Heme Relaxation Research Articles

Ultrafast Heme Relaxation Dynamics Probing the Unfolded States of Cytochrome c Induced by Liposomes: Effect of Charge of Phospholipids.

The ubiquitous electron transfer heme protein, Cytochrome c (Cyt c) catalyzes the peroxidation of cardiolipin (CL) in the early stage of apoptosis, where Cyt c undergoes conformational changes leading to the partial unfolding of the protein. Here the interaction dynamics of Cyt c with liposomes having different charges [CL, - 2; POPG (2-Oleoyl-1-palmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt), -1; and POPC (2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine), 0] leading to various degrees of partial unfolding is investigated with steady state optical spectroscopy and femtosecond time-resolved pump-probe spectroscopy. The signature of the partial unfolding of the protein was observed in the absorption, fluorescence, and CD spectra of Cyt c-liposome complexes with an increase of lipid/protein (L/P) ratio, and the protein was refolded by the addition of 0.1 M of NaCl. The femtosecond transient absorption spectra of the complexes were measured by selectively exciting the heme and tryptophan (Trp) at 385 and 280 nm, respectively. Though significant changes were not observed in the excited state relaxation dynamics of the heme in liposomes by exciting at 385 nm, the 280 nm excitation exhibited a systematic increase of the excited state relaxation dynamics leading to the increase of lifetime of Trp and global conformational relaxation dynamics with the increase of anionic charge of the lipids. This reveals the decrease of efficiency of fluorescence resonance energy transfer from Trp to heme due to the increase of distance between them upon increase of partial unfolding of the proteins by liposomes. Such observation exhibits the Trp as a marker amino acid to reflect the dynamics of partial unfolding of the protein rising from the change in the tertiary structure and axial ligand interaction of the heme proteins in liposomes. The relaxation dynamics of the complexes in the presence of salt are similar to that of the protein alone, reflecting that the refolding of the protein and the interactions are dominated by electrostatic interaction rather than the hydrophobic interaction.

Ultrafast Heme Dynamics of Ferric Cytochrome c in Different Environments: Electronic, Vibrational, and Conformational Relaxation.

The excited-state dynamics of ferric cytochrome c (Cyt c), an important electron-transfer heme protein, in acidic to alkaline medium and in its unfolded form are investigated by using femtosecond pump-probe spectroscopy, exciting the heme and Tryptophan (Trp) to understand the electronic, vibrational, and conformational relaxation of the heme. At 390 nm excitation, the electronic relaxation of heme is found to be ≈150 fs at different pH values, increasing to 480 fs in the unfolded form. Multistep vibrational relaxation dynamics of the heme, including fast and slow processes, are observed at pH 7. However, in the unfolded form and at pH 2 and 11, fast phases of vibrational relaxation dominate, revealing the energy dissipation occurring through the covalent bond interaction between the heme and the nearest amino acids. A significant shortening of the excited-state lifetime of Trp is observed at various pH values at 280 nm excitation due to resonance energy transfer to the heme. The longer time constant (25 ps) observed in the unfolded form is attributed to a complete global conformational relaxation of Cyt c.

Resonance Raman Spectra of an O2-Binding H-NOX Domain Reveal Heme Relaxation upon Mutation

Resonance Raman and electronic absorption spectra were measured for the wild type Heme-Nitric oxide/OXygen binding domain from Thermoanaerobacter tengcongensis (Tt H-NOX WT) and three other Tt H-NOX proteins containing mutations at key conserved residues to determine the heme conformation in solution. The most dramatic changes in heme conformation occurred in the O2-bound forms, and the single Tt H-NOX P115A mutation was sufficient to generate a significant relaxation of the chromophore. Clear evidence of heme relaxation in the Tt H-NOX I5L, P115A, and I5L/P115A mutants in solution is demonstrated by the observation of reduced resonance Raman intensities for several out-of-plane low frequency modes (e.g., γ11, γ12, γ13, and γ15) in the 400-750 cm−1 region known to be sensitive to ruffling and saddling deformations, as well as increased vibrational frequencies for the core heme skeletal stretching modes, ν3, ν2, and ν10.

