Transient resonance Raman spectroscopy shows unrelaxed heme following CO photodissociation from cytochrome-c peroxidase

The 7 ns 436 nm pulses of an H2-shifted YAG laser have been used to photolyze the CO adduct of cytochrome-c peroxidase and produce the resonance Raman spectrum of the photoproduct. A 3 cm-1 downshift, relative to the spectrum of reduced enzyme, was observed for the porphyrin C-N breathing mode, v4. The downshift diminishes with decreasing CO /protein ratio, implying, in conjunction with a recent study of CO binding, that the unrelaxed heme is associated with adduct having a tilted, H-bonded FeCO unit. The downshift is eliminated when the phosphate buffer concentration is increased from 0.01 to 0.1 M. It is proposed that the heme relaxation under study involves a transition between two conformations, B and A, differing in the disposition of the distal residues, and having different v4 frequencies for unligated Fe(II) heme. Conformation B allows H-bonding to bound CO, and is favored at high CO and phosphate concentrations, while conformation A, which is unfavorable to CO H-bonding, is favored at low CO and phosphate concentrations. The recently reported absence of unrelaxed frequencies in the 7 ns photo-product of the CO adduct of horseradish peroxidase has been confirmed, and is attributed to lower stability for conformation B and a smaller A - B v4 difference.     

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)     

Time-resolved resonance Raman study on ultrafast structural relaxation and vibrational cooling of photodissociated carbonmonoxy myoglobin

A localized small structural change is converted to a higher order conformational change of protein and extends to a mesoscopic scale to induce a physiological function. To understand such features of protein, ultrafast dynamics of myoglobin (Mb) following photolysis of carbon monoxide were investigated. Recent results are summarized here with a stress on structural and vibrational energy relaxation. The core expansion of heme takes place within 2 ps but the out of plane displacement of the heme iron and the accompanying protein conformational change occur in 10 and 100 s of the picosecond regimes, respectively. Unexpectedly, it was found from UV resonance Raman spectra that Trp7 in the N-terminal region and Tyr151 in the C-terminal region undergo appreciable structural changes upon ligand binding-dissociation while Tyr104, Tyr146, and Trp14 do not. Because of the communication between the movements of these surface residues and the heme iron, the rate of spectral change of the iron-histidine (Fe- His) stretching band after CO photodissociation is influenced by the viscosity of solvent. Temporal changes of the anti-Stokes Raman intensity demonstrated immediate generation of vibrationally excited heme upon photodissociation and its decay with a time constant of 1-2 ps.     

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.     

Response of the local heme environment of (carbonmonoxy)hemoglobin to protein dehydration

The effects of protein dehydration upon the equilibrium and dynamic properties of the heme active site in human hemoglobin (HbA) have been probed by resonance Raman scattering. Spectra of equilibrium carbonmonoxy-HbA and the photolytic heme transient species generated within 10 ns of ligand photolysis have been obtained from thin films of protein in various stages of dehydration. These data provide detailed information concerning the response of the heme and its bonding interactions with both the proximal histidine and carbon monoxide as a function of protein hydration. For protein hydration levels of 0.4-1.0 g of H2O/g of protein, our results indicate that the C = O stretching mode of carbonmonoxy-HbA is dramatically affected by protein hydration levels, thus corroborating the infrared results of Brown et al. [Brown, W. E., Sutcliffe, J. W., & Pulsinelli, P. D. (1983) Biochemistry 22, 2914-2923]. However, we find that both heme skeletal modes and the Fe-C bond strength are largely insensitive to dehydration. Moreover, the proximal pocket geometry (as reflected in the behavior of the Fe-proximal histidine stretching mode) immediately following ligand photolysis was found to be very similar to that of R-state solution hemoglobin. At protein hydration levels below the theoretical monolayer limit, small changes in the resonance Raman spectra of both equilibrium HbCO and the transient heme species generated subsequent to ligand photolysis are detected. These include broadening of the Fe-C stretching mode in equilibrium HbCO and a small shift to lower frequency of the Fe-His mode in the photolytic transient species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Time-resolved circular dichroism and absorption studies of the photolysis reaction of (carbonmonoxy)myoglobin.

