Biomedical subjects

B R Cowen

Publications and source records attributed to B R Cowen.

9 recordsLinked to original sources

PubMed1994

Femtosecond coherent transient infrared spectroscopy of reaction centers from Rhodobacter sphaeroides.

Protein and cofactor vibrational dynamics associated with photoexcitation and charge separation in the photosynthetic reaction center were investigated with femto-second (300-400 fs) time-resolved infrared (1560-1960 cm-1) spectroscopy. The experiments are in the coherent transient limit where the quantum uncertainty principle governs the evolution of the protein vibrational changes. No significant protein relaxation accompanies charge separation, although the electric field resulting from charge separation modifies the polypeptide carbonyl spectra. The potential energy surfaces of the "special pair" P and the photoexcited singlet state P* and environmental perturbations on them are similar as judged from coherence transfer measurements. The vibrational dephasing time of P* modes in this region is 600 fs. A subpicosecond transient at 1665 cm-1 was found to have the kinetics expected for a sequential electron transfer process. Kinetic signatures of all other transient intermediates, P, P*, and P+, participating in the primary steps of photosynthesis were identified in the difference infrared spectra.

Kinetics↗

PubMed1993

Picosecond infrared studies of the dynamics of the photosynthetic reaction center.

The changes in the vibrational transitions of the protein and redox cofactors of the photosynthetic reaction center were examined by picosecond infrared spectroscopy. The spectra in the vibrational mid-infrared region (1800-1550 cm-1) of hydrated and partially dehydrated reaction centers were investigated from 50 ps to 4 ns after photoinitiation of the electron transfer. Features in the infrared difference spectra were identified with both protein and redox cofactor vibrational modes and correlated with electron transfer events whose kinetics were measured in the infrared and visible regions. The observed protein response is confined to a few amide I transitions (1644 cm-1, 1661 cm-1, 1665 cm-1) and carboxylic residues (1727 cm-1). About 85% of the observed signal corresponded to alterations in the cofactor-associated ester and keto carbonyls. The amide I and carboxylic transitions appeared prior to 50 ps, suggesting that the primary electron transfer event is coupled with a specific piece of the protein backbone and to glutamic or aspartic residues nearby the special pair. Infrared absorption changes accompanying bacteriochlorophyll-dimer cation formation dominated the signal at all times investigated. Infrared spectral changes observed in hydrated and partially dehydrated reaction centers were distinctly different; a band at 1665 cm-1 with a spectral width of 6 cm-1 in the hydrated protein, corresponding to a protein amide I bleach, was not present in the dehydrated film. These differences are discussed in terms of the markedly different electron transfer kinetics observed in the presence of water.

Electron Transport↗

PubMed1992

Picosecond dynamics of bacteriorhodopsin, probed by time-resolved infrared spectroscopy.

The photoinduced reaction cycle of bacteriorhodopsin (BR) has been studied by means of a recently developed picosecond infrared spectroscopic method at ambient temperature. BR - K difference spectra between 1560 and 1700 cm-1 have been recorded at delay times from 100 ps to 14 ns. The spectrum remains unchanged during this period. The negative difference OD band at 1660 cm-1 indicates the peptide backbone responds within 50 ps. A survey in the region of carboxylic side chain absorption around 1740 cm-1 reveals that perturbations of those groups, present in low-temperature FTIR spectra, are not observable within 10 ns, suggesting a slow conformational change.

Bacteriorhodopsins↗

PubMed1991

Ligand binding to heme proteins: connection between dynamics and function.

Ligand binding to heme proteins is studied by using flash photolysis over wide ranges in time (100 ns-1 ks) and temperature (10-320 K). Below about 200 K in 75% glycerol/water solvent, ligand rebinding occurs from the heme pocket and is nonexponential in time. The kinetics is explained by a distribution, g(H), of the enthalpic barrier of height H between the pocket and the bound state. Above 170 K rebinding slows markedly. Previously we interpreted the slowing as a "matrix process" resulting from the ligand entering the protein matrix before rebinding. Experiments on band III, an inhomogeneously broadened charge-transfer band near 760 nm (approximately 13,000 cm-1) in the photolyzed state (Mb*) of (carbonmonoxy)myoglobin (MbCO), force us to reinterpret the data. Kinetic hole-burning measurements on band III in Mb* establish a relation between the position of a homogeneous component of band III and the barrier H. Since band III is red-shifted by 116 cm-1 in Mb* compared with Mb, the relation implies that the barrier in relaxed Mb is 12 kJ/mol higher than in Mb*. The slowing of the rebinding kinetics above 170 K hence is caused by the relaxation Mb*----Mb, as suggested by Agmon and Hopfield [(1983) J. Chem. Phys. 79, 2042-2053]. This conclusion is supported by a fit to the rebinding data between 160 and 290 K which indicates that the entire distribution g(H) shifts. Above about 200 K, equilibrium fluctuations among conformational substates open pathways for the ligands through the protein matrix and also narrow the rate distribution. The protein relaxations and fluctuations are nonexponential in time and non-Arrhenius in temperature, suggesting a collective nature for these protein motions. The relaxation Mb*----Mb is essentially independent of the solvent viscosity, implying that this motion involves internal parts of the protein. The protein fluctuations responsible for the opening of the pathways, however, depend strongly on the solvent viscosity, suggesting that a large part of the protein participates. While the detailed studies concern MbCO, similar data have been obtained for MbO2 and CO binding to the beta chains of human hemoglobin and hemoglobin Zürich. The results show that protein dynamics is essential for protein function and that the association coefficient for binding from the solvent at physiological temperatures in all these heme proteins is governed by the barrier at the heme.

