Time-resolved optical spectroscopy and structural dynamics following photodissociation of carbonmonoxyhemoglobin

Murray, Lionel P.; Hofrichter, James; Henry, Eric R.; Eaton, William A.

doi:10.1016/0301-4622(88)87025-x

Cited by 84 publications

(70 citation statements)

References 83 publications

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“…One percent recombination is what would be expected if the geminate rebinding were an exponential process with a yield of -40% and a rebinding rate coefficient of =50 ns, the values reported for aqueous HbCO by Hofrichter and coworkers (12,34). The picosecond IR geminate recombination measurements connect reasonably well with the nanosecond studies of Hofrichter and coworkers (12,34) so that the kinetics for rebinding are not far from exponential over 5 decades in time, from 1 ps to 100 ns. Since the CO is now known to be near the heme for much of this time, the barrier to recombination must form rapidly.…”

Section: Resultssupporting

confidence: 72%

Direct observations of ligand dynamics in hemoglobin by subpicosecond infrared spectroscopy.

Anfinrud

Han²,

Hochstrasser³

1989

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

188

146

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The photodissociation of CO from HbCO at ambient temperature is studied by means of a femtosecond IR technique. The bleaching of the FeCO absorption and the appearance of a new IR absorption near that of free CO are both observed at 300 fs after optical excitation. The bleach does not recover on the time scale of a few picoseconds but does recover by ;7--4% within 1 ns, which suggests that a barrier to recombination is formed within a few picoseconds. The CO spectrum does not change significantly between 300 fs and 1 ns, suggesting that the CO quickly finds some locations in the heme pocket that are not more than a few angstroms from the iron. The de-ligated CO appears in its ground vibrational level. There is evidence that 85 ± 10% of this CO remains in the heme pocket at 1 ns; it probably resides there for 50 ns. The flow of excess vibrational energy from the heme to the solvent was directly observed in the IR experiments. The heme cools within 1-2 ps while thermal disruption of the surrounding solvent structure requires ""30 ps.Ultrafast spectroscopic methods have contributed much to understanding the photophysics and structural dynamics that occur after the photodissociation of oxy-and carboxyhemoglobin (1-3). The motion of the iron out of the heme plane has been discussed in some detail already, and evidence from optical spectroscopy (3) and Raman scattering (4, 5) suggests that the heme is partially or fully domed within 350 fs and perhaps as rapidly as 50 fs. This time frame for motion of the iron and its associated proximal histidine is also found in theoretical simulations of the heme relaxation (6). On the other hand, very little is yet known about the properties of the ligand generated by photolysis in ambient temperature solution.In the normal operation of hemoglobin, 02 diffuses into the protein from the solvent and bonds to the iron. Questions regarding ligand diffusion into and escape from the heme pocket as well as binding within the protein therefore have central importance to the function of hemoglobin. The hemoglobin structure, as determined from x-ray analysis (7), has no channel large enough to facilitate passage of a ligand between the solvent and heme. Hence, diffusion to the heme requires significant globin motions (7-9). Although transient optical methods are sensitive, the electronic spectrum of hemes cannot respond to changes in the location of a relatively inert diatomic molecule within the protein. Thus, experiments that can observe the ligand directly and monitor any changes in its structure or environment are expected to extend significantly our understanding of hemoprotein function. Such measurements on free CO would enable us to address some new issues for hemoglobin under ambient aqueous conditions. For example, additional information could be obtained on the rate of generation of CO and on the pathways of photodissociation (10, 11), the efficiency of subnanosecond geminate recombination of CO to iron could be brought into relation with results from nanosecond (12) and m...

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Section: Resultssupporting

confidence: 72%

Direct observations of ligand dynamics in hemoglobin by subpicosecond infrared spectroscopy.

