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E R Henry

Publications and source records attributed to E R Henry.

35 records · Page 2Linked to original sources
PubMed1991

Dynamics of the quaternary conformational change in trout hemoglobin.

The kinetics of conformational changes in trout hemoglobin I have been characterized over the temperature range 2-65 degrees C from time-resolved absorption spectra measured following photodissociation of the carbon monoxide complex. Changes in the spectra of the deoxyheme photoproduct were used to monitor changes in the protein conformation. Although the deoxyheme spectral changes are only about 8% of the total spectral change due to ligand rebinding, a combination of high-precision measurements and singular value decomposition of the data permits a detailed analysis of both their amplitudes and relaxation rates. Systematic variation of the degree of photolysis was used to alter the distribution of liganded tetramers, permitting the assignment of the spectral relaxation at 20 microseconds to the R----T quaternary conformational change of the zero-liganded and singly liganded molecules and spectral relaxations at about 50 ns and 2 microseconds to tertiary conformational changes within the R structure. Analysis of the effect of photoselection by the linearly polarized excitation pulse indicates that a major contribution to the apparent geminate rebinding in the 50-ns relaxation arises from rotational diffusion of molecules containing unphotolyzed heme-CO complexes. The activation enthalpy and activation entropy for the R0----T0 transition are +7.4 kcal/mol and -12 cal mol-1 K-1. Using the equilibrium data, delta H = +29.4 kcal/mol and delta S = +84.4 cal mol-1 K-1 [Barisas, B. G., & Gill, S. J. (1979) Biophys. Chem. 9, 235-244], the activation parameters for the T0----R0 transition are calculated to be delta H = +37 kcal/mol and delta S = +73 cal mol-1 K-1. The similarity of the equilibrium and activation parameters for the T0----R0 transition indicates that the transition state is much more R-like than T-like. This result suggests that in the path from T0 to R0 the subunits have already almost completely rearranged into the R configuration when the transition state is reached, while in the path from R0 to T0 the subunits remain in a configuration close to R in the transition state. The finding of an R-like transition state explains why the binding of ligands causes much smaller changes in the R----T rates than in the T----R rates.

Amino Acid Sequence↗
PubMed1991

Crystals of haemoglobin with the T quaternary structure bind oxygen noncooperatively with no Bohr effect.

The relationship between the structure and function of haemoglobin has mainly been studied by comparing its X-ray crystal structures with its function in solutions. To make a direct comparison we have studied the functional properties of haemoglobin in single crystals, an approach that has been an important part of the investigation of several enzyme mechanisms. Here we report on the oxygen binding by single crystals of human haemoglobin grown in solutions of polyethylene glycol. Unlike haemoglobin crystals formed in concentrated salt solution, which crack and become disordered on oxygenation, crystals grown in polyethylene glycol remain intact. X-ray studies have shown that the T (deoxy) quaternary structure of haemoglobin in this crystal at pH 7.0 is maintained at atmospheric oxygen pressure, and that the salt-bridges are not broken. We find striking differences between oxygen binding by haemoglobin in this crystal and by haemoglobin in solution. Not only is oxygenation of the crystal noncooperative, but the oxygen affinity is independent of pH in the range 6.0-8.5, and is much lower than that of the T state in solution. The lack of cooperativity without a change in quaternary structure is predicted by the two-state allosteric model of Monod, Wyman and Changeux. The absence of a Bohr effect without breakage of salt-bridges is predicted by Perutz's stereochemical mechanism. In contrast to the X-ray result that oxygen binds only to the alpha haems, our measurements show that the alpha haems have only a slightly higher affinity than the beta haems.

Allosteric Regulation↗
PubMed1991

Application of linear free energy relations to protein conformational changes: the quaternary structural change of hemoglobin.

The transition state for the R in equilibrium with T quaternary conformational change of hemoglobin has thermodynamic properties much closer to those of the R conformation than to those of the T conformation. This finding is based on a comparison of activation and equilibrium enthalpy and entropy changes and on the observation of a linear free energy relationship between quaternary rate and equilibrium constants. A previous theoretical study [Janin, J. & Wodak, S. J. (1985) Biopolymers 24, 509-526], using a highly simplified energy function, suggests that the R-like transition state is the result of a reaction pathway with the maximum buried surface area between alpha beta dimers.