Binding Sites↗

PubMed1991

Carboxy Mb at pH 3. Time-resolved resonance Raman study at cryogenic temperatures.

Cryogenic samples of MbCO at pH3 are studied using nanosecond and picosecond time-resolved resonance Raman spectroscopy. It is observed that under excitation conditions sufficient to completely photodissociate MbCO at pH7, the pH3 sample at 10 ns remains substantially unphotolyzed even at 15 K. The similarity in the optical and resonance Raman spectra of MbCO at pH3 with that of pH7 indicates that at pH3 the iron remains six-coordinate and low-spin. The Fe-CO stretch frequency is consistent with a more upright CO orientation. The absence of the v(Fe-His) band in the 30 ps photoproduct Raman spectrum suggests that the Fe-His(F8) bond is broken within 30 ps of photodissociation. Other Raman bands, though, are not consistent with a normal four-coordinate heme for the photoproduct, Mb*. Suggested possible interpretations include a four-coordinate heme highly perturbed by the close lying protonated proximal histidine or a five-coordinate heme with the Fe-His bond significantly weakened. The partial photolysis monitored at 30 ps and 100 K indicates either a significant amount of geminate recombination within 30 ps or low quantum yield or photolysis. The time course for CO recombination is monitored via the Raman spectra from 30 ps to 3 ns at 100 K and 160 K. Of the fraction of protein-ligand pairs that remain photodissociated at 30 ps, 50% recombine by approximately 250 ps at 100 K and 160 K, supporting the flash photolysis rebinding data of Cowen et al. (Cowen, B. R. 1990. Ph. D. thesis. University of Illinois at Urbana-Champaign; Cowen, B. R., D. Braunstein, H. Frauenfelder, P. J. Steinbach, and R. D. Young. 1989. Biophys. J. 55:55a. [Abstr.].) The conclusions from these resonance Raman studies are extended to solution phase studies at ambient temperatures.

Animals↗

PubMed1990

Conformational substates and motions in myoglobin. External influences on structure and dynamics.

Myoglobin, a simppe dioxygen-storage protein, is a good laboratory for the investigation of the connection between protein structure, dynamics, and function. Fourier-transform infrared spectroscopy on carbon-monoxymyoglobin (MbCO) shows three major CO bands. These bands are excellent probes for the investigation of the structure-function relationship. They have different CO binding kinetics and their CO dipoles form different angles with respect to the heme normal, implying that MbCO exists in three major conformational substates, A0, A1, and A3. The entropies and enthalpies of these substates depend on temperature above approximately 180 K and are influenced by pH, solvent, and pressure. These results suggest that even a protein as simple as Mb can assume a small number of clearly different structures that perform the same function, but with different rates. Moreover, protein structure and dynamics depend strongly on the interaction of the protein with its environment.

Fourier Analysis↗

PubMed1990

Inhomogeneous broadening in spectral bands of carbonmonoxymyoglobin. The connection between spectral and functional heterogeneity.

