Resonance Raman evidence for the mechanism of the allosteric control of O2-binding in a cobalt-substituted monomeric insect hemoglobin.

The substitution of iron for cobalt in the monomeric insect hemoglobin CTT (Chironomus thummi thummi) III does not alter the Bohr effect for O2-binding. The cobalt substitution in this hemoglobin allows us to identify not only the O-O and Co-O2 stretching mode but also the Co-O-O bending mode by resonance Raman spectroscopy. The assignments were made via 16O2/18O2 isotope exchange. The modes associated with the Co-O-O moiety are pH-dependent. These pH-induced changes of the resonance Raman spectra are correlated with the t = r conformation transition. At high pH (high-affinity state) two unperturbed O-O stretching modes are observed at 1,068 cm-1 (major component) and 1,093 cm-1 (minor component) for the 18O2 complex. These frequencies correspond to split modes at 1,107 cm-1 and 1,136 cm-1 and an unperturbed mode at approximately 1,153 cm-1 for the 16O2 complex. At low pH (low-affinity state) the minor component becomes the major component and vice versa. The Co-O2 stretching frequency varies for approximately 520 cm-1 (pH 5.5) to 537 cm-1 (pH 9.5) indicating a stronger (hence shorter) Co-O2 bond in the high-affinity state. On the other hand, the O-O bond is weakened upon the conversion of the low- to the high-affinity state. The Co-O-O bending mode changes from 390 cm-1 (pH 9.5) to 374 cm-1 (pH 5.5). In the deoxy form the resonance Raman spectra are essentially pH-insensitive except for a vinyl mode at 414 cm-1 (pH 5.5), which is shifted to 416 cm-1 (pH 5.5).     

Haem-rotational disorder in monomeric allosteric cyano-Met insect haemoglobins monitored by resonance Raman spectroscopy

The haem-rotational disorder (insertion of haem into globin rotated about the alpha, gamma-meso axis by 180 degrees) has been investigated in the cyano-Met form of the monomeric allosteric insect haemoglobins, CTT III and CTT IV, by resonance Raman spectroscopy. The effect of haem disorder on the resonance Raman spectra has been observed in proto-IX, deutero-IX, and meso-IX CTTs. Most importantly, in the absence of overlapping vinyl vibrations, we have identified two Fe-C-N bending vibrations at 401 cm-1 and 422 cm-1 (pH 9.5) for 57Fe deutero-IX CTT IV ligated with 13C15N-, which are attributed to the two haem-rotational components. One Fe-C-N bending mode at 422 cm-1 shows a pH-induced shift to 424 cm-1 (pH 5.5) indicating the t----r conformational transition, whereas the other bending mode is pH-insensitive, representing a non-allosteric component. By replacing the unsymmetrical porphyrins with the "symmetrical" protoporphyrin-III we eliminate the haem disorder. Then, sharpening of the Fe-N epsilon(His) (at 313 cm-1) and Fe-CN (at 453 cm-1) stretching modes is observed and a single Fe-C-N bending mode (at 412 cm-1) appears. In cyano-Met proto-IX CTT III two vinyl bending vibrations at 412 cm-1 and 591 cm-1 assigned by deuteration of the vinyl groups also reflect the haem disorder. The 412 cm-1 vinyl vibration is intensity-enhanced via through-space coupling with one of the Fe-C-N bending modes (at 412 cm-1). In the cyano-Met form of proto-III CTT III this vinyl vibration is shifted to 430 cm-1 resulting in a dramatic drop in intensity. It is most likely that the specific vinyl-protein interaction at position 4 in one of the haem-rotational components is the origin of the coupling between the Fe-C-N and vinyl bending modes. The Fe-N epsilon(proximal His) and the Fe-CN stretching vibrations as well as the Fe-C-N bending vibration have been identified by 54Fe/57Fe and 13C15N/12C15N/13C14N/12C14N isotope exchange.     

Mechanism of the control of dioxygen binding in a dimeric cobalt-substituted insect hemoglobin. Resonance Raman evidence for cobalt-axial-ligand bond changes