Allosteric Regulation↗
PubMed1989

Photocycles of bacteriorhodopsin in light- and dark-adapted purple membrane studied by time-resolved absorption spectroscopy.

Nanosecond time-resolved absorption spectra have been measured throughout the photocycle of bacteriorhodopsin in both light-adapted and dark-adapted purple membrane (PM). The data from dark-adapted samples are interpretable as the superposition of two photocycles arising independently from the all-trans and 13-cis retinal isomers that coexist in the dark-adapted state. The presence of a photocycle in dark-adapted PM which is indistinguishable from that observed for light-adapted PM under the same experimental conditions is demonstrated by the observation of the same five relaxation rates associated with essentially identical changes in the photoproduct spectra. This cycle is attributed to the all-trans component. The cycle of the 13-cis component is revealed by scaling the data measured for the light-adapted sample and subtracting it from the data on the dark-adapted mixture. At times less than 1 ms, the resulting difference spectra are nearly time-independent. The peak of the difference spectrum is near 600 nm, although there appears to be a slight (approximately 2 nm) blue-shift in the first few microseconds. Subsequently the amplitude of this spectrum decays and the peak of the difference spectrum shifts in two relaxations. Most of the amplitude of the photoproduct difference spectrum (approximately 80%) decays in a single relaxation having a time constant of approximately 35 ms. The difference spectrum remaining after this relaxation peaks at approximately 590 nm and is indistinguishable from the classical light-dark difference spectrum, which we find, in experiments performed on a much longer time scale, to peak at 588 nm. The decay of this remaining photo-product is not resolvable in the nanosecond kinetic experiments, but dark adaptation of a completely light-adapted sample is found to occur exponentially with a relaxation time of approximately 2,000 s under the conditions of our experiments.

Bacteriorhodopsins↗
PubMed1988

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

A summary is presented of our current understanding of the kinetics of ligand rebinding and conformational changes at room temperature following photodissociation of the carbon monoxide complex of hemoglobin with pulsed lasers. The events which occur subsequent to excitation have been followed over 12 decades in time, from about 100 fs to the completion of ligand rebinding at about 100 ms. Experiments with picosecond and subpicosecond lasers by others, together with molecular dynamics simulations, indicate that by 1 ns the deoxyhemoglobin photoproduct is in a thermally equilibrated ground electronic state, so that subsequent processes are unaffected by the initial laser excitation. The principal results have been obtained from time-resolved optical absorption spectroscopy using a sensitive nanosecond laser spectrometer. Five relaxations have been observed which are interpreted as geminate rebinding at about 50 ns that competes with motion of the ligand away from the heme which produces a tertiary conformational change, a second tertiary conformational change at 0.5-1 microseconds, transition from the R to T quaternary structure at about 20 microseconds, and overall bimolecular rebinding of ligands from the solvent to the R and T quaternary structures at about 200 microseconds and 10 ms. Assuming that the dissociation pathway in photolysis experiments is the reverse of the association pathway, we find that for the R state there is a 40% probability that the ligand will bind to the heme after entering the protein, and a 60% probability that it will return to the solvent. Studies on the alpha-subunit of an iron-cobalt hybrid hemoglobin indicate that carbon monoxide enters the protein at the same rate for both R and T quaternary structures. For the alpha-subunit in the T state the probability of binding after entry is much lower, and the ligand returns to the solvent more than 99% of the time, accounting for the 60-fold overall lower association rate. This decreased probability of binding results from a decreased rate of binding to the heme from within the protein, and not an increased rate of return to the solvent. There are still unresolved problems on the basic structural description of carbon monoxide binding and dissociation, particularly the functional significance of the tertiary relations in both the R and T states, and the precise number of kinetic barriers within the protein.

Algorithms↗
PubMed1988

The effect of quaternary structure on the kinetics of conformational changes and nanosecond geminate rebinding of carbon monoxide to hemoglobin.

