Metastable Species of Hemoglobin: Room Temperature Transients and Cryogenically Trapped Intermediates

Ondrias, M. R.; Friedman, Joel M.; Rousseau, Denis L.

doi:10.1126/science.6836305

Cited by 32 publications

(21 citation statements)

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“…By this method changes in the properties of photodissociated heme proteins were detected many years ago with optical absorption techniques (4)(5)(6), and studies of the metastable species have been since extended with many other techniques. The relationship between the structure, as inferred from the resonance Raman spectra, of the lowtemperature photoproduct and the transient photoproduct generated at 300 K was discussed recently, and in carbon monoxide hemoglobin, HbCO, it was observed that the differences in the Raman spectra between the stabilized photoproduct and the deoxy preparation at 80 K were the same as those obtained transiently at room temperature with 10-nsec pulses (7). These findings led us to investigate the low-temperature (1.6-100 K) photodissociation of carbon monoxide myoglobin, MbCO, with resonance Raman scattering to determine the properties of the unrelaxed photoproduct and its relationship to the room-temperature transient photoproduct.…”

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confidence: 90%

Metastable photoproducts from carbon monoxide myoglobin.

Rousseau¹,

Argade²

1986

Proc. Natl. Acad. Sci. U.S.A.

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The photoproduct of carbon monoxide myoglobin generated at 4 K and lower has a resonance Raman spectrum characteristic of a high-spin heme but in which the high-frequency core size-sensitive lines are at lower frequency than those in the deoxy preparation. Such differences are not detected in the photoproduct generated at higher temperatures (50 K) or in that generated at room temperature with 10-nsec pulses. The data indicate that at the low temperature (4 K), the heme in the photoproduct is not fully relaxed, and from the data we conclude that the photoproduct has an expanded porphyrin core. We infer that the core size exceeds that in deoxymyoglobin because the rigid protein prevents the highspin iron atom from moving to its full out-of-plane displacement at the very low temperatures.To gain an understanding of the basic mechanisms of ligand association in heme proteins, the elementary steps in the ligand binding and release process must be uncovered. An approach to the elucidation of these steps is to photodissociate a bound ligand and follow the relaxation of the deoxy heme that has been generated. At room temperature the evolution of this photoproduct occurs over a wide temporal scale ranging from the subpicosecond absorption of the photon (1) to structural relaxation of the protein residues, which may occur on a time scale of 10s of microseconds (2,3). The specific relaxation pathway of the photoproduct is very complex and depends on the protein as well as the ligand.An alternate approach in the identification of the intermediate structures in the photoproduct relaxation is through the use of cryogenic temperatures, where metastable states may be isolated (4). By this method changes in the properties of photodissociated heme proteins were detected many years ago with optical absorption techniques (4-6), and studies of the metastable species have been since extended with many other techniques. The relationship between the structure, as inferred from the resonance Raman spectra, of the lowtemperature photoproduct and the transient photoproduct generated at 300 K was discussed recently, and in carbon monoxide hemoglobin, HbCO, it was observed that the differences in the Raman spectra between the stabilized photoproduct and the deoxy preparation at 80 K were the same as those obtained transiently at room temperature with 10-nsec pulses (7). These findings led us to investigate the low-temperature (1.6-100 K) photodissociation of carbon monoxide myoglobin, MbCO, with resonance Raman scattering to determine the properties of the unrelaxed photoproduct and its relationship to the room-temperature transient photoproduct. The experiments were carried out by freezing CO-bound Mb to low-temperature and then directing a laser beam on the sample. The laser photodissociates the CO, resulting in a deoxy heme trapped in the frozen protein matrix. At the lowest temperatures we find significant differences in the resonance Raman spectra ofthe photoproduct and the equilibrium deoxy species. We interpret these results...

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confidence: 90%

Metastable photoproducts from carbon monoxide myoglobin.

Rousseau¹,

Argade²

1986

Proc. Natl. Acad. Sci. U.S.A.

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“…[1][2][3]13 This band exhibits timedependent spectral shifts after MbCO photolysis, and this has been taken to be an indicator of protein conformational changes in response to bond breaking and recombination. [4][5][6]13,14 The absorption and Stark spectra of deoxyMb are presented in Figure 1, 15 and the values of parameters extracted from an analysis of the Stark spectra are summarized in Table 1. The Stark spectrum shows clear features for bands I and III, as well as for the Q and B bands; the broad negative base line offset below about 16 000 cm -1 may be associated with the Stark effect on band II.…”

