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H Frauenfelder

Publications and source records attributed to H Frauenfelder.

At least 37 records · Page 2Linked to original sources
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↗
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

Orientation of carbon monoxide and structure-function relationship in carbonmonoxymyoglobin.

Fourier transform infrared spectroscopy of the CO stretch bands in carbonmonoxymyoglobin (MbCO) reveals three major bands implying that MbCO exists in three major substates, A0, A1, and A3. After photolysis at low temperatures the CO is in the heme pocket, and the resulting CO stretch bands represent the B substates. Photoselection experiments determine the orientation of CO in the A (bound) and B (photolyzed) substates: Small fractions of MbCO are photolyzed at 10 K with linearly polarized light at 540 nm. The resulting linear dichroism in the A and B IR bands yields the tilt angle between the heme normal and CO. The average angles are as follows: alpha (A0) = 15 degrees +/- 3 degrees; alpha (A1) = 28 degrees +/- 2 degrees, and alpha (A3) = 33 degrees +/- 4 degrees. The A bands are inhomogeneously broadened; the angle alpha shows a wavenumber dependence within the A bands. The wavenumber dependence is interpreted as a distribution of the tilt angle within the individually inhomogeneous A substates, thus providing a structural parameter to characterize the distribution of the conformational substates. The B substates exhibit no induced linear dichroism; in the photolyzed substates the ligand is randomly oriented with respect to the heme plane. The present results together with earlier data on static and kinetic properties of CO binding to Mb establish relations among spectroscopic, structural, energetic, and functional parameters.

Carbon Monoxide↗
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↗
PubMed1987

Recombination of carbon monoxide to ferrous horseradish peroxidase types A and C.

The recombination of carbon monoxide to isoenzymes A2 and C of horseradish peroxidase (HRP) was studied as a function of temperature (2 to 320 K) and pH (5 to 8.3) with flash photolysis and infrared difference absorption. At low temperatures three geminate recombination processes are observed. One of these internal processes, denoted by I*, is exponential in time with a rate coefficient that deviates strongly from an Arrhenius behavior below 100 K, implying phonon-assisted tunneling. The two other processes, denoted by I, are non-exponential in time and related to different carbonyl isomers, as shown by the infrared difference spectra. The existence of three internal processes indicates that HRP differs considerably from myoglobin where only one internal process, I, is seen. Moreover, the internal processes in HRP are faster than process I in myoglobin. At 300 K, only one recombination process from the solvent is observed and it is very slow (lambda s approximately 1 s-1 at 1 atm CO (1 atm = 101,325 Pa)), much slower than the corresponding association process in myoglobin. Since process I is fast, but binding from the solvent is slow, the barrier at the heme cannot be responsible for the small association rate. The infrared absorption difference spectra of the amide I/II bands indicate that photolysis and recombination trigger a two-step structural change. The slow recombination rate at 300 K can thus be explained by the large Gibbs energy of the conformational transition that is necessary to let CO move into the heme pocket. The partition coefficient for the CO in the heme pocket and the solvent is extremely small, while bond formation with the heme iron occurs in less than 100 nanoseconds.

Carbon Monoxide↗
PubMed1987

Thermal expansion of a protein.

The thermal expansion of a protein, metmyoglobin, was investigated by analysis of the refined X-ray crystal structures at 80 and 255-300 K. On heating from 80 to 300 K, the volume occupied by myoglobin increases by approximately 3%. The linear thermal expansion coefficient is estimated to be 115 X 10(-6) K-1. This value is more than twice as large as that of liquid water but less than that of benzene. As the temperature is raised, the internal volume change does not come from the large, atom-sized internal cavities in the structure but from an increase in the small, subatomic free volumes between atoms. The largest expansion occurs in the region of the CD and GH corners; both these regions move away from the center of the protein. The remainder of the expansion results from the lengthening of contacts between segments of secondary structure.

Animals↗
PubMed1986

Ligand binding to heme proteins: relevance of low-temperature data.

Binding of carbon monoxide to the beta chain of adult human hemoglobin has been studied by flash photolysis over the time range from about 100 ps to seconds and the temperature range from 40 to 300 K. Below about 180 K, binding occurs directly from the pocket (process I) and is nonexponential in time. Above about 180 K, some carbon monoxide molecules escape from the pocket into the protein matrix. Above about 240 K, escape into the solvent becomes measurable. Process I can be observed up to 300 K. The low-temperature data extrapolate smoothly to 300 K, proving that the results obtained below 180 K provide functionally relevant information. The experiments show again that the binding process even at physiological temperatures is regulated by the final binding step at the heme iron and that measurements at high temperatures are not sufficient to fully understand the association process.

Carbon Monoxide↗
PubMed1985

Rate theories and puzzles of hemeprotein kinetics.

The binding of dioxygen and carbon monoxide to heme proteins such as myoglobin and hemoglobin has been studied with flash photolysis. At temperatures below 200 K, binding occurs from within the heme pocket and, contrary to expectation, with nearly equal rates for both ligands. This observation has led to a reexamination of the theory of the association reaction taking into account friction, protein structure, and the nature of electronic transitions. The rate coefficients for the limiting cases of large and small friction are found with simple arguments that use characteristic lengths and times. The arguments indicate how transition state theory as well as calculations based on nonadiabatic perturbation theory, which is called the Golden Rule, may fail. For ligand-binding reactions the data suggest the existence of intermediate states not directly observed so far. The general considerations may also apply to other biomolecular processes such as electron transport.

Carbon Monoxide↗
PubMed1985

Dynamics of dioxygen and carbon monoxide binding to soybean leghemoglobin.

The association of dioxygen and carbon monoxide to soybean leghemoglobin (Lb) has been studied by laser flash photolysis at temperatures from 10 to 320 K and times from 50 ns to 100 s. Infrared spectra of the bound and the photodissociated state were investigated between 10 and 20 K. The general features of the binding process in leghemoglobin are similar to the ones found in myoglobin. Below about 200 K, the photodissociated ligands stay in the heme pocket and rebinding is not exponential in time, implying a distributed enthalpy barrier between pocket and heme. At around 300 K, ligands migrate from the solvent through the protein to the heme pocket, and a steady state is set up between the ligands in the solvent and in the heme pocket. The association rate, lambda on, is mainly controlled by the final binding step at the heme, the bond formation with the heme iron. Differences between Lb and other heme proteins show up in the details of the various steps. The faster association rate in Lb compared to sperm whale myoglobin (Mb) is due to a faster bond formation. The migration from the solvent to the heme pocket is much faster in Lb than in Mb. The low-temperature binding (B----A) and the infrared spectra of CO in the bound state A and the photodissociated state B are essentially solvent-independent in Mb, but depend strongly on solvent in Lb. These features can be correlated with the x-ray structure.

Carbon Monoxide↗