The rebinding kinetics of CO to myoglobin after flash photolysis is nonexponential in time below approximately 180 K; the kinetics is governed by a distribution of enthalpic barriers. This distribution results from inhomogeneities in the protein conformation, referred to as conformational substates. Hole-burning experiments on the Soret and IR CO-stretch bands test the assumption that an inhomogeneous distribution of conformational substates results in inhomogeneously broadened spectra. CO was slowly photolyzed at different wavelengths in the Soret band at 10 K. Both the Soret band and the CO-stretch band A1, centered at 1,945 cm-1, shift during photolysis, demonstrating that different wavelengths excite different parts of the distributed population. We have also done kinetic hole-burning experiments by measuring peak shifts in the Soret and A1 bands as the CO molecules rebind. The shifts indicate that the spectral and enthalpic distributions are correlated. In the A1 band, the spectral and enthalpic distributions are highly correlated while in the Soret the correlation is weak. From the peak shifts in the spectral and kinetic hole-burning experiments the inhomogeneous broadening is estimated to be approximately 15% of the total width in the Soret band and approximately 60% in A1. We have previously measured the tilt angle alpha between the bound CO and the heme normal (Ormos, P., D. Braunstein, H. Frauenfelder, M. K. Hong, S.-L. Lin, T. B. Sauke, and R. D. Young. 1988. Proc. Natl. Acad. Sci. USA. 85:8492-8496) and observed a wave number dependence of the tilt angles within the CO-stretch A bands. Thus the spectral and enthalpic distributions of the A bands are coupled to a heterogeneity of the structure.

Binding Sites↗

PubMed1988

Ligand binding to synthetic mutant myoglobin (His-E7----Gly): role of the distal histidine.

Low-temperature flash photolysis with IR and visible spectroscopy was used to probe the influence of the distal histidine His-64(E7) of sperm-whale myoglobin (Mb) on the orientation of bound carbon monoxide (CO) and on the kinetics of CO rebinding. The synthesis and high-level expression of a sperm-whale myoglobin gene in Escherichia coli permits the efficient substitution of the distal histidine through site-directed mutagenesis. Substitution of His-E7 with glycine [GlyE7]Mb bound with CO (CO[GlyE7]Mb) results in one broad bound-CO IR stretch band, v(C-O), centered at 1973 cm-1 at 10 K, in contrast to three distinct bands for native and synthetic wild-type MbCO at 1966, 1945, and 1929 cm-1. After flash photolysis at 10 K, the unbound state of CO[GlyE7]Mb exhibits two CO stretch bands, whereas MbCO has three. Fourier transform IR spectroscopy measurements of the linear dichroism after photoselective flash photolysis of CO bound to [GlyE7]Mb at 10 K reveals the bound CO to be oriented at an angle of alpha = 20 degrees +/- 2 degrees with respect to the heme normal. Flash photolysis data from 10 to 300 K provide evidence for a larger distal pocket and a smaller enthalpy barrier (by approximately 4 kJ/mol) for [GlyE7]MbCO as compared with wild-type MbCO. These results reinforce the notion that the dominant control of the binding step at the heme iron comes from the proximal side through the protein structure.

Animals↗

PubMed1987

Rebinding and relaxation in the myoglobin pocket.

The infrared stretching bands of carboxymyoglobin (MbCO) and the rebinding of CO to Mb after photodissociation have been studied in the temperature range 10-300 K in a variety of solvents. Four stretching bands imply that MbCO can exist in four substates, A0-A3. The temperature dependences of the intensities of the four bands yield the relative binding enthalpies and and entropies. The integrated absorbances and pH dependences of the bands permit identification of the substates with the conformations observed in the X-ray data (Kuriyan et al., J. Mol. Biol. 192 (1986) 133). At low pH, A0 is hydrogen-bonded to His E7. The substates A0-A3 interconvert above about 180 K in a 75% glycerol/water solvent and above 270 K in buffered water. No major interconversion is seen at any temperature if MbCO is embedded in a solid polyvinyl alcohol matrix. The dependence of the transition on solvent characteristics is explained as a slaved glass transition. After photodissociation at low temperature the CO is in the heme pocket B. The resulting CO stretching bands which are identified as B substates are blue-shifted from those of the A substates. At 40 K, rebinding after flash photolysis has been studied in the Soret, the near-infrared, and the integrated A and B substates. All data lie on the same rebinding curve and demonstrate that rebinding is nonexponential in time from at least 100 ns to 100 ks. No evidence for discrete exponentials is found. Flash photolysis with monitoring in the infrared region shows four different pathways within the pocket B to the bound substates Ai. Rebinding in each of the four pathways B----A is nonexponential in time to at least 10 ks and the four pathways have different kinetics below 180 K. From the time and temperature dependence of the rebinding, activation enthalpy distributions g(HBA) and preexponentials ABA are extracted. No pumping from one A substate to another, or one B substate to another, is observed below the transition temperature of about 180 K. If MbCO is exposed to intense white light for 10-10(3) s before being fully photolyzed by a laser flash, the amplitude of the long-lived states increases. The effect is explained in terms of a hierarchy of substates and substate symmetry breaking. The characteristics of the CO stretching bands and of the rebinding processes in the heme pocket depend strongly on the external parameters of solvent, pH and pressure. This sensitivity suggests possible control mechanisms for protein reactions.

Carbon Monoxide↗