To determine the effect of quaternary structure on the individual kinetic steps in the binding of carbon monoxide to the alpha subunit of hemoglobin, time-resolved absorption spectra were measured after photodissociation of carbon monoxide from a hemoglobin tetramer in which cobalt was substituted for iron in the beta subunits. Cobalt porphyrins do not bind carbon monoxide. Spectra were measured in the Soret region at room temperature after time delays that varied from a few nanoseconds to the completion of ligand rebinding at about 100 ms. The results show that the liganded molecule, alpha(Fe-CO)2 beta(Co)2, is in the R state, but can be almost completely switched into the T state by the allosteric effectors inositol hexaphosphate and bezafibrate. The geminate yield, which is the probability that the ligand rebinds to the heme from within the protein, is found to be 40% for the R state and less than 1% for the T state. According to the simplest kinetic model, these results indicate that carbon monoxide enters the protein in the R and T quaternary conformations at the same rate, and that the 60-fold decrease in the overall binding rate, of carbon monoxide to the alpha subunit in the T state compared to the R state is almost completely accounted for by the decreased probability of binding after the ligand has entered the protein. The results further suggest that the low probability for the T state results from a decreased binding rate to the heme and not from an increased rate of return of the ligand to the solvent.

Allosteric Regulation↗
PubMed1987

Molecular dynamics simulations of fluorescence polarization of tryptophans in myoglobin.

The fluorescence of heme proteins is influenced by energy transfer from the excited tryptophan to the heme. Molecular dynamics simulations of the tryptophan and heme motions in sperm whale myoglobin were used to calculate the fluorescence intensity and anisotropy decays. The side chains underwent both small rapid orientational fluctuations and large infrequent transitions between conformations. The predicted motions of the tryptophans and the heme produce large fluctuations in the instantaneous rate of energy transfer, but no stable conformations in which energy transfer is suppressed were found. The calculated fluorescence anisotropies exhibited a large subpicosecond decay, corresponding to nondiffusive side-chain motions. The calculations adequately predict the observed fluorescence decay curve for myoglobin and the total anisotropy decay at 16-ps time resolution. The subnanosecond decays of anisotropy for tryptophan-14 in tuna myoglobin are not reproduced by the calculation.

Animals↗
PubMed1986

Molecular dynamics simulations of cooling in laser-excited heme proteins.

In transient optical experiments the absorbed photon raises the vibrational temperature of the chromophore. In heme proteins at room temperature conversion of a 530-nm photon into vibrational energy is estimated to raise the temperature of the heme by 500-700 K. Cooling of the heme is expected to occur mainly by interacting with the surrounding protein. We report molecular dynamics simulations for myoglobin and cytochrome c in vacuo that predict that this cooling occurs on the ps time scale. The decay of the vibrational temperature is nonexponential with about 50% loss occurring in 1-4 ps and with the remainder in 20-40 ps. These results predict the presence of nonequilibrium vibrational populations that would introduce ambiguity into the interpretation of transient ps absorption and Raman spectra and influence the kinetics of sub-ns geminate recombination.

Biophysical Phenomena↗
PubMed1985

Geminate recombination of n-butyl isocyanide to myoglobin.

Transient optical absorption spectra of myoglobin were measured following photolysis of the n-butyl isocyanide complex with 10-ns laser pulses at room temperature. The data were analyzed by using singular value decomposition to give the kinetics of ligand rebinding and spectral changes. Geminate recombination phases were observed at 30 ns and 1 microsecond following photodissociation. These processes were accompanied by simultaneous changes in the shape of the Soret band which indicate changes in protein conformation. These spectral changes are not present in the geminate recombination of photolyzed complexes of myoglobin with the diatomic ligands oxygen and carbon monoxide. This difference in behavior, as well as the slower overall association rate of n-butyl isocyanide to myoglobin, can be rationalized as arising from distortion of the protein structure by the larger isocyanide ligand along the binding pathway.

Animals↗
PubMed1985

Spectroscopic studies of protein-heme interactions accompanying the allosteric transition in methemoglobins.