Time-resolved circular dichroism (TRCD) and absorption spectroscopy are used to follow the photolysis reaction of (carbonmonoxy)myoglobin (MbCO). Following the spectral changes associated with the initial loss of CO, a subtle change is observed in the visible absorption spectrum of the Mb product on a time scale of a few hundred nanoseconds. No changes are seen in the CD spectrum of Mb in the visible and near-UV regions subsequent to the loss of CO. The data suggest the existence of an intermediate found after ligand loss from MbCO that is similar in structure to the final Mb product.     

Resonance Raman spectroscopic evidence for heme iron-hydroxide ligation in peroxidase alkaline forms

Horseradish peroxidase will convert from a five-coordinate high-spin heme at neutral pH to a six-coordinate low-spin heme at alkaline pH. Though alkaline forms of other heme proteins such as hemoglobin and myoglobin are known to contain a heme-ligated hydroxide, alkaline horseradish peroxidase has been considered not to contain a ligated hydroxide. Several alternatives have been proposed which would be stronger field ligands than a hydroxide ion. In this report we provide resonance Raman evidence, using Soret excitation, that alkaline horseradish peroxidase does in fact contain a heme iron-ligated hydroxyl group. The band was located for isoenzymes C and A-1 by its sensitivity to 18O substitution and confirmed with 54Fe, 57Fe, and 2H. An isoenzyme of turnip peroxidase was investigated and found to also contain a ligated hydroxide at alkaline pH. The observed peroxidase Fe(III)-OH frequencies are 15-25 cm-1 higher than the corresponding frequencies of alkaline methemoglobin and metmyoglobin and correlate with changes in spin-state distribution. This is explained in the context of hydrogen bonding to a distal histidine which results in increased ligand field strength facilitating the formation of low-spin hemes. It has been demonstrated that the ferryl/ferric redox potential of horseradish peroxidase is markedly lowered at alkaline pH (Hayashi, Y., and Yamazaki, I. (1979) J. Biol. Chem. 254, 9101-9106). These observations are rationalized in terms of oxidation of a ligated ferric hydroxyl group facilitated through base catalysis by a distal histidine.     

Resonance Raman interrogation of the consequences of heme rotational disorder in myoglobin and its ligated derivatives

Resonance Raman spectroscopy is employed to characterize heme site structural changes arising from conformational heterogeneity in deoxyMb and ligated derivatives, i.e., the ferrous CO (MbCO) and ferric cyanide (MbCN) complexes. The spectra for the reversed forms of these derivatives have been extracted from the spectra of reconstituted samples. Dramatic changes in the low-frequency spectra are observed, where newly observed RR modes of the reversed forms are assigned using protohemes that are selectively deuterated at the four methyl groups or at the four methine carbons. Interestingly, while substantial changes in the disposition of the peripheral vinyl and propionate groups can be inferred from the dramatic spectral shifts, the bonds to the internal histidyl imidazole ligand and those of the Fe-CO and Fe-CN fragments are not significantly affected by the heme rotation, as judged by lack of significant shifts in the nu(Fe-N(His)), nu(Fe-C), and nu(C-O) modes. In fact, the apparent lack of an effect on these key vibrational parameters of the Fe-N(His), Fe-CO, and Fe-CN fragments is entirely consistent with previously reported equilibrium and kinetic studies that document virtually identical functional properties for the native and reversed forms.     

Copper(II) protoporphyrin IX as a reporter group for the heme environment in myoglobin.

Copper(II) protoporphyrin IX has been introduced into apomyoglobin, and its utility as a reporter group of the heme environment has been examined. The Soret and visible absorption bands and electron spin resonance spectrum show that the Cu(II) is five coordinate, probably through coordination to the F-8 proximal histidine. The resonance Raman spectrum does not indicate any appreciable distortion from the solution conformation of copper(II) protoporphyrin IX dimethyl ester in CS2. The ultraviolet circular dichroism shows no alteration of the helical content of the globin from that of metmyoglobin. The circular dichroism of the porphyrin transitions suggests that the packing of the amino acid side chains around the porphyrin is different than that in the native metmyoglobin.     