Resonance Raman spectroscopy has been used to investigate the allosteric control mechanism for O2 binding in a cobalt-substituted dimeric insect hemoglobin (CTT II), which exhibits a large Bohr effect due to a pH-induced transition between two ligand affinity states. Substitution of cobalt for iron in CTT II does not modify the Bohr effect, but permits the resonance enhancement (hence the detection) of Raman lines corresponding to the vibrations of the axial ligand-cobalt bonds. Using 16O2/18O2 isotope substitution the O-O and Co-O2 stretching and the Co-O-O bending mode have been assigned to the two affinity states of this hemoglobin: v (O-O) changes from 1152 cm-1 (pH 5.5; t conformation) to about 1125 cm-1 (pH 9.5, r conformation), v (Co-O2) from 512 cm-1 (pH 5.5) to 537 cm-1 (pH 9.5) and delta (Co-O-O) from 378 cm-1 (pH 5.5) to 390 cm-1 (pH 9.5). The Co-N epsilon (His) stretching mode has also been detected changing from 313 cm-1 (pH 5.5) to 307 cm-1 (pH 9.5). For the first time, reciprocal behaviour between the Co-N epsilon and Co-O2 bonds and between the Co-O2 and the O-O bonds in an allosteric hemoglobin are demonstrated. Furthermore, the pH sensitivity of a vinyl bending mode in the range of 411-415 cm-1 has been investigated and shown also to reflect the t in equilibrium with r conformation transition.     

Resonance Raman evidence for an unusually strong exogenous ligand-metal bond in a monomeric nitrosyl manganese hemoglobin

Resonance Raman spectroscopy has been employed to determine the vibrational modes of monomeric nitrosyl manganese Chironomus thummi thummi hemoglobin (CTT IV). This insect hemoglobin has no distal histidine. By applying various isotope-labeled nitric oxides (14N16O, 15N16O, 14N18O), we have identified the Mn11-NO stretching model at 628 cm-1, the Mn11-N-O bending mode at 574 cm-1 and the N-O stretching mode at 1735 cm-1. The results suggest a strong Mn11-NO bond and a weak N-O bond. The vinyl group substitution does not influence the nu (Mn11-NO), delta (Mn11-N-O) and nu (N-O) vibrations. The Mn11-NO stretching frequency is insensitive to distal histidine interactions with NO, whereas the N-O stretching frequency is sensitive. Nitric oxide also binds to Met manganese CTT IV to form an Mn111. NO complex which undergoes a slow but complete autoreduction resulting in the Mn11.NO species. In manganese meso-IX CTT IV, the Mn111. NO Mn11. NO conversion alters the intensities of the porphyrin ring modes at 342, 360, 1587 and 1598 cm-1, but shifts the frequencies at 1504 and 1633 cm-1 (in Mn111.NO) to 1497 and 1630 cm-1 (in Mn11. NO), respectively. The unshifted marker line at 1378 cm-1 reflects the fact that the pi electron densities of the porphyrin ring are the same in the two complexes.     

Evidence for allosteric transitions in secondary structure induced by superhelical stress

Previous studies suggest that the global secondary structures of native supercoiled and equilibrium linear DNAs may differ somewhat. Recent evidence also indicates that metastable secondary structure commonly persists following complete relaxation of the superhelical stress by intercalating dyes or by the action of topoisomerase I. In this work, the torsion constants (alpha) of pBR322, pUC8 and M13mp7 (replicative form) DNAs are determined by time-resolved fluorescence polarization anisotropy at various times subsequent to linearization. In all three cases, the torsion constants are relatively low immediately after linearization, and evolve for eight to ten weeks before reaching their apparent equilibrium values. It is shown in detail how the persistence of metastable secondary structure, subsequent to relaxation of superhelical stress, necessarily implies that one or more transitions in equilibrium secondary structure are induced as the superhelix density is varied from zero to native, or vice versa. Samples of pUC8 dimer (5434 base-pairs) with different superhelix densities are prepared by the action of topoisomerase I in the presence of various amounts of ethidium. Their median linking number differences are determined by standard band counting methods. The translational diffusion coefficient (Do) and the plateau diffusion coefficient (Dplat) characterizing internal motions over short distances (225 A) are determined by dynamic light-scattering. The torsion constant (alpha) between base-pairs and the circular dichroism spectrum are also measured for each sample. Curves of Dplat, Do, alpha and molar ellipticity ([theta]) (at the minimum near 250 nm) versus superhelix density (sigma) are constructed. The curve of Do versus sigma is very similar to that for sedimentation coefficient versus sigma for simian virus 40 (SV40) and polyoma DNAs. The curves of Dplat, Do, alpha and [theta] versus sigma show that, with increasing negative superhelix density, a structural transition occurs near sigma = -0.020 to an intermediate state with low torsion constant, and a second structural transition occurs near sigma = -0.035 to a state that exhibits more normal properties by sigma = -0.048. These data are consistent with the hypothesis that supercoiling induces two successive allosteric transitions to alternative global secondary structures. The data are much less consistent with the hypothesis that supercoiling induces some radical secondary structure at one or a few sites of small extent at sigma = -0.020, and at other sites at sigma = -0.035, or with hypotheses based on changes in tertiary structure alone.(ABSTRACT TRUNCATED AT 400 WORDS)     

Raman signatures of ligand binding and allosteric conformation change in hexameric insulin.