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Stark-Effect Spectroscopy of the Heme Charge-Transfer Bands of Deoxymyoglobin

et al. 1999

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We report the Stark spectra of the Soret, Q, and charge-transfer bands of deoxymyoglobin. The data show that band III has charge-transfer character but that the magnitude of the charge displacement is significantly smaller than expected or than what is observed for band I or even the Soret or Q bands. The data also show that symmetry breaking of iron-protoporphyrin IX in myoglobin produces a larger difference dipole moment in the Soret transition when compared to the dimethyl ester of protoporphyrin IX. The results lend further credence to the hypothesis that the electrostatic environment of the heme pocket in myoglobin is important in modifying the properties of the heme transitions, and they provide a basis for quantitative analysis of the static and time-dependent band shifts observed in response to environmental electrostatic perturbations and ligand binding dynamics.The electronic absorption of hemes has been investigated for more than 100 years. Ligand and substituent effects have been systematically assigned, and the focus in recent years has shifted toward static and dynamic band shifts that characterize biological function. [1][2][3][4][5][6] The origins of these band shifts can be roughly divided into specific chemical effects, e.g., changes in ligand or heme substituents, and more global environmental shifts, typically originating in changes in the electrostatic field around the chromophore. The latter effects are Stark shifts due to the interaction between the change in dipole moment (∆µ) and polarizability (∆R) of the chromophore with the electrostatic field of the ordered environment. To understand these electrostatic shifts quantitatively, it is necessary to characterize ∆µ and ∆R for each type of transition, and this can be achieved by measuring the effect of an externally applied electric field, the Stark-effect spectrum, for each transition. 7,8 The absorption spectra of heme and other metalloporphyrins are dominated by the B (Soret) and Q absorption bands that arise from configuration interaction (CI) of four orbitals, the nearly degenerate pair a 1u , a 2u HOMO and the doubly degenerate e 1g LUMO. 9,10 The result of CI is an intense Soret transition (λ max ≈ 435 nm in deoxymyoglobin, Mb) and a weak Q band (λ max ≈ 556 nm) that gains most of its intensity through vibronic coupling with the Soret band. 11 Although these bands dominate the spectra, there are other less prominent bands which are of interest. These include the four ligand-to-metal charge transfer (CT) and d-d electronic absorption transitions (bands I-IV) which have been assigned in the near-IR region of deoxyhemoglobin based on single-crystal absorption, CD, and MCD spectroscopy. 12 Band III is relatively narrow and easily resolved, and it has been shown that there are interesting correlations between the band position and ligand rebinding kinetics at cryogenic temperature in Mb. [1][2][3]13 This band exhibits timedependent spectral shifts after MbCO photolysis, and this has been taken to be an indicator of protein conformational...

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“…Sci. USA 81 (1984) Friedman, Rousseau, and collaborators (20)(21)(22)(23)(24)(25) (27), it is now feasible to study the magnetic properties of photodissociated heme proteins. Such experiments permit a definite answer to the question of the spin state in Mb*.…”

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Comparison of the magnetic properties of deoxy- and photodissociated myoglobin.

Röder¹,

Berendzen²,

Bowne³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

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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. Heme proteins perform a wide variety of functions, from oxygen storage to detoxification. Since they all share the same prosthetic group, a detailed understanding of the function of one heme protein can provide insight into a broader field. The binding of small ligands such as dioxygen and CO to ferrous Mb and Hb is probably the simplest such biological process and it has been studied with a variety of tools (1-4). Flash photolysis experiments over a wide range of time and temperature have established that the controlling reaction in ligand binding occurs at the heme group and involves the heme iron (5, 6). In the discussion of the crucial association and dissociation step it is generally assumed that the ligandfree (deoxy) form of the ferrous heme is in the high-spin state (S = 2) while the ligand-bound form is in the low-spin state (S = 0). Consequently, association and dissociation of 02 and CO are assumed to be accompanied by a spin change of 2.The assignment of the spin states is based on the pioneering magnetic susceptibility measurements of Pauling and Coryell (7) on Hb and related proteins. MbCO was shown to be diamagnetic by Theorell and Ehrenberg (8). The magnetic properties of deoxy Hb and Mb have been studied by Nakano et al. (9). In addition to verifying the spin state they characterized the electronic fine structure of the iron in these heme proteins.Ligand binding not only modifies the electronic structure of the heme iron but also induces distinct changes in the conformation of the heme and its environment (10). X-ray crystallography shows that in deoxy Mb (11) the iron ion lies out of the mean heme plane by...

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Metastable Species of Hemoglobin: Room Temperature Transients and Cryogenically Trapped Intermediates

Cited by 32 publications

References 21 publications

Metastable photoproducts from carbon monoxide myoglobin.

Metastable photoproducts from carbon monoxide myoglobin.

Stark-Effect Spectroscopy of the Heme Charge-Transfer Bands of Deoxymyoglobin

Comparison of the magnetic properties of deoxy- and photodissociated myoglobin.

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