Ligand binding to heme proteins. VI. Interconversion of taxonomic substates in carbonmonoxymyoglobin.

The kinetic properties of the three taxonomic A substates of sperm whale carbonmonoxy myoglobin in 75% glycerol/buffer are studied by flash photolysis with monitoring in the infrared stretch bands of bound CO at nu(A0) approximately 1967 cm-1, nu(A1) approximately 1947 cm-1, and nu(A3) approximately 1929 cm-1 between 60 and 300 K. Below 160 K the photodissociated CO rebinds from the heme pocket, no interconversion among the A substates is observed, and rebinding in each A substate is nonexponential in time and described by a different temperature-independent distribution of enthalpy barriers with a different preexponential. Measurements in the electronic bands, e.g., the Soret, contain contributions of all three A substates and can, therefore, be only approximately modeled with a single enthalpy distribution and a single preexponential. The bond formation step at the heme is fastest for the A0 substate, intermediate for the A1 substate, and slowest for A3. Rebinding between 200 and 300 K displays several processes, including geminate rebinding, rebinding after ligand escape to the solvent, and interconversion among the A substates. Different kinetics are measured in each of the A bands for times shorter than the characteristic time of fluctuations among the A substates. At longer times, fluctuational averaging yields the same kinetics in all three A substates. The interconversion rates between A1 and A3 are determined from the time when the scaled kinetic traces of the two substates merge. Fluctuations between A1 and A3 are much faster than those between A0 and either A1 or A3, so A1 and A3 appear as one kinetic species in the exchange with A0. The maximum-entropy method is used to extract the distribution of rate coefficients for the interconversion process A0 <--> A1 + A3 from the flash photolysis data. The temperature dependencies of the A substate interconversion processes are fitted with a non-Arrhenius expression similar to that used to describe relaxation processes in glasses. At 300 K the interconversion time for A0 <--> A1 + A3 is 10 microseconds, and extrapolation yields approximately 1 ns for A1 <--> A3. The pronounced kinetic differences imply different structural rearrangements. Crystallographic data support this conclusion: They show that formation of the A0 substate involves a major change of the protein structure; the distal histidine rotates about the C(alpha)-C(beta) bond, and its imidazole sidechain swings out of the heme pocket into the solvent, whereas it remains in the heme pocket in the A1 <--> A3 interconversion. The fast A1 <--> A3 exchange is inconsistent with structural models that involve differences in the protonation between A1 and A3.     

Ligand binding to heme proteins. V. Light-induced relaxation in proximal mutants L89I and H97F of carbonmonoxymyoglobin.

We have studied the proximal mutants L89I and H97F of MbCO with FTIR and temperature-derivative spectroscopy at temperatures between 10 and 160 K. The mutations give rise only to minor alterations of the stretch spectra of the bound and photodissociated CO ligand. The most pronounced difference is a larger population in the A3 substate at approximately 1930 cm-1 in the mutants. The barrier distributions, as determined by temperature-derivative spectroscopy, are very similar to native MbCO after short illumination. Extended illumination leads to substantial increases of the rebinding barriers in native MbCO and the proximal mutants. A larger fraction of light-relaxed states is found in the proximal mutants, implying that the conformational energy landscape has been modified to more easily allow light-induced transitions. These and other spectroscopic data imply that the large changes in the binding properties are brought about by a light-induced conformational relaxation involving the structure at the heme iron. Similarities with spectral hole-burning studies and physical models are discussed.     

Proton electron nuclear double resonance from nitrosyl horse heart myoglobin: the role of His-E7 and Val-E11

Electron nuclear double resonance (ENDOR) spectroscopy has been used to study protons in nitrosyl horse heart myoglobin (MbNO). (1)H ENDOR spectra were recorded for different settings of the magnetic field. Detailed analysis of the ENDOR powder spectra, using computer simulation, based on the "orientation-selection" principle, leads to the identification of the available protons in the heme pocket. We observe hyperfine interactions of the N(HisF8)-Fe(2+)-N(NO) complex with five protons in axial and with eight protons in the rhombic symmetry along different orientations, including those of the principal axes of the g-tensor. Protons from His-E7 and Val-E11 residues are identified in the two symmetries, rhombic and axial, exhibited by MbNO. Our results indicate that both residues are present inside the heme pocket and help to stabilize one particular conformation.     