Resonance Raman Spectra of an O2-Binding H-NOX Domain Reveal Heme Relaxation upon Mutation

Resonance Raman spectra were measured for the wild type Heme-Nitric oxide/OXygen binding domain from Thermoanaerobacter tengcongensis (Tt H-NOX WT) and three other Tt H-NOX proteins containing mutations at key conserved residues to determine the heme conformation in solution. The most dramatic changes in heme conformation occurred in the O2-bound forms, and the single Tt H-NOX P115A mutation was sufficient to generate a significant relaxation of the chromophore. Clear evidence of heme relaxation in the Tt H-NOX I5L, P115A, and I5L/P115A mutants in solution is demonstrated by the observation of reduced resonance Raman intensities for several out-of-plane low frequency modes (e.g., gamma11, gamma12, gamma13, and gamma15) in the 400-750 cm(-1) region known to be sensitive to ruffling and saddling deformations, as well as increased vibrational frequencies for the core heme skeletal stretching modes, nu3, nu2, and nu10. In addition, all three mutants exhibited some degree of heme conformational heterogeneity based on several broad skeletal markers (e.g., nu10) in the high frequency region. These results are comparable to those observed by Olea et al. for Tt H-NOX P115A in crystal form, where four different heme structures were determined from a single unit cell. On the basis of the resonance Raman spectra, it is clear that the actual heme conformation for Tt H-NOX P115A in solution is considerably more relaxed than that of the WT protein, with increased flexibility within the protein pocket, allowing for rapid sampling of alternate conformations.

Measurements of Heme Relaxation and Ligand Recombination in Strong Magnetic Fields

Heme cooling signals and diatomic ligand recombination kinetics are measured in strong magnetic fields (up to 10 T). We examined diatomic ligand recombination to heme model compounds (NO and CO), myoglobin (NO and O(2)), and horseradish peroxidase (NO). No magnetic field induced rate changes in any of the samples were observed within the experimental detection limit. However, in the case of CO binding to heme in glycerol and O(2) binding to myoglobin, we observe a small magnetic field dependent change in the early time amplitude of the optical response that is assigned to heme cooling. One possibility, consistent with this observation, is that there is a weak magnetic field dependence of the nonradiative branching ratio into the vibrationally hot electronic ground state during CO photolysis. Ancillary studies of the "spin-forbidden" CO binding reaction in a variety of heme compounds in the absence of magnetic field demonstrate a surprisingly wide range for the Arrhenius prefactor. We conclude that CO binding to heme is not always retarded by unfavorable spin selection rules involving a double spin-flip superexchange mechanism. In fact, it appears that the small prefactor ( approximately 10(9) s(-1)) found for CO rebinding to Mb may be anomalous, rather than the general rule for heme-CO rebinding. These results point to unresolved fundamental issues that underlie the theory of heme-ligand photolysis and rebinding.

Ligand Dynamics in an Electron Transfer Protein: PICOSECOND GEMINATE RECOMBINATION OF CARBON MONOXIDE TO HEME IN MUTANT FORMS OF CYTOCHROME c

Substitution of the heme coordination residue Met-80 of the electron transport protein yeast iso-1-cytochrome c allows external ligands like CO to bind and thus increase the effective redox potential. This mutation, in principle, turns the protein into a quasi-native photoactivable electron donor. We have studied the kinetic and spectral characteristics of geminate recombination of heme and CO in a series of single M80X (X = Ala, Ser, Asp, Arg) mutants, using femtosecond transient absorption spectroscopy. In these proteins, all geminate recombination occurs on the picosecond and early nanosecond time scale, in a multiphasic manner, in which heme relaxation takes place on the same time scale. The extent of geminate recombination varies from >99% (Ala, Ser) to approximately 70% (Arg), the latter value being in principle low enough for electron injection studies. The rates and extent of the CO geminate recombination phases are much higher than in functional ligand-binding proteins like myoglobin, presumably reflecting the rigid and hydrophobic properties of the heme environment, which are optimized for electron transfer. Thus, the dynamics of CO recombination in cytochrome c are a tool for studying the heme pocket, in a similar way as NO in myoglobin. We discuss the differences in the CO kinetics between the mutants in terms of the properties of the heme environment and strategies to enhance the CO escape yield. Experiments on double mutants in which Phe-82 is replaced by Asp or Gly as well as the M80D substitution indicate that such steric changes substantially increase the motional freedom-dissociated CO.

