Structure-dynamics-function relationships in Asian elephant (Elephas maximus) myoglobin. An optical spectroscopy and flash photolysis study on functionally important motions

Cupane, Antonio; Leone, Maurizio; Vitrano, Eugenio; Cordone, Lorenzo; Hiltpold, U.R.; Winterhalter, Kaspar H.; Yu, Weiming; Iorio, Ernesto E. Di

doi:10.1016/s0006-3495(93)81311-0

Cited by 31 publications

(26 citation statements)

References 47 publications

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“…The reference value CO (ϱ) can be measured directly from the spectrum at the longest delay from photolysis, i.e., 20-200 ms depending on the temperature. The temperature dependence of CO (ϱ) measured in the present experiment closely parallels the analogous behavior obtained from static measurements on equilibrium MbCO (35). The reference value deoxy (ϱ) cannot be measured directly from the spectra because at long times the deoxy component becomes vanishingly small.…”

Section: Resultssupporting

confidence: 82%

“…2). Indeed, at 120 K and 350 ns after photolysis virtually no CO molecules had yet rebound to the heme iron, so that N Ϸ 1; moreover, at 432 nm the signal-to-noise ratio is optimal and ⌬A max , measured in static spectra, is almost temperature independent (35).…”

Section: Resultsmentioning

confidence: 99%

“…Values of deoxy (equilibrium) have been taken from ref. 35, after suitable calibration of the different spectrometers used.…”

Section: Resultsmentioning

confidence: 99%

“…The other parameters have been taken from previous works (35,36) and have been kept constant in the fittings. This choice is justified by the fact that spectral variations involve essentially intensity variations, peak shifts, and band broadenings; it is also justified ''a posteriori'' by the excellent quality of the fits.…”

Section: Methodsmentioning

confidence: 99%

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Different relaxations in myoglobin after photolysis

Levantino

Cupane

Zimányi

et al. 2004

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

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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.T he complexity of proteins is reflected in their heterogeneous structure: they exhibit a distribution of conformational substates (CS) (1, 2). As a consequence, all physical parameters that are determined or influenced by the structure are in general also distributed.Ligand rebinding to myoglobin (Mb) after flash photolysis has produced a large amount of information about the nature of protein reactions. However, even this apparently simple reaction is surprisingly complicated, and a full understanding still has not been achieved. If CO is the ligand (state A, carbon monoxide Mb, MbCO), photolysis breaks the bond between the heme iron and CO, the protein goes to a metastable intermediate (Mb*), and CO moves away from the heme. The subsequent rebinding of CO can be followed spectroscopically.Below Ϸ180 K the population of CS is frozen: the protein ensemble is distributed with distributed properties. After photolysis, the ligand cannot escape from the heme pocket; it rebinds to the heme iron from a docking site (state B) in a single step (geminate rebinding). From the x-ray structure of the Mb*ϩCO photoproduct at low temperatures (3-5) the overall structure of the B state is known: the photolyzed CO lies on top of the heme, parallel to it, and at a distance of Ϸ4 Å from the iron. The simple B3A reaction is not exponential in time: proteins in different CS have different activation enthalpies for rebinding (1). The reaction can be described by a temperature-independent distribution of enthalpies, g(H), up to Ϸ180 K (6). Absorption measurements on Mb have demonstrated that its spectral lines are inhomogeneously broadened (7-9). This spectral heterogeneity is coupled to the enthalp...

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Section: Resultssupporting

confidence: 82%

Section: Resultsmentioning

confidence: 99%

“…Values of deoxy (equilibrium) have been taken from ref. 35, after suitable calibration of the different spectrometers used.…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Different relaxations in myoglobin after photolysis

Levantino

Cupane

Zimányi

et al. 2004

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

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“…Motions within BR as a function of hydration have been characterized further by quasielastic neutron scattering (11,12). Other experimental and theoretical studies also have provided evidence that biological activity in proteins is correlated with a dynamical transition from a harmonic to a softer, anharmonic atomic fluctuation regime (13)(14)(15)(16)(17)(18), suggesting that flexibility is required for function (17). Before the present work, experiments had only explored this transition in the global behavior of a few proteins.…”

mentioning

confidence: 92%

Dynamics of different functional parts of bacteriorhodopsin: H- ² H labeling and neutron scattering

Réat

Patzelt

Ferrand

et al. 1998

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

152

121

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We show that dynamics of specific amino acids within a protein can be characterized by neutron spectroscopy and hydrogen-deuterium labeling, and we present data on the motions of a selected set of groups within bacteriorhodopsin (BR), the retinal-based proton pump in the purple membrane of halophilic Archaea. Elastic incoherent neutron scattering experiments allow the definition of motions in the nano-to picosecond time scale and have revealed a dynamical transition from a harmonic to a softer, anharmonic atomic f luctuation regime in the global behavior of proteins. Biological activity in proteins is correlated with this transition, suggesting that f lexibility is required for function. Elastic incoherent neutron scattering is dominated by H atom scattering, and to study the dynamics of a selected part of BR, fully deuterated purple membrane with BR containing Hretinal, H-tryptophan, and H-methionine was prepared biosynthetically in Halobacterium salinarum. These amino acids cluster in the functional center of the protein. In contrast to the protein globally, the thermal motions of the labeled atoms were found to be shielded from solvent melting effects at 260 K. Above this temperature, the labeled groups appear as more rigid than the rest of the protein, with a significantly smaller mean square amplitude of motion. These experimental results quantify the dynamical heterogeneity of BR (which meets the functional requirements of global f lexibility), on the one hand, to allow large conformational changes in the molecule and of a more rigid region in the protein, on the other, to control stereo-specific selection of retinal conformations.