Ligand binding to heme proteins: II. Transitions in the heme pocket of myoglobin.

Phenomena occurring in the heme pocket after photolysis of carbonmonoxymyoglobin (MbCO) below about 100 K are investigated using temperature-derivative spectroscopy of the infrared absorption bands of CO. MbCO exists in three conformations (A substrates) that are distinguished by the stretch bands of the bound CO. We establish connections among the A substates and the substates of the photoproduct (B substates) using Fourier-transform infrared spectroscopy together with kinetic experiments on MbCO solution samples at different pH and on orthorhombic crystals. There is no one-to-one mapping between the A and B substates; in some cases, more than one B substate corresponds to a particular A substate. Rebinding is not simply a reversal of dissociation; transitions between B substates occur before rebinding. We measure the nonequilibrium populations of the B substates after photolysis below 25 K and determine the kinetics of B substate transitions leading to equilibrium. Transitions between B substates occur even at 4 K, whereas those between A substates have only been observed above about 160 K. The transitions between the B substates are nonexponential in time, providing evidence for a distribution of substates. The temperature dependence of the B substate transitions implies that they occur mainly by quantum-mechanical tunneling below 10 K. Taken together, the observations suggest that the transitions between the B substates within the same A substate reflect motions of the CO in the heme pocket and not conformational changes. Geminate rebinding of CO to Mb, monitored in the Soret band, depends on pH. Observation of geminate rebinding to the A substates in the infrared indicates that the pH dependence results from a population shift among the substates and not from a change of the rebinding to an individual A substate.     

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.     

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.     

Ligand binding to cytochrome c and other related haem proteins and peptides. Part III. Temperature dependence studies

The temperature dependency of ligand binding processes lend support to the proposed mechanisms and the factors affecting ligand binding reported earlier in this series. The free energy contribution from each factor affecting ligand binding was estimated for a number of haem proteins. The structures of the haem proteins used, as conveyed from ligand binding data, are in agreement with the structures of these haem proteins as determined by other methods (e.g. X-ray crystallography, NMR, etc.). Therefore, ligand binding could be used as a facile probe to investigate some of the structural and functional properties of haem proteins. In this respect, it was concluded that the structure of native cytochrome c at pH 10 is similar to the structure of carboxymethyl-Met 80 cytochrome c between pH 7 and 10.     

Ligand binding to proteins: the binding landscape model.

Models of ligand binding are often based on four assumptions: (1) steric fit: that binding is determined mainly by shape complementarity; (2) native binding: that ligands mainly bind to native states; (3) locality: that ligands perturb protein structures mainly at the binding site; and (4) continuity: that small changes in ligand or protein structure lead to small changes in binding affinity. Using a generalization of the 2D HP lattice model, we study ligand binding and explore these assumptions. We first validate the model by showing that it reproduces typical binding behaviors. We observe ligand-induced denaturation, ANS and heme-like binding, and "lock-and-key" and "induced-fit" specific binding behaviors characterized by Michaelis-Menten or more cooperative types of binding isotherms. We then explore cases where the model predicts violations of the standard assumptions. For example, very different binding modes can result from two ligands of identical shape. Ligands can sometimes bind highly denatured states more tightly than native states and yet have Michaelis-Menten isotherms. Even low-population binding to denatured states can cause changes in global stability, hydrogen-exchange rates, and thermal B-factors, contrary to expectations, but in agreement with experiments. We conclude that ligand binding, similar to protein folding, may be better described in terms of energy landscapes than in terms of simpler mass-action models.     

FTIR and resonance Raman studies of nitric oxide binding to H93G cavity mutants of myoglobin.