Extended subnanosecond structural dynamics of myoglobin revealed by Laue crystallography.

Work carried out over the last 30 years unveiled the role of structural dynamics in controlling protein function. Cavity networks modulate structural dynamics trajectories and are functionally relevant; in globins they have been assigned a role in ligand migration and docking. These findings raised renewed interest for time-resolved structural investigations of myoglobin (Mb), a simple heme protein displaying a photosensitive iron-ligand bond. Photodissociation of MbCO generates a nonequilibrium population of protein structures relaxing over a time range extending from picoseconds to milliseconds. This process triggers ligand migration to matrix cavities with clear-cut effects on the rate and yield of geminate rebinding. Here, we report subnanosecond time-resolved Laue diffraction data on the triple mutant YQR-Mb [Leu-29(B10)Tyr, His-64(E7)Gln, Thr-67(E10)Arg] that depict the sequence of structural events associated with heme and protein relaxation from 100 ps to 316 ns and above. The photodissociated ligand rapidly (<0.1 ns) populates the Xe-binding cavity distal to the heme. Moreover, the heme relaxation toward the deoxy configuration is heterogeneous, with a slower phase ( approximately ns) evident in these experiments. Damping of the heme response appears to result from a strain exerted by the E-helix via the CD-turn; Phe-43(CD1), in close contact with heme, opposes tilt until the strain is relieved. A comparison with crystallographic data on wild-type Mb and mutants Leu(29)Phe or Leu(29)Trp suggests that the internal structure controls the rate and amplitude of the relaxation events. A correlation between structural dynamics as unveiled by Laue crystallography and functional properties of Mb is presented.

Dynamics of Nitric Oxide Rebinding and Escape in Horseradish Peroxidase

Ultrafast kinetic measurements of NO rebinding to horseradish peroxidase (HRP) are reported for the first time. The geminate kinetics are found to be exponential for all HRP samples studied. The ferric forms of HRP have NO geminate recombination time constants in the range of 15-30 ps, while the ferrous form has a time constant of approximately 7 ps. The simple exponential NO geminate kinetics found for HRP demonstrate that heme relaxation is not the underlying source of the nonexponential NO rebinding in myoglobin (Mb). The NO ligand escape rates from HRP are also determined, and they are found to depend dramatically on the presence or absence of the competitive inhibitor benzohydroxamic acid (BHA). The kinetic results indicate that, in contrast to Mb, there is direct solvent access to the distal heme pocket of HRP.

Vibrational Energy Relaxation of “Tailored” Hemes in Myoglobin Following Ligand Photolysis Supports Energy Funneling Mechanism of Heme “Cooling”

In a previous molecular dynamics simulation study, the kinetic energy relaxation of photolyzed heme in solvated carbonmonoxymyoglobin was found to be a single exponential decay process with the relaxation time constant 5.9 ps [Sagnella, D. E.; Straub, J. E. J. Phys. Chem. B 2001, 105, 7057]. The strong electrostatic interaction of the isopropionate side chains and the solvating water molecules was shown to be the single most important “doorway” for dissipation of excess kinetic energy in the heme. In this work, the results of a molecular dynamics simulation study of heme “cooling” in two modified myoglobins, in which (1) the two isopropionate side chains in the heme are replaced by hydrogen or (2) the proximal histidine is replaced by glycine, His93Gly, are presented. For each “tailored” protein, the relaxation of the heme's excess kinetic energy is found to be a single exponential decay process. For the His93Gly mutant protein, the relaxation time is found to be 5.9 ps, in agreement with the relaxation t...

