Spectroscopic Evidence for Nanosecond Protein Relaxation after Photodissociation of Myoglobin−CO † (original) (raw)
Related papers
Journal of Biological Chemistry, 1997
The role of the protein structural change monitored by absorption band shifts following flash photolysis of CO from myoglobin is discussed in terms of structurefunction relationships. Evidence is presented that the Soret band shift does not depend primarily on the covalent linkage of the heme iron to the protein by using the mutation H93G(L) in which the proximal histidine 93 is replaced by glycine and an exogenous ligand L, which coordinates the heme iron but is not covalently bound to the globin. While CO rebinding kinetics depend strongly on the nature of the exogenous ligand L in H93G(L), the magnitude and time evolution of the Soret band shift in a viscous buffer on the nanosecond time scale are hardly perturbed in all cases studied. Comparison of the Soret band and charge transfer Band III shifts demonstrates that both have a similar time dependence on the nanosecond to microsecond time scale following flash photolysis in viscous solvents. We conclude that the nonexponential kinetics of protein relaxation probed by transient absorption band position shifts involves primarily distal coordinates prior to ligand escape. This result agrees with earlier measurements of Soret band shifts in distal pocket mutants of myoglobin (1). We suggest that the band shifts are primarily a response to changes in the electrostatic field around the heme (a transient Stark shift) associated with changes in protein structure that occur following ligand photodissociation.
Protein relaxation in the photodissociation of myoglobin−CO complexes
Photochemical & Photobiological Sciences, 2003
Laser-induced optoacoustic spectroscopy has been applied to the study of the photodissociation of myoglobin-CO complexes. Time-resolved optoacoustic signals have been measured from aqueous solutions of horse myoglobin-CO complex (hMbCO) at pH 3.5 and 8, and of sperm whale myoglobin-CO complex (swMbCO) at pH 8, in the temperature range 273-300 K. The signal of hMbCO at pH 8 exhibits three components. The first, which is faster than 20 ns and is associated with a reaction enthalpy of 61 kJ mol Ϫ1 , corresponds to Fe-CO bond breakage. The second component has a decay time of 80 ns at 293 K and is associated with an exothermic protein relaxation (Ϫ13 kJ mol Ϫ1) and a volume change of Ϫ3 ml mol Ϫ1. The relaxation, which involves a state where the photodissociated CO is still in a protein docking site, is thermally activated, with an activation enthalpy of 51 kJ mol Ϫ1. The third component has a decay time of 800 ns at 293 K and an activation enthalpy of 39 kJ mol Ϫ1 , and is associated with an endothermic process (26 kJ mol Ϫ1) and an expansion of 19 ml mol Ϫ1. This process is ascribed to the migration of the photodissociated CO to the bulk solvent. At acidic pH, the latter process becomes faster (230 ns) and the volume change decreases. These features are correlated with the presence of an open form of the protein. swMbCO exhibits two components only, due to the overlap of the two fastest processes. The first involves a reaction enthalpy of 49 kJ mol Ϫ1 and a volume contraction of Ϫ4.9 ml mol Ϫ1. The second component (900 ns at 293 K, activation enthalpy 45 kJ mol Ϫ1) is associated with a reaction enthalpy of 38 kJ mol Ϫ1 and a volume expansion of 15.3 ml mol Ϫ1. These experimental findings have been interpreted by means of a new model, which also takes into account both laser flash photolysis results and structural information. The model is based on a two-dimensional scheme which describes both protein relaxation and the CO pathway following photodissociation.
Time-resolved studies of carbonmonoxy myoglobin photolysis: protein relaxation and ligand trajectory
Quantum Electronics International Conference, 1998
We are conducting nanosecond time-resolved crystallographic studies at the ID9 beamline of the ESRF on two heme proteins: sperm whale myoglobin (Mb) and dimeric hemoglobin (HbI) from the clam Scapharca inaequivalvis. The goals of these studies are to understand the evolution of the photo-induced structural changes and their propagation from the active site through the protein and to determine the
Protein relaxation without a geminate phase in nanosecond photodissociated CO carp hemoglobin
Chemical Physics Letters, 1999
Transient heme-protein interactions upon passing from ligated to deligated carp hemoglobin were observed through time-resolved optical spectra following nanosecond CO photodissociation. The spectral evolution of the heme, in the nanosecond and microsecond time ranges, shows a protein conformational relaxation and the absence of a geminate CO recombination in carp hemoglobin. The comparison of the phenomena in carp and human hemoglobin implies that the physical basis of the geminate rebinding in human hemoglobin should involve an out-of-equilibrium protein conformation, close to a dissipative structure defined by the thermodynamics of Prigogine. q 1999 Elsevier Science B.V. All rights reserved.