Nitric oxide (NO) binds to the myoglobin (Mb) cavity mutant, H93G, forming either a five- or six-coordinate Fe-NO complex. The H93G mutation eliminates the covalent attachment between the protein and the proximal ligand, allowing NO to bind H93G possibly from the proximal side of the heme rather than the typical diatomic binding pocket on the distal side. The question of whether NO binds on the distal or proximal side was addressed by FTIR spectroscopy of the N-O vibrational frequency nuN(-O) for a set of Mb mutants that perturb the electrostatic environment of the heme pocket. Vibrational spectra of five- and six-coordinate MbNO complexes indicate that nu(N-O) shifts (by as much as 26 cm(-1)) to higher energies for the distal mutants H64V and H64V/H93G relative to the energies of wild-type and H93G MbNO, while nu(N-O) is not affected by the proximal side mutation S92A/H93G. This result suggests that NO binds on the distal side of heme in the five- and six-coordinate MbNO complexes of H93G. Additionally, values of the Fe-NO vibrational frequency nu(Fe-NO) as measured by resonance Raman spectroscopy are reported for the distal and proximal double mutants of H93G. These results suggest that nu(Fe-NO) is not very sensitive to mutations that perturb the electrostatic environment of the heme pocket, leading to the observation that nu(N-O) and nu(Fe-NO) are not quantitatively correlated for the MbNO complexes presented here. Furthermore, nu(N-O) and nu(Fe-NO) do not correlate well with equilibrium constants for imidazole binding to the five-coordinate MbNO complexes of the H93G double mutants. The data presented here do not appear to support the presence of pi-back-bonding or an inverse trans effect of NO binding in Mb mutants that alter the electrostatic environment of the heme pocket.     

Structural comparisons of heme binding proteins.

Of the 82 three dimensionally characterized residues of cytochrome c551, 49 are found to be structurally and topologically equivalent to the globin fold and 41 are equivalent to the cytochrome b5 fold, with a respective root mean square separation of 3.5 and 4.9 A between equivalenced Calpha atoms. The common fold represents a central heme binding core, corresponding to the middle exon of certain globin genes. After superposition of the protein folds, the heme irons are found to be separated by 5.4 and 1.6 A, while their heme normals are inclined by 6 degrees and 32 degrees, respectively. Furthermore, the heme "face", determined by the asymmetric attachment of the vinyl and propionyl side chains, is directed similarly in all three heme proteins. The heme itself is rotated by 72 degrees and 116 degrees about its normal, respectively. The minimum base change per codon for the three pairwise comparisons corresponds to the expected value of random sequence comparisons. While all three heme proteins may have diverged from a common ancestor, their similarity may have arisen from the requirements of heme binding or the utilization of a particularly stable fold. Known structures within commonly accepted divergent families were superimposed in order to discriminate better between convergence and divergence. Minimum base changes per codon, number of deletions and insertions, percentage of equivalenced residues, precision of heme superposition, and root mean square separation of equivalenced Calpha atoms were tested as measures of evolutionary relationships.     

Ligand binding and protein relaxation in heme proteins: a room temperature analysis of NO geminate recombination

Ultrafast absorption spectroscopy is used to study heme-NO recombination at room temperature in aqueous buffer on time scales where the ligand cannot leave its cage environment. While a single barrier is observed for the cage recombination of NO with heme in the absence of globin, recombination in hemoglobin and myoglobin is nonexponential. Examination of hemoglobin with and without inositol hexaphosphate points to proximal constraints as important determinants of the geminate rebinding kinetics. Molecular dynamics simulations of myoglobin and heme-imidazole subsequent to ligand dissociation were used to investigate the transient behavior of the Fe-proximal histidine coordinate and its possible involvement in geminate recombination. The calculations, in the context of the absorption measurements, are used to formulate a distinction between nonexponential rebinding that results from multiple protein conformations (substates) present at equilibrium or from nonequilibrium relaxation of the protein triggered by a perturbation such as ligand dissociation. The importance of these two processes is expected to depend on the time scale of rebinding relative to equilibrium fluctuations and nonequilibrium relaxation. Since NO rebinding occurs on the picosecond time scale of the calculated myoglobin relaxation, a time-dependent barrier is likely to be an important factor in the observed nonexponential kinetics. The general implications of the present results for ligand binding in heme proteins and its time and temperature dependence are discussed. It appears likely that, at low temperatures, inhomogeneous protein populations play an important role and that as the temperature is raised, relaxation effects become significant as well.     