Optical absorption band III of deoxyheme proteins as a probe of their structure and dynamics

Theory of the temperature dependence of a non-Frank–Condon optical absorption band in the case of weak anharmonicity is developed and applied to the interpretation of band III properties of deoxyheme proteins. It is obtained that dependence of the transition dipole moment on the displacement of the iron out of the heme plane, the weak anharmonic coupling between the heme doming and the iron–histidine vibration, and the glass–liquid phase transition cause notable and very specific temperature and pressure dependence of band III. It follows from this study that intensity of band III is sensitive to the iron–porphyrin distance, whereas its position can be affected by both the changes in this distance and protein electric field on the heme. Because the iron–porphyrin distance is affected by the protein, the band intensity and position (and not only width) must be sensitive to the protein structure and dynamics. In particular, intensity of band III can be used as a probe of protein dynamics, its dependence on the solvent viscosity, and protein and heme relaxation in the flash-photolysis experiments.

Dissociation and Recombination between Ligands and Heme in a CO-sensing Transcriptional Activator CooA: A FLASH PHOTOLYSIS STUDY

CooA from Rhodospirillum rubrum is a transcriptional activator in which a heme prosthetic group acts as a CO sensor and regulates the activity of the protein. In this study, the electronic relaxation of the heme, and the concurrent recombination between ligands and the heme at approximately 280 K were examined in an effort to understand the environment around the heme and the dynamics of the ligands. Upon photoexcitation of the reduced CooA at 400 nm, electronic relaxation of the heme occurred with time constants of 0.8 and 1.7 ps. The ligand rebinding was substantially completed with a time constant of 6.5 ps, followed by a slow relaxation process with a time constant of 173 ps. In the case of CO-bound CooA, relaxation of the excited heme occurred with two time constants, 1.1 and 2.4 ps, which were largely similar to those with reduced CooA. The subsequent CO recombination process was remarkably fast compared with that of other CO-bound heme proteins. It was well described as a biphasic geminate recombination process with time constants of 78 ps (60%) and 386 ps (30%). About 10% of the excited heme remained unligated at 1.9 ns. The dynamics of rebinding of CO thus will help us to understand how the physiologically relevant diatomic molecule approaches the heme binding site in CooA with picosecond resolution.

Cryogenic stabilization of myoglobin photoproducts.

The low frequency resonance Raman spectra of photodissociated carbon monoxymyoglobin at cryogenic temperatures (4-77 K) differ from those of deoxymyoglobin. Intensity differences occur in several low frequency porphyrin modes, and intensity and frequency differences occur in the iron-histidine stretching mode. This mode appears at about 225 cm-1 in deoxymyoglobin. At the lowest temperature studied, approximately 4 K, the frequency of the iron-histidine stretching mode in the photoproduct is approximately 233 cm-1, and the intensity is very low. When the temperature of the photoproduct is increased, the intensity of the mode increases, but its frequency is unchanged. The differences between the photoproduct and the deoxy preparation persist to 77 K, the highest temperature studied, and are independent of whether samples are frozen in phosphate buffer or a 50:50 ethylene glycol/phosphate buffer mixture. It is proposed that the frequency of the iron-histidine stretching mode is governed by the tilt angle of the histidine with respect to the normal to the heme plane, and the intensity of the mode is governed by the overlap between the sigma orbital of the iron-histidine bond and the pi orbital of the porphyrin macrocycle. This model can account for differences between the resonance Raman spectra of the photoproduct and the deoxy preparations of both hemoglobin and myoglobin. Furthermore, by considering the F-helix motions in going from 6-coordinate to 5-coordinate hemoglobin and myoglobin, the heme relaxation of these proteins at room temperature with 10-ns pulses can be explained. Based on the findings reported here, low temperature relaxation pathways for both hemoglobin and myoglobin are proposed.

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

The 7 ns 436 nm pulses of an H 2-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, v 4. 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 v 4 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 confimred, and is attributed to lower stability for conformation B and a smaller A – B v 4 difference.

NMR relaxation of protein and water protons in methemoglobin solutions

Hemoglobin (Hb) proton spins rapidly equilibrate among themselves after an initial excitation, and relax toward thermal equilibrium as a unit. In the diamagnetic form, spin diffusion to nearby methyl relaxation sinks can account for this. For metHb, four strong heme relaxation centers dominate, and spin diffusion must occur over long distances. A sizeable difference in protein T1 is found between H2O and D2O solutions, much more than for diamagnetic Hb, consistent with internal H2O acting as a spin carrier to the heme.