Hexameric insulin is an allosteric protein that undergoes transitions between three conformational states (T(6), T(3)R(3), and R(6)). These allosteric states are stabilized by the binding of ligands to the phenolic pockets and by the coordination of anions to the His B10 metal sites. Raman difference (RD) spectroscopy is utilized to examine the binding of phenolic ligands and the binding of thiocyanate, p-aminobenzoic acid (PABA), or 4-hydroxy-3-nitrobenzoic acid (4H3N) to the allosteric sites of T(3)R(3) and R(6). The RD spectroscopic studies show changes in the amide I and III bands for the transition of residues B1-B8 from a meandering coil to an alpha helix in the T-R transitions and identify the Raman signatures of the structural differences among the T(6), T(3)R(3), and R(6) states. Evidence of the altered environment caused by the approximately 30 A displacement of phenylalanine (Phe) B1 is clearly seen from changes in the Raman bands of the Phe ring. Raman signatures arising from the coordination of PABA or 4H3N to the histidine (His) B10 Zn(II) sites show these carboxylates give distorted, asymmetric coordination to Zn(II). The RD spectra also reveal the importance of the position and the type of substituents for designing aromatic carboxylates with high affinity for the His B10 metal site.     

NMR studies of the heme pocket conformations of monomeric hemoglobins from Glycera dibranchiata. Implications for ligand binding

Two-dimensional 1H-NMR methods have been used to assign side-chain resonances for the tryptophan residues and for several amino acids located in the heme pockets of the carbon monoxide complexes of the major monomeric hemoglobins from Glycera dibranchiata. The NMR spectra reveal a high degree of conservation of the heme pocket structure in the different hemoglobins. However some conformational differences are evident and residues at positions B10 and G8 on the distal side of the heme pocket are not conserved. From the present NMR studies it appears that the monomeric G. dibranchiata hemoglobin examined by X-ray crystallography [Padlan, E. A. & Love, W. (1974) J. Biol. Chem. 249, 4067-4078] corresponds to HbC. Except that the orientation of the heme in solution is the reverse of that reported in the crystal structure, there is a close correspondence between the heme pocket structure in the crystal and in solution. The proximal histidine coordination geometry is almost identical in the CO complexes of the three monomeric hemoglobins studied. Distal residues are strongly implicated in determining the observed kinetic differences in ligand binding reactions. In particular, steric crowding of the ligand binding site in hemoglobin A is probably a major factor in the slower kinetics of this component.     

Resonance Raman assignment and evidence for noncoupling of individual 2- and 4-vinyl vibrational modes in a monomeric cyanomethemoglobin

We have investigated the resonance Raman spectra of monomeric insect cyanomethemoglobins (CTT III and CTT IV) reconstituted with (1) protohemes IX selectively deuterated at the 4-vinyl as well as the 2,4-divinyls, (2) monovinyl-truncated hemes such as pemptoheme (2-hydrogen, 4-vinyl) and isopemptoheme (2-vinyl, 4-hydrogen), (3) symmetric hemes such as protoheme III (with 2- and 3-vinyls) and protoheme XIII (with 1- and 4-vinyls), and (4) hemes without 2- and 4-vinyls such as mesoheme IX, deuteroheme IX, 2,4-dimethyldeuteroheme IX, and 2,4-dibromodeuteroheme IX. Evidence is presented that the highly localized vinyl C = C stretching vibrations at the 2- and 4-positions of the heme in these cyanomet CTT hemoglobins are noncoupled and inequivalent; i.e., the 1631- and 1624-cm-1 lines have been assigned to 2-vinyl and 4-vinyl, respectively. The elimination of the 2-vinyl (in pemptoheme) or the 4-vinyl (in isopemptoheme) does not affect the C = C stretching frequency of the remaining vinyl. Furthermore, two low-frequency vinyl bending modes at 412 and 591 cm-1 exhibit greatly different resonance Raman intensities between 2-vinyl and 4-vinyl. The observed intensity at 412 cm-1 is primarily derived from 4-vinyl, whereas the 591-cm-1 line results exclusively from the 2-vinyl. Again, there is no significant coupling between 2-vinyl and 4-vinyl for these two bending modes.     

Resonance Raman evidence for substrate reorginization in the active site of papain.