Resonance Raman, optical absorption, and circular dichroism spectroscopic techniques have been used to examine the effect of the addition of inositol hexaphosphate (IHP) to a series of carp and human methemoglobin derivatives. Markers of spin equilibrium in the high-frequency region (1450-1650 cm-1) of the resonance Raman spectrum yield high/low-spin ratios consistent with direct magnetic susceptibility measurements. Changes in the low-frequency region (100-600 cm-1) of the resonance Raman spectrum appear to correlate with the quaternary structure transition. Changes in the ultraviolet absorption spectra and the circular dichroism spectra also appear to be related to the quaternary structure change. By using the resonance Raman spin markers, we find that those derivatives of carp methemoglobin which are in spin equilibrium have a larger ratio of high-spin to low-spin populations than the corresponding derivatives of human methemoglobin. Upon the addition of IHP to the methemoglobins the spin equilibrium is shifted toward a larger high-spin population. This change in equilibrium is larger for the carp protein than for the human protein. We obtain an IHP-induced change in the free energy difference between the high-spin and low-spin states of 300 cal/mol for those human methemoglobins in which a quaternary structure change occurs and 600 cal/mol for carp methemoglobins. Our data are consistent with a quaternary structure change induced by IHP in all the carp methemoglobins studied (F-, H2O, SCN-, NO2-, N3-, and CN-) and in the F-, H2O, and SCN- derivatives of the human protein but not in the NO2-, N3-, and CN- derivatives. The Fe-CN stretching mode has been identified by isotopic substitution and found to be unchanged in frequency in carp CN- metHb when the quaternary structure is changed. On the basis of our results we conclude that the protein forces at the heme due to the addition of IHP do not significantly affect the position of the iron atom with respect to the heme plane. Rather, the changes in spin equilibrium may be caused by protein-induced changes in the orientation of the proximal histidine or tertiary structure changes in the heme pocket which affect the porphyrin macrocycle. Either of these changes, or a combination thereof, leads to changes in the iron d orbital energies and concomitant changes in the spin equilibrium.

Allosteric Regulation↗
PubMed1985

Nanosecond optical spectra of iron-cobalt hybrid hemoglobins: geminate recombination, conformational changes, and intersubunit communication.

Hybrid hemoglobins were prepared in which cobalt was substituted for the heme iron in either the alpha or beta subunits. Transient optical absorption spectra were measured at room temperature for these hybrids at time intervals between 0 and 50 ms following photodissociation of the carbon monoxide complex with 10-ns laser pulses. The cobalt porphyrins do not bind carbon monoxide, making it possible to investigate the time-resolved response of the cobalt-containing subunits to photodissociation of carbon monoxide in the iron-containing subunits. At the same time the response of the iron-containing subunits to the photolysis event can be studied, permitting an independent determination of the kinetics of ligand rebinding and conformational changes in the alpha and beta subunits of an intact tetramer. The data were analyzed by using singular-value decomposition to obtain the kinetic progress curve for ligand rebinding, the deoxyheme and cobalt porphyrin spectral changes, and the time course of these spectral changes. The geminate rebinding kinetics following photodissociation of alpha(Co)2 beta(Fe-CO)2 were very similar to those found unsubstituted hemoglobin, alpha(Fe-CO)2 beta(Fe-CO)2, indicating equivalence of the geminate kinetics for alpha and beta subunits within the R-state tetramer. The results for alpha(Fe-CO)2 beta(Co)2 were consistent with this conclusion, even though the analysis was complicated by the presence of comparable populations of R- and T-state species. Comparison of the deoxyheme spectral changes and relaxation times among the three molecules indicated that both alpha and beta subunits contribute to the deoxyheme spectral changes that signal tertiary and quaternary conformational changes in the unsubstituted tetramer. The response of the cobalt porphyrins to photodissociation was similar in the two hybrids. No structural changes were detected in the cobalt-containing subunits until the second tertiary conformational change in the iron-containing subunits observed at 1-2 microseconds. Much larger structural changes, as judged by the amplitude of the spectral changes, occurred in the cobalt-containing subunits concomitant with the R----T quaternary change at about 20 microseconds.

Cobalt↗
PubMed1985

Molecular dynamics simulation of photodissociation of carbon monoxide from hemoglobin.

