UVRR spectral pattern, with comparable enhancement of the amide modes II, III and S. When the ring is diminished to eight (epsilon -caprolactam) or seven (2-azacyclooctanone) members, this pattern is replaced by a single strong band near 1497 cm(-1), characteristic of the Cz-N stretch of a cis-amide vibration (amide IIc). Further shrinkage of the ring decreases the amide IIc frequency. It is lowered over 100 cm(-1) to 1389 cm(-1) in the case of a 4-membered ring (2-azaidine), reflecting diminution of the Cz-N bond order due to ring strain and pyramidalization of the C and N atoms. At the same time the amide Ic (Cz=O stretching) frequency increases, reflecting the localization of the Cz=O double bond. Also the sensitivity to hydrogen-deuterium exchange reverses for amide Ic and IIc modes as ring size decreases. The UV absorption maximum, which is red-shifted for cis-relative to trans-amides, shifts increasingly to the blue as the ring size decreases, again reflecting localization of the pi bonding. In the case of amides with 5- and 6-membered rings (2-pyrrolidinone and delta-eloctam) multiple UVRR bands are seen in the amide IIc region, whose relative intensities are temperature-dependent. These are assigned to conformers in which different members of the ring are out of the mean plane, resulting in variable perturbations of the amide bond. The cyclic dipeptides cyclo(Gly-Gly) and cyclo(Gly-Pro) have perturbed amide IIc frequencies, reflecting the kinematic mixing of the amide coordinates into in- and out-of-phase modes. Excitation profiles reveal electronic mixing as well, with the transition dipoles adding for the in-phase and cancelling for the out-of-phase modes.     

UV resonance Raman studies of alpha-nitrosyl hemoglobin derivatives: relation between the alpha 1-beta 2 subunit interface interactions and the Fe-histidine bonding of alpha heme.

Human alpha-nitrosyl beta-deoxy hemoglobin A, alpha(NO)beta(deoxy), is considered to have a T (tense) structure with the low O(2) affinity extreme and the Fe-histidine (His87) (Fe-His) bond of alpha heme cleaved. The Fe-His bonding of alpha heme and the intersubunit interactions at the alpha 1-beta 2 contact of alpha(NO)-Hbs have been examined under various conditions with EPR and UV resonance Raman (UVRR) spectra excited at 235 nm, respectively. NOHb at pH 6.7 gave the UVRR spectrum of the R structure, but in the presence of inositol-hexakis-phosphate (IHP) for which the Fe-His bond of the alpha heme is broken, UVRR bands of Trp residues behaved half-T-like while Tyr bands remained R-like. The half-ligated nitrosylHb, alpha(NO)beta(deoxy), in the presence of IHP at pH 5.6, gave T-like UVRR spectra for both Tyr and Trp, but binding of CO to its beta heme (alpha(NO)beta(CO)) changed the UVRR spectrum to half-T-like. Binding of NO to its beta heme (NOHb) changed the UVRR spectrum to 70% T-type for Trp but almost R-type for Tyr. When the pH was raised to 8.2 in the presence of IHP, the UVRR spectrum of NOHb was the same as that of COHb. EPR spectra of these Hbs indicated that the Fe-His bond of alpha(NO) heme is partially cleaved. On the other hand, the UVRR spectra of alpha(NO)beta(deoxy) in the absence of IHP at pH 8.8 showed the T-like UVRR spectrum, but the EPR spectrum indicated that 40-50% of the Fe-His bond of alpha hemes was intact. Therefore, it became evident that there is a qualitative correlation between the cleavage of the Fe-His bond of alpha heme and T-like contact of Trp-beta 37. We note that the behaviors of Tyr and Trp residues at the alpha 1-beta 2 interface are not synchronous. It is likely that the behaviors of Tyr residues are controlled by the ligation of beta heme through His-beta 92(F8)-->Val-beta 98(FG5)-->Asp-beta 99(G1 )-->Tyr-alpha 42(C7) or Tyr-beta 145(HC2).     

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.     