Resonance Raman spectra were obtained for the acylenzyme 4-dimethylamino-3-nitro(alpha-benzamido)cinnamoyl-papain prepared using the chromophoric substrate methyl 4-dimethylamino-3-nitro(alpha-benzamido)cinnamate. These spectra contained vibrational spectral data of the acyl residue while covalently attached to the active site and could be used to follow directly acylation and deacylation kinetics. Spectra were obtained at pH values ranging from those where the acyl-enzyme is relatively stable (pH 3.0, tau 1/2 congruent to 800 s) to those where it is relatively unstable (pH 9.2, tau 1/2 congruent to 223 s). Throughout this range acyl-enzyme spectra differed completely from that of the free substrate or the product (4-dimethylamino-3-nitro(alpha-benzamido)cinnamic acid) indicating that a structural change occurred on combination with the active site. The spectra are consistent with rearrangement of the alpha-benzamido group in the bound substrate, -NH--C(==O)Ph becoming --N==C(--OX)Ph, where the bonding to oxygen is unknown. Superimposed on these large differences, small changes in acyl-enzyme spectra also occurred as pH was raised to decrease the half-life. All of the above spectral perturbations are consistent with a structural change in the acyl-enzyme which precedes the rate-determining step in deacylation. Thus, deacylation proceeds from an acyl residue structure differing from that of the substrate in solution. Upon acid denaturation the spectrum characteristic of the intermediate reverts to one closely resembling the substrate, demonstrating that a functioning active site is necessary to produce the observed differences. Spectra in D2O of native acyl-enzyme were identical with those in H2O, indicating that the observed differences in rate constant were not due to solvent-induced structural changes. Activated papain purified by crystallization or by affinity chromatography formed the acyl-enzyme. However, the kinetics of formation and deacylation differed between these materials, as did the spectral properties. Small differences in active-site structure are considered to be responsible for this effect, and it is suggested that such spectral perturbations may be useful in directly relating small differences in structure of the substrate in the active site with corresponding differences in kinetics.     

Raman spectroscopic evidence for structural changes in poly-L-lysine induced by an approximately 50 mT static magnetic field

We have explored the mechanism of coupling of an approximately 50 mT static magnetic field with the α helices of poly-L-lysine. Structural changes in poly-L-lysine were determined by Raman spectroscopy. Our testable hypothesis is that static magnetic fields of this magnitude can couple with the α-helical segments of the polypeptide, and, as a result, the structure of the polypeptide is significantly altered. Our model further suggests that a static magnetic field can promote protein unfolding and can prevent refolding. © 1996 Wiley-Liss, Inc.     

Resonance Raman evidence for the activation of dioxygen in horseradish oxyperoxidase

Resonance Raman spectroscopy has been employed to investigate the molecular bases for the markedly different properties of horseradish oxyperoxidase and oxymyoglobin. The porphyrin core of oxyperoxidase is slightly more expanded with the iron atom closer to the porphyrin plane, and there is greater iron d pi-to-oxygen pi backbonding compared to oxymyoglobin. The iron-oxygen (stretching or bending) bands are observed at 570 and 562 cm-1, respectively, for oxymyoglobin and oxyperoxidase, and the iron-His stretching bands have been tentatively identified at 276 and 289 cm-1, respectively. It is suggested that the stronger iron-His bond in oxyperoxidase facilitates greater iron d pi-to-oxygen pi backdonation by raising the energy of the iron d pi orbitals closer to the energy of the oxygen pi orbitals. This weakens the O-O bond and activates dioxygen for use as an electron acceptor in the peroxidase-oxidase reaction.     

The structural bases for the unique ligand binding properties of Glycera dibranchiata hemoglobins. A resonance Raman study

The hemoglobin of the marine annelid Glycera dibranchiata possesses several unique features: the hemoglobin consists of multiple monomeric and polymeric components, quaternary structure is lacking, the distal histidine is replaced by leucine in at least one monomeric constituent, and 4) the protein exhibits extremely rapid ligand binding kinetics. The effect of these structural modifications on the ligand binding process has been evaluated using resonance Raman spectroscopy to examine the vibrational modes of the porphyrin macrocycle in deoxy and carbonmonoxy equilibrium species of hemoglobin G. dibranchiata in both the unseparated monomeric and polymeric forms and in a single monomeric component designated Fraction II. Significant differences relative to hemoglobin were found in porphyrin pi electron density, vinyl environment, low frequency vibrational modes, and, in particular, the Fe-proximal histidine stretching mode. Spectra of the deoxy heme transients generated within 10 ns of ligand photolysis have also been examined. These clearly indicate large differences in the heme pocket dynamics subsequent to CO photolysis in G. dibranchiata hemoglobins relative to other hemoglobins. The significance of these results in terms of the kinetics and thermodynamics of ligand binding is discussed.     