A molecular dynamics simulation of the photodissociation of carbon monoxide from the alpha subunit of hemoglobin is described. To initiate photodissociation, trajectories of the liganded molecule were interrupted, the iron-carbon monoxide bond was broken, and the parameters of the iron-nitrogen bonds were simultaneously altered to produce a deoxyheme conformation. Heme potential functions were used that reproduce the energies and forces for the iron out-of-plane motion obtained from quantum mechanical calculations. The effect of the protein on the rate and extent of the displacement of the iron from the porphyrin plane was assessed by comparing the results with those obtained for an isolated complex of heme with imidazole and carbon monoxide. The half-time for the displacement of the iron from the porphyrin plane was found to be 50-150 fs for both the protein and the isolated complex. These results support the interpretation of optical absorption studies using 250-fs laser pulses that the iron is displaced from the porphyrin plane within 350 fs in both hemoglobin and a free heme complex in solution.

Animals↗
PubMed1983

Geminate recombination of carbon monoxide to myoglobin.

Transient absorption spectra of myoglobin, following photolysis of the carbon monoxide complex at room temperature, were measured using a newly developed, sensitive nanosecond absorption spectrometer. The Soret spectrum of the immediate photoproduct is almost identical to that of deoxymyoglobin at equilibrium, suggesting that the heme group has changed from a planar to a domed structure in less than about 3 ns. About 4% of the photodissociated carbon monoxide molecules rebind to the hemes to which they were initially bound, with a relaxation time of 180 ns. Duddell et al. (1980) observed a geminate yield of 27% and a relaxation time of approximately 55 ns for the photolysis of oxymyoglobin. Comparison of the two results using the simplest kinetic model suggests that the 30-fold more rapid overall association rate for the reaction of oxygen with myoglobin compared to carbon monoxide results mainly from faster binding at the heme, with a small contribution from more rapid entry of oxygen into the protein from the solvent. The data on carbon monoxide are also compared with predictions from low-temperature studies of Frauenfelder and co-workers. This comparison points to the need for further experiments to demonstrate the correspondence between the ligand rebinding processes observed at high and low temperatures.

Animals↗
PubMed1983

Nanosecond absorption spectroscopy of hemoglobin: elementary processes in kinetic cooperativity.

A nanosecond absorption spectrometer has been used to measure the optical spectra of hemoglobin between 3 ns and 100 ms after photolysis of the CO complex. The data from a single experiment comprise a surface, defined by the time-ordered set of 50-100 spectra. Singular value decomposition is used to represent the observed spectra in terms of a minimal set of basis spectra and the time course of their amplitudes. Both CO rebinding and conformational changes are found to be multiphasic. Prior to the quaternary structural change, two relaxations are observed that are assigned to geminate recombination followed by a tertiary structural change. These relaxations are interpreted in terms of a kinetic model that points out their potential role in kinetic cooperativity. The rapid escape of CO from the heme pocket compared with the rate of rebinding observed for both R and T quaternary states shows that the quaternary structure controls the overall dissociation rate by changing the rate at which the Fe--CO bond is broken. A comparable description of the control of the overall association rates must await a more complete experimental description of the kinetics of the quaternary T state.

Allosteric Regulation↗
PubMed1980

Raman difference spectroscopy of tertiary and quaternary structure changes in methaemoglobins.

There have been several resonance Raman scattering investigations of the effect of inositol hexaphosphate (IHP) on methaemoglobins. In those studies the sensitivity for detecting frequency differences was limited to 1-2 cm-1, and consequently frequency differences were not detected although spectral intensity differences due to changes in spin equilibria were. Shelnutt et al. recently reported on the observation of frequency differences induced by changes in the quaternary structure of chemically modified deoxyhaemoglobins. An improved Raman difference spectroscopic technique with 0.1 cm-1 sensitivity allowed the detection of these differences. We report here the application of this technique to a series of methaemoglobins with and without the addition of IHP. In addition to the intensity changes resulting from changes in the spin equilibria, we have observed frequency differences. In all liganded methaemo-globins that we examined a decrease in frequency of the mode in the 1,370 cm-1 region was observed on addition of IHP. In those in which a quaternary structure change is known to occur the frequency difference is greater than 0.5 cm-1. In those in which no quaternary structure change occurs [metHbA(CN-) and methHbA(N-3)] the frequency difference is smaller (approximately 0.15 cm-1).

Hemoglobin A↗