Resonance Raman evidence of chloride binding to the heme iron in myeloperoxidase

The resonance Raman spectra of ferric derivatives of myeloperoxidase at pH 8 show ligand-dependent differences. The data are consistent with the resting enzyme and the chloride and fluoride derivatives all having 6-coordinated high-spin configurations. At pH 4 we find that the resting enzyme is susceptible to photodegradation from our low power incident laser beam. Chloride binding inhibits this denaturation. Our data support direct binding of chloride to the enzyme under physiological conditions.     

Pressure effects on the proximal heme pocket in myoglobin probed by Raman and near-infrared absorption spectroscopy.

The influence of high pressure on the heme protein conformation of myoglobin in different ligation states is studied using Raman spectroscopy over the temperature range from 30 to 295 K. Photostationary experiments monitoring the oxidation state marker bands demonstrate the change of rebinding rate with pressure. While frequency changes of vibrational modes associated with rigid bonds of the porphyrin ring are <1 cm(-1), we investigate a significant shift of the iron-histidine mode to higher frequency with increasing pressure (approximately 3 cm(-1) for deltaP = 190 MPa in Mb). The observed frequency shift is interpreted structurally as a conformational change affecting the tilt angle between the heme plane and the proximal histidine and the out-of-plane iron position. Independent evidence for iron motion comes from measurements of the redshift of band III in the near-infrared with pressure. This suggests that at high pressure the proximal heme pocket and the protein are altered toward the bound state conformation, which contributes to the rate increase for CO binding. Raman spectra of Mb and photodissociated MbCO measured at low temperature and variable pressure further support changes in protein conformation and are consistent with glasslike properties of myoglobin below 160 K.     

1H NMR resonance assignments in a paramagnetic heme protein by two-dimensional spectroscopy: heme resonances in equine met-azido myoglobin

Specific heme protons for the majority of resonances in the downfield resolved region of equine met-azido myoglobin have been assigned using solely the two-dimensional 1H NMR experiments NOESY and COSY. Metazido myoglobin provides a useful test case for the applicability of these techniques to paramagnetic proteins for the following reasons. First met-azido myoglobin is a mixed spin-state protein, with significantly shorter relaxation times and broadened lines relative to pure low-spin systems (eg., met-cyano myoglobin). Second, met-azido hemoglobin and met-azido myoglobin are important as models for the physiological forms of hemoglobin. Third, a few sperm whale met-azido myoglobin resonances have been previously assigned, which permits a comparison of assignments for these similar proteins, and a check of the method presented here.     

Picosecond Kerr-gated time-resolved resonance Raman spectroscopy of the [Ru(phen)(2)dppz](2+) interaction with DNA

To investigate the basis of the 'light-switch' effect, the solvent dependence of the Kerr-gated picosecond-time resolved resonance Raman (TR(3)) spectra of [Ru(bpy)(2)dppz](2+), [Ru(phen)(2)dppz](2+), and the modified complex [Ru(phen)(2)cpdppzOMe](2+) and a dimer [mu-C4(cpdppz)(2)-(phen)(4)Ru(2)](4+) were studied. The investigation focussed on comparing the behaviour of [Ru(phen)(2)dppz](2+) in acetonitrile, ethanol, H(2)O, D(2)O, and DNA. The data are consistent with a model wherein excitation induces metal-to-ligand charge transfer (MLCT) to any of the ligands (termed the 'precursor' state) which, by interligand electron transfer (ILET), produces an excited state localised on the dppz ligand, MLCT(1). In water this state relaxes with a characteristic time of approximately 6 ps to a non-emissive state (MLCT(2)). The TR(3) spectra in water, acetonitrile and DNA are all distinctly different. However, the early (4 ps) water spectrum resembles the spectrum in DNA. This interesting observation suggests that the DNA-bound excited state of the complex can be thought of as a model for the initial, poorly solvated state in water.     