The Photoexcited Triplet State as a Probe of Chromophore-Protein Interaction in Myoglobin
Biophysical Journal, 1998
The photoexcited metastable triplet state of Mg 2ϩ -mesoporphyrin IX (MgMPIX) or Mg 2ϩ -protoporphyrin IX (MgPPIX) located in the heme pocket of horse myoglobin (Mb) was investigated by optical and electron paramagnetic resonance (EPR) spectroscopy, and its properties were compared with the model complexes, MgMPIX, MgPPIX, and Mg 2ϩ etioporphyrin I (MgETIOI), in noncoordinating and coordinating organic glasses. Zero-field splitting parameters, line shape, and Jahn-Teller distortion in the temperature range of 3.8 -110 K are discussed in terms of porphyrin-protein interactions. The triplet line shapes for MgMPIXMb and MgPPIXMb show no temperature-dependent spectral line shape changes suggestive of Jahn-Teller dynamics, and it is concluded that the energy splitting is Ͼ Ͼ150 cm Ϫ1 , suggesting symmetry breaking from the anisotropy of internal electric fields of the protein, and consistent with previous predictions (Geissinger et al. 1995. J. Phys. Chem. 99:16527-16529). Both MgMPIXMb and MgPPIXMb demonstrate electron spin polarization at low temperature, and from the polarization pattern it can be concluded that intersystem crossing occurs predominantly into in-plane spin sublevels of the triplet state. The splitting in the Q 0,0 absorption band and the temperature dependence and splitting of the photoexcited triplet state of myoglobin in which the iron was replaced by Mg 2ϩ are interpreted in terms of effects produced by electric field asymmetry in the heme pocket.
Different relaxations in myoglobin after photolysis
Proceedings of the National Academy of Sciences, 2004
To clarify the interplay of kinetic hole-burning (KHB), structural relaxation, and ligand migration in myoglobin (Mb), we measured time-resolved absorption spectra in the Soret region after photolysis of carbon monoxide Mb (MbCO) in the temperature interval 120 -260 K and in the time window 350 ns to 200 ms. The spectral contributions of both photolyzed (Mb*) and liganded Mb (MbCO) have been analyzed by taking into account homogeneous bandwidth, coupling to vibrational modes, and static conformational heterogeneity. We succeeded in separating the ''time-dependent'' spectral changes, and this work provides possibilities to identify the events in the process of ligand rebinding. KHB is dominant at T <190 K in both the Mb* and the MbCO components. For MbCO, conformational substates interconversion at higher temperatures tends to average out the KHB effect. At 230 -260 K, whereas almost no shift is observed in the MbCO spectrum, a shift of the order of Ϸ80 cm ؊1 is observed in Mb*. We attribute this shift to protein relaxation coupled to ligand migration. The time dependence of the Mb* spectral shift is interpreted with a model that enables us to calculate the highly nonexponential relaxation kinetics. Fits of stretched exponentials to this kinetics yield Kohlrausch parameter values of 0.25, confirming the analogy between proteins and glasses.
Structural and dynamic properties of the heme pocket in myoglobin probed by optical spectroscopy
Biopolymers, 1988
We report the optical absorption spectra of sperm whale deoxy-, oxy-, and carbonmonoxymyoglobin in the temperature range 300-20 K and in &5' % glycerol or ethylene glycol-water mixtures. By lowering the temperature, all bands exhibit half-width narrowing and peak frequency shift; moreover, the near-ir bands of deoxymyoglobin show a marked increase of the integrated intensities. Opposed to what has already been reported for human hemoglobin, the temperature dependence of the first moment of the investigated bands does not follow the behavior predicted by the harmonic Franck-Condon approximation and is sizably affected by the solvent composition; this solvent effect is larger in liganded than in nonligmded myoglobin. However, for all the observed bands the behavior of the second moment can be quite well rationalized in terms of the harmonic Ranck-Condon approximation and is not dependent on solvent composition. On the basis of these data we put forward some suggestions concerning the structural and dynamic properties of the heme pocket in myoglobin and their dependence upon solvent composition. We also discuss the different behaviok of myoglobin and hemoglobin in terms of the different heme pocket structures and deformabilities of the two proteins.