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Conformational substates of the Fe2+-His F8 linkage in deoxymyoglobin and hemoglobin probed in parallel by the Raman band of the Fe-His stretching vibration and the near-infrared absorption band III

Gilch

Schweitzer‐Stenner

Dreybrodt

et al. 1996

Int. J. Quantum Chem.

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mWe measured the Y~~-~,~ Raman band of horse heart deoxymyoglobin and human deoxyhemoglobin as a function of temperature between 10 and 300 K. A self-consistent spectral analysis of the deoxymyoglobin Raman band reveals that it is underlied by three different sublines with frequencies at f l , = 209 cm-', i12 = 217 cm-', and R, = 225 cm-' and an identical half-width of 13 cm-l. All these parameters were found to be independent of temperature. These sublines were attributed to different conformational substates of the Fe2+-His F8 linkage, which comprise different out-off-plane displacements of the heme iron and tilt angles of the Fe2+-N,(His F8) bond. The intensity ratio 1J12 exhibits a van't Hoff behavior between 150 and 300 K, bends over in a region between 150 and 80 K, and remains constant at lower temperature. In contrast, I2/I1 shows a maximum at 170 K and approaches a constant value at 80 K. These data can be fitted by a modified van't Hoff expression, which accounts for the freezing into nonequilibrium distributions of substrates in a temperature interval AT around a distinct temperature TI and also for the linear temperature dependence of the protein's specific heat. The fits to the above intensity ratios yield a freezing temperature of TI = 117 K and a transition region of AT = 55 K. The v~~-~~~ Raman band of human deoxyhemoglobin was decomposed into seven sublines with frequencies at 195,202,211,218,226,234, and 240 cm-', with half-widths of 12 cm-'. While the low-frequency sublines are strong at GILCH ET AL.300 K, the high-frequency sublines dominate the band at cryogenic temperatures. In comparison, we also investigated the temperature dependence of the near-infrared band I11 at 760 nm. Band I11 of deoxymyoglobin can be decomposed into two subbands which are 165 cm-l apart. The ratio of their absorption cross sections shows a temperature dependence which parallels that of the ratio I 3 / ( I 2 + I,) of the corresponding Raman sublines. Band I11 of deoxyhemoglobin was decomposed into three subbands, the absorption cross sections of which also depend on temperature , similar to what has been observed for the v~~-~~~ subbands. These observations provide strong evidence that the frequency positions of the subbands of band I11 and the v~~-~~~ sublines are governed by the same coordinates. For both proteins investigated, the frequency positions and the half-widths of the band I11 subbands depend significantly on temperature. This is rationalized in terms of an earlier proposed model (Cupane et al., Eur. Biophys. J. 21; 385 1993) which assumes that the corresponding electronic transition is coupled to a bath of low-frequency modes. Our data indicate that these modes are harmonic below 130 K but become anharmonic above this temperature. This onset of anharmonic motions is interpreted as resulting from conformational transitions within the protein which affect the prostethic group via heme-protein coupling.

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Structure-dynamics-function relationships in Asian elephant (Elephas maximus) myoglobin. An optical spectroscopy and flash photolysis study on functionally important motions

Cited by 31 publications

References 47 publications

Different relaxations in myoglobin after photolysis

Different relaxations in myoglobin after photolysis

Dynamics of different functional parts of bacteriorhodopsin: H- ² H labeling and neutron scattering

Conformational substates of the Fe2+-His F8 linkage in deoxymyoglobin and hemoglobin probed in parallel by the Raman band of the Fe-His stretching vibration and the near-infrared absorption band III

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Structure-dynamics-function relationships in Asian elephant (Elephas maximus) myoglobin. An optical spectroscopy and flash photolysis study on functionally important motions

Cited by 31 publications

References 47 publications

Different relaxations in myoglobin after photolysis

Different relaxations in myoglobin after photolysis

Dynamics of different functional parts of bacteriorhodopsin: H- 2 H labeling and neutron scattering

Conformational substates of the Fe2+-His F8 linkage in deoxymyoglobin and hemoglobin probed in parallel by the Raman band of the Fe-His stretching vibration and the near-infrared absorption band III

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Dynamics of different functional parts of bacteriorhodopsin: H- ² H labeling and neutron scattering