Anfinrud

Han²,

Hochstrasser³

1989

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

188

146

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“…3 illustrates the wealth of information contained in the grating components of the MbCO response at 20°C for the full range of time scales studied in this experiment. The Im part is simple and shows the same trends as previous transient absorption studies (26), i.e., essentially a constant offset that decays with CO recombination. In contrast, the Re part of the signal is dramatically different.…”

Section: Resultssupporting

confidence: 55%

Diffractive optics-based heterodyne-detected four-wave mixing signals of protein motion: From “protein quakes” to ligand escape for myoglobin

Dadusc

Ogilvie

Schulenberg

et al. 2001

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

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Ligand transport through myoglobin (Mb) has been observed by using optically heterodyne-detected transient grating spectroscopy. Experimental implementation using diffractive optics has provided unprecedented sensitivity for the study of protein motions by enabling the passive phase locking of the four beams that constitute the experiment, and an unambiguous separation of the Real and Imaginary parts of the signal. Ligand photodissociation of carboxymyoglobin (MbCO) induces a sequence of events involving the relaxation of the protein structure to accommodate ligand escape. These motions show up in the Real part of the signal. The ligand (CO) transport process involves an initial, small amplitude, change in volume, reflecting the transit time of the ligand through the protein, followed by a significantly larger volume change with ligand escape to the surrounding water. The latter process is well described by a single exponential process of 725 ؎ 15 ns at room temperature. The overall dynamics provide a distinctive signature that can be understood in the context of segmental protein fluctuations that aid ligand escape via a few specific cavities, and they suggest the existence of discrete escape pathways.

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“…It should be noted that the rebinding time scale is on the order of 10 Ϫ1 s, indicating that rebinding of CO molecules from the solvent to the deoxy proteins is being observed; in fact, the time scale of geminate rebinding is, at room temperature, on the order of 50 ns, while tertiary and quaternary conformational relaxations occur in time scales ranging from 1 to 200 s (40).…”

Section: Resultsmentioning

confidence: 99%

Modification of α-Chain or β-Chain Heme Pocket Polarity by Val(E11) → Thr Substitution Has Different Effects on the Steric, Dynamic, and Functional Properties of Human Recombinant Hemoglobin

Cupane

Leone

Militello

et al. 1997

Journal of Biological Chemistry

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The dynamic and functional properties of mutant deoxyhemoglobins in which either the ␤-globin Val 67 (E11) or the ␣-globin Val 62 (E11) is replaced by threonine have been investigated through the thermal evolution of the Soret absorption band in the temperature range 300 to 20 K and through the kinetics of CO rebinding after flash photolysis at room temperature. The conformational properties of the modified ␣ chain and ␤ chain distal heme pockets were also studied through x-ray crystallography and molecular modeling. The data obtained with the various techniques consistently indicate that the polar isosteric mutation in the distal side of the ␣ chain heme pocket has a larger effect on the investigated properties than the analogous mutation on the ␤ chain. We attribute the observed differences to the presence of a water molecule in the distal heme pocket of the modified ␣ chains, interacting with the hydroxyl of the threonine side chain. This is indicated by molecular modeling which showed that the water molecule present in the ␣ chain distal heme pocket can bridge by H bonding between Thr 62 (E11) and His 58 (E7) without introducing any unfavorable steric interactions. Consistent with the dynamic and functional data, the presence of a water molecule in the distal heme pocket of the modified ␤ chains is not observed by x-ray crystallography.

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Time-resolved optical spectroscopy and structural dynamics following photodissociation of carbonmonoxyhemoglobin

Cited by 84 publications

References 83 publications

Direct observations of ligand dynamics in hemoglobin by subpicosecond infrared spectroscopy.

Direct observations of ligand dynamics in hemoglobin by subpicosecond infrared spectroscopy.

Diffractive optics-based heterodyne-detected four-wave mixing signals of protein motion: From “protein quakes” to ligand escape for myoglobin

Modification of α-Chain or β-Chain Heme Pocket Polarity by Val(E11) → Thr Substitution Has Different Effects on the Steric, Dynamic, and Functional Properties of Human Recombinant Hemoglobin

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