A1 reduction in intact cyanobacterial photosystem I particles studied by time-resolved step-scan Fourier transform infrared difference spectroscopy and isotope labeling

Time-resolved step-scan Fourier transform infrared (FTIR) difference spectroscopy, with 5 mus time resolution, has been used to produce P700(+)A(1)(-)/P700A(1) FTIR difference spectra in intact photosystem I particles from Synechococcus sp. 7002 and Synechocystis sp. 6803 at 77 K. Corresponding spectra were also obtained for fully deuterated photosystem I particles from Synechococcus sp. 7002 as well as fully (15)N- and (13)C-labeled photosystem I particles from Synechocystis sp. 6803. Static P700(+)/P700 FTIR difference spectra at 77 K were also obtained for all of the unlabeled and labeled photosystem I particles. From the time-resolved and static FTIR difference spectra, A(1)(-)/A(1) FTIR difference spectra were constructed. The A(1)(-)/A(1) FTIR difference spectra obtained for unlabeled trimeric photosystem I particles from both cyanobacterial strains are very similar. There are some mode frequency differences in spectra obtained for monomeric and trimeric PS I particles. However, the spectra can be interpreted in an identical manner, with the proposed band assignments being compatible with all of the data obtained for labeled and unlabeled photosystem I particles. In A(1)(-)/A(1) FTIR difference spectra obtained for unlabeled photosystem I particles, negative bands are observed at 1559 and 1549-1546 cm(-)(1). These bands are assigned to amide II protein vibrations, as they downshift approximately 86 cm(-)(1) upon deuteration and approximately 13 cm(-)(1) upon (15)N labeling. Difference band features at 1674-1677(+) and 1666(-) cm(-)(1) display isotope-induced shifts that are consistent with these bands being due to amide I protein vibrations. The observed amide modes suggest alteration of the protein backbone (possibly in the vicinity of A(1)) upon A(1) reduction. A difference band at 1754(+)/1748(-) cm(-)(1) is observed in unlabeled spectra from both strains. The frequency of this difference band, as well as the observed isotope-induced shifts, indicate that this difference band is due to a 13(3) ester carbonyl group of chlorophyll a species, most likely the A(0) chlorophyll a molecule that is in close proximity to A(1). Thus A(1) reduction perturbs A(0), probably via a long-range electrostatic interaction. A negative band is observed at 1693 cm(-)(1). The isotope shifts associated with this band are consistent with this band being due to the 13(1) keto carbonyl group of chlorophyll a, again, most likely the 13(1) keto carbonyl group of the A(0) chlorophyll a that is close to A(1). Semiquinone anion bands are resolved at approximately 1495(+) and approximately 1414(+) cm(-)(1) in the A(1)(-)/A(1) FTIR difference spectra for photosystem I particles from both cyanobacterial strains. The isotope-induced shifts of these bands could suggest that the 1495(+) and 1414(+) cm(-)(1) bands are due to C-O and C-C modes of A(1)(-), respectively.     

Differences in changes of the alpha1-beta2 subunit contacts between ligand binding to the alpha and beta subunits of hemoglobin A: UV resonance raman analysis using Ni-Fe hybrid hemoglobin

The alpha1-beta2 subunit contacts in the half-ligated hemoglobin A (Hb A) have been explored with ultraviolet resonance Raman (UVRR) spectroscopy using the Ni-Fe hybrid Hb under various solution conditions. Our previous studies demonstrated that Trpbeta37, Tyralpha42, and Tyralpha140 are mainly responsible for UVRR spectral differences between the complete T (deoxyHb A) and R (COHb A) structures [Nagai, M., Wajcman, H., Lahary, A., Nakatsukasa, T., Nagatomo, S., and Kitagawa, T. (1999) Biochemistry, 38, 1243-1251]. On the basis of it, the UVRR spectra observed for the half-ligated alpha(Ni)beta(CO) and alpha(CO)beta(Ni) at pH 6.7 in the presence of IHP indicated the adoption of the complete T structure similar to alpha(Ni)beta(deoxy) and alpha(deoxy)beta(Ni). The extent of the quaternary structural changes upon ligand binding depends on pH and IHP, but their characters are qualitatively the same. For alpha(Ni)beta(Fe), it is not until pH 8.7 in the absence of IHP that the Tyr bands are changed by ligand binding. The change of Tyr residues is induced by binding of CO, but not of NO, to the alpha heme, while it was similarly induced by binding of CO and NO to the beta heme. The Trp bands are changed toward R-like similarly for alpha(Ni)beta(CO) and alpha(CO)beta(Ni), indicating that the structural changes of Trp residues are scarcely different between CO binding to either the alpha or beta heme. The ligand induced quaternary structural changes of Tyr and Trp residues did not take place in a concerted way and were different between alpha(Ni)beta(CO) and alpha(CO)beta(Ni). These observations directly indicate that the phenomenon occurring at the alpha1-beta2 interface is different between the ligand binding to the alpha and beta hemes and is greatly influenced by IHP. A plausible mechanism of the intersubunit communication upon binding of a ligand to the alpha or beta subunit to the other subunit and its difference between NO and CO as a ligand are discussed.