Fourier transform infrared (FTIR) spectroscopy in the CO stretch bands combined with temperature derivative spectroscopy (TDS) was used to characterize intermediate states obtained by photolysis of two sperm whale mutant myoglobins, YQR (L29(B10)Y, H64(E7)Q, T67(E10)R) and YQRF (with an additional I107(G8)F replacement). Both mutants assume two different boundstate conformations, A 0 and A 3 , which can be distinguished by their different CO bands near 1965 and 1933 cm ؊1 . They most likely originate from different conformations of the Gln-64 side chain. Within each A substate, a number of photoproduct states have been characterized on the basis of the temperature dependence of recombination in TDS experiments. Different locations and orientations of the ligand within the protein can be distinguished by the infrared spectra of the photolyzed CO. Recombination from the primary docking site, B, near the heme dominates below 50 K. Above 60 K, ligand rebinding occurs predominantly from a secondary docking site, C, in which the CO is trapped in the Xe4 cavity on the distal side, as shown by crystallography of photolyzed YQR and L29W myoglobin CO. Another kinetic state (C) has been identified from which rebinding occurs around 130 K. Moreover, a population appearing above the solvent glass transition at ϳ180 K (D state) is assigned to rebinding from the Xe1 cavity, as suggested by the photoproduct structure of the L29W sperm whale myoglobin mutant. For both the YQR and YQRF mutants, rebinding from the B sites near the heme differs for the two A substates, supporting the view that the return of the ligand from the C, C, and D states is not governed by the recombination barrier at the heme iron but rather by migration to the active site. Comparison of YQR and YQRF shows that access to the Xe4 site (C) is severely restricted by introduction of the bulky Phe side chain at position 107.
Comparison of the magnetic properties of deoxy- and photodissociated myoglobin
Proceedings of the National Academy of Sciences, 1984
The magnetic susceptibility of photodissociated carbon monoxy myoglobin has been measured over the temperature range from 1.7 to 25 K at 10 and 50 kG with a superconducting susceptometer. The spin and the crystal field parameters of the iron ion were extracted by a spin Hamiltonian approach. Under equivalent conditions the magnetic susceptibility of deoxy myoglobin was measured. In both experiments the CO-bound protein was used as a diamagnetic reference. Above about 5 K the metastable photolysed state and the equilibrium deoxy form of myoglobin are magnetically indistinguishable and can be fitted with S = 2 and g = 2. The transition from spin 0 to spin 2 and the conformational changes known to accompany the electronic change thus also occur after photolysis at low temperature. At temperatures below 5 K, differences become apparent, indicating a somewhat smaller zero-field splitting in the photoproduct as compared to the ligand-free state at equilibrium. In qualitative agreement with observations made by other techniques, the data imply that even at 1.7 K substantial structural relaxation occurs in the heme region of myoglobin after photodissociation. The results are important for the interpretation of the ligand binding kinetics after flash photolysis at low temperature and contribute to the understanding of the relationship between electronic structure and function in heme proteins.
Ligand binding to heme proteins: II. Transitions in the heme pocket of myoglobin
Biophysical Journal, 1993
Phenomena occurring in the heme pocket after photolysis of carbonmonoxymyoglobin (MbCO) below about 100 K are investigated using temperature-derivative spectroscopy of the infrared absorption bands of CO. MbCO exists in three conformations (A substates) that are distinguished by the stretch bands of the bound CO. We establish connections among the A substates and the substates of the photoproduct (B substates) using Fourier-transform infrared spectroscopy together with kinetic experiments on MbCO solution samples at different pH and on orthorhombic crystals. There is no one-to-one mapping between the A and B substates; in some cases, more than one B substate corresponds to a particular A substate. Rebinding is not simply a reversal of dissociation; transitions between B substates occur before rebinding. We measure the nonequilibrium populations of the B substates after photolysis below 25 K and determine the kinetics of B substate transitions leading to equilibrium. Transitions between B substates occur even at 4 K, whereas those between A substates have only been observed above about 160 K. The transitions between the B substates are nonexponential in time, providing evidence for a distribution of substates. The temperature dependence of the B substate transitions implies that they occur mainly by quantum-mechanical tunneling below 10 K. Taken together, the observations suggest that the transitions between the B substates within the same A substate reflect motions of the CO in the heme pocket and not conformational changes. Geminate rebinding of CO to Mb, monitored in the Soret band, depends on pH. Observation of geminate rebinding to the A substates in the infrared indicates that the pH dependence results from a population shift among the substates and not from a change of the rebinding to an individual A substate. INTRODUCTION1 Myoglobin is a globular protein consisting of 153 amino acids and a heme (Fe-protoporphyrin IX) as the prosthetic group. Textbooks state that the function of Mb is the reversible binding of small ligands such as dioxygen (02) and carbon monoxide (CO) at the heme iron (Stryer, 1988). One could expect such a binding process to be simple. It was indeed originally described as a one-step process (Antonini and Brunori, 1971). Flash photolysis experiments performed over wide ranges in time and temperature imply, however, that the binding process is far from simple (Austin et al., 1975). Four phenomena, in particular, produce complexity: 1. multiple wells along the reaction coordinate, 2. confor-mational substates, 3. protein relaxation after photodissociation, and 4. thermal fluctuations. In the following, we briefly describe how these phenomena affect the ligand binding to myoglobin. Multiple wells along the reaction coordinate In the simplest model, CO in the solvent binds to the heme iron in one step. Flash photolysis data, however, show evidence for multiple processes. A model that describes the salient features of the kinetic data uses three wells (states) along the reaction coordinate, A i± B S.