Engineering His(E7) affects the control of heme reactivity in Aplysia limacina myoglobin

Aplysia limacina myoglobin lacks the distal histidine (His (E7)) and displays a ligand stabilization mechanism based on Arg(E10). The double mutant Val(E7)His-Arg(E10)Thr has been prepared to engineer the role of His(E7), typical of mammalian myoglobins, in a different globin framework. The 2.0 A crystal structure of Val(E7)His-Arg(E10)Thr met-Mb mutant reveals that the His(E7) side chain points out of the distal pocket, providing an explanation for the observed failure to stabilize the Fe(II) bound oxygen in the ferrous myoglobin. Moreover, spectroscopic analysis together with kinetic data on azide binding to met-myoglobin are reported and discussed in terms of the presence of a water molecule at coordination distance from the heme iron.     

FeNO structure in distal pocket mutants of myoglobin based on resonance Raman spectroscopy

FeNO vibrational frequencies were investigated for a series of myoglobin mutants using isotope-edited resonance Raman spectra of (15/14)NO adducts, which reveal the FeNO and NO stretching modes. The latter give rise to doublet bands, as a result of Fermi resonances with coincident porphyrin vibrations; these doublets were analyzed by curve-fitting to obtain the nuNO frequencies. Variations in nuNO among the mutants correlate with the reported nuCO variations for the CO adducts of the same mutants. The correlation has a slope near unity, indicating equal sensitivity of the NO and CO bonds to polar influences in the heme pocket. A few mutants deviate from the correlation, indicating that distal interactions differ for the NO and CO adducts, probably because of the differing distal residue geometries. In contrast to the strong and consistent nuFeC/nuCO correlation found for the CO adducts, nuFeN correlates only weakly with nuNO, and the slope of the correlation depends on which residue is being mutated. This variability is suggested to arise from steric interactions, which change the FeNO angle and therefore alter the Fe-NO and N-O bond orders. This effect is modeled with Density Functional Theory (DFT) and is rationalized on the basis of a valence isomer bonding model. The FeNO unit, which is naturally bent, is a more sensitive reporter of steric interactions than the FeCO unit, which is naturally linear. An important additional factor is the strength of the bond to the proximal ligand, which modulates the valence isomer equilibrium. The FeNO unit is bent more strongly in MbNO than in protein-free heme-NO complexes because of a combination of a strengthened proximal bond and distal interactions.     

Distal heme pocket conformers of carbonmonoxy derivatives of Ascaris hemoglobin: evidence of conformational trapping in porous sol-gel matrices

We report the ligand dependence of the conformer distribution in the distal heme pocket of Ascaris suum hemoglobin (Hb) studied by resonance Raman spectroscopy. The heme-bound CO is used as a spectroscopic antenna to probe the original distribution of conformers in the dioxygen derivative of Ascaris Hb, by utilizing sol-gel encapsulation. The first step is to encapsulate the dioxygen derivative in the porous sol-gel and let the gel age, thus trapping the equilibrium conformational distribution of Ascaris dioxygen Hb. In the second step, the dioxygen ligand is replaced by CO. The sol-gel environment impedes any large scale movements, drastically slowing down the conformational relaxation triggered by the ligation change, essentially "locking in" the initial quaternary and even tertiary structure of the protein. Studying the Fe-CO frequencies of the latter sample allows evaluation of the distribution of the distal heme pocket conformers that was originally associated with the dioxygen derivative. Extending the study to the Ascaris mutants allows for examination of the effect of specific residues in the distal pocket on the conformational distribution. The choice of mutants was largely based on the anticipated variation in hydrogen bonding patterns. The results show that the sol-gel encapsulation can slow or prevent re-equilibration within the distal heme pocket of Ascaris Hb and that the distribution of distal heme pocket conformers for the CO derivative of Ascaris Hb in the sol-gel is highly dependent on the history of the sample. Additionally, we report a detailed study of the CO complex of the mutants in solution for assignment of the various heme pocket conformers, and we present a comparison of the sol-gel data with solution data. The results support a picture in which the dioxygen derivative biases the population strongly toward a tightly packed configuration that favors the network of strong hydrogen bonding interactions, and suggest that Ascaris Hb is uniquely designed for dioxygen capture.