Crystalline ligand transitions in lamprey hemoglobin. Structural evidence for the regulation of oxygen affinity

The hemoglobins of the Sea Lamprey (Petromyzon marinus) exist in an equilibrium between low affinity oligomers, stabilized by proton binding, and higher affinity monomers, stabilized by oxygen binding. Recent crystallographic analysis revealed that dimerization is coupled with key changes at the ligand binding site with the distal histidine sterically restricting ligand binding in the deoxy dimer but with no significant structural rearrangements on the proximal side. These structural insights led to the hypothesis that oxygen affinity of lamprey hemoglobin is distally regulated. Here we present the 2.9-A crystal structure of deoxygenated lamprey hemoglobin in an orthorhombic crystal form along with the structure of these crystals exposed to carbon monoxide. The hexameric assemblage in this crystal form is very similar to those observed in the previous deoxy structure. Whereas the hydrogen bonding network and packing contacts formed in the dimeric interface of lamprey hemoglobin are largely unaffected by ligand binding, the binding of carbon monoxide induces the distal histidine to swing to positions that would preclude the formation of a stabilizing hydrogen bond with the bound ligand. These results suggest a dual role for the distal histidine and strongly support the hypothesis that ligand affinity in lamprey hemoglobin is distally regulated.     

Resonance Raman spectra of plastocyanin and pseudoazurin: evidence for conserved cysteine ligand conformations in cupredoxins (blue copper proteins)

New resonance Raman (RR) spectra at 15 K are reported for poplar (Populus nigra) and oleander (Oleander nerium) plastocyanins and for Alcaligenes faecalis pseudoazurin. The spectra are compared with those of other blue copper proteins (cupredoxins). In all cases, nine or more vibrational modes between 330 and 460 cm-1 can be assigned to a coupling of the Cu-S(Cys) stretch with Cys ligand deformations. The fact that these vibrations occur at a relatively constant set of frequencies is testimony to the highly conserved ground-state structure of the Cu-Cys moiety. Shifts of the vibrational modes by 1-3 cm-1 upon deuterium exchange can be correlated with N-H...S hydrogen bonds from the protein backbone to the sulfur of the Cys ligand. There is marked variability in the intensities of these Cys-related vibrations, such that each class of cupredoxin has its own pattern of RR intensities. For example, plastocyanins from poplar, oleander, French bean, and spinach have their most intense feature at approximately 425 cm-1; azurins show greatest intensity at approximately 410 cm-1, stellacyanin and ascorbate oxidase at approximately 385 cm-1, and nitrite reductase at approximately 360 cm-1. These variable intensity patterns are related to differences in the electronic excited-state structures. We propose that they have a basis in the protein environment of the copper-cysteinate chromophore. A further insight into the vibrational spectra is provided by the structures of the six cupredoxins for which crystallographic refinements at high resolution are available (plastocyanins from P. nigra, O. nerium, and Enteromorpha prolifera, pseudoazurin from A. faecalis, azurin from Alcaligenes denitrificans, and cucumber basic blue protein). The average of the Cu-S(Cys) bond lengths is 2.12 +/- 0.05 A. Since the observed range of bond lengths falls within the precision of the determinations, this variation is considered insignificant. The Cys ligand dihedral angles are also highly conserved. Cu-S gamma-C beta-C alpha is always near -170 degrees and S gamma-C beta-C alpha-N near 170 degrees. As a result, the Cu-S gamma bond is coplanar with the Cys side-chain atoms and part of the polypeptide backbone. The coplanarity accounts for the extensive coupling of Cu-S stretching and Cys deformation modes as seen in the RR spectrum. The conservation of this copper-cysteinate conformation in cupredoxins may indicate a favored pathway for electron transfer.     

Resonance Raman studies of hemoglobins reconstituted with mesoheme. Unperturbed iron-histidine stretching frequencies in a functionally altered hemoglobin

Hybrid hemoglobins, containing mesoheme in one type of subunit and protoheme in the partner subunits, have been studied by resonance Raman spectroscopy. These hybrids have been studied in both the met hybrid and fully reduced, deoxy forms. Judicious choice of laser excitation frequency permits selective enhancement of modes associated with each type of subunit; i.e., either meso- or protoheme-containing subunit. The assignments of low-frequency modes of meso- and protoheme are briefly discussed with special reference to the iron-histidine linkage. Despite functional differences between the hybrids, no significant changes in the strength of the iron-histidine linkages are detected by resonance Raman spectroscopy. These results are discussed with reference to recent high-resolution NMR studies of these same hybrids.