Ligand binding to cytochrome c and other related haem proteins and peptides. Part I. Equilibrium studies

Investigation of ligand binding to native cytochrome c, carboxymethyl-Met 80-cytochrome c, myoglobin and haemhexapeptide revealed that the binding of exogenous ligands is modulated by the following factors:

• 1.Hydrophobicity of the haem environment.
• 2.Haem accessibility to exogenous ligands, termed the haem crevice ‘open-closed’ parameter.
• 3.Steric interactions between the protein and the bound ligand.

     

Fast reactions in carbon monoxide binding to heme proteins.

Using fast flash photolysis, we have measured the binding of CO to carboxymethylated cytochrome c and to heme c octapeptide as a function of temperature (5 degrees-350 degreesK) over an extended time range (100 ns(-1) ks). Experiments used a microsecond dye laser (lambda = 540 nm), and a mode-locked frequency-doubled Nd-glass laser (lambda = 530 nm). At low temperatures (5 degrees-120 degreesK) the rebinding exhibits two components. The slower component (I) is nonexponential in time and has an optical spectrum corresponding to rebiding from an S = 2, CO-free deoxy state. The fast component (I*) is exponential in time with a lifetime shorter than 10 mus and an optical spectrum different from the slow component. In myoglobin and the separated alpha and beta chains of hemoglobin, only process I is visible. The optical absorption spectrum of I* and its time dependence suggest that it may correspond to recombination from an excited state in which the iron has not yet moved out of the heme plane. The temperature dependences of both processes have been measured. Both occur via quantum mechanical tunneling at the lowest temperatures and via over-the-barrier motion at higher temperatures.     

Ligand binding and self-association of proteins

Summary This review deals with the quantitative analysis of protein self-association and ligand binding when there is a significant mutual influence of the two processes. The particular points of interest are the evaluation of the pertinent equilibrium constants and the prediction and interpretation of concentration profiles in transport experiments, including gel elution and sedimentation velocity. The case of dimertetramer equilibrium with four binding sites for ligand is considered in detail. Three representative experimental studies are described which deal with hemoglobin, phosphorylase b, and tubulin.     

Comparison of the evolution of heme binding and NAD binding proteins.

Of the 85 three-dimensionally characterized residues of cytochrome b5, 51 are structurally and topologically equivalent to the globin fold. When these proteins have been superimposed, the heme irons are found to be less than 1.4 A separated and the heme normals are inclined by less than 9.5 degrees. Comparison of minimum base changes per codon between heme binding and NAD binding proteins are of the same order.     

Ligand binding studies of engineered cytochrome P-450d wild type, proximal mutants, and distal mutants

Interactions of various axial ligands with cytochrome P-450d wild type, proximal mutants (Lys453Glu, Ile460Ser), and putative distal mutants (Glu318Asp, Thr319Ala, Thr322Ala) expressed in yeast were studied with optical absorption spectroscopy. P-450d wild type and all five mutants were purified essentially as the high-spin form, but the putative distal mutants contained about 5% low-spin form. Bindings of metyrapone and 4-phenylimidazole to the wild type and all mutants formed nitrogen-bound low-spin forms. In contrast, binding of 2-phenylimidazole to the wild type and most of mutants formed oxygen-bond low-spin forms except for the mutant Glu318Asp in which the nitrogen-bound low-spin form was formed. By analogy with the distal structure of P-450cam, it was thus suggested that Glu318 of P-450d, which corresponds with Asp251 of P-450cam, somehow interacts with 2-phenylimidazole over the heme plane. Addition of 1-butanol and acetanilide, a substrate of P-450d, to the wild type and mutants caused the spin change to the low-spin form. The order of dissociation constants of these oxygen ligands to P-450d was wild type greater than proximal mutants greater than putative distal mutants. Spectral analyses showed that the binding of acetanilide is the same as that of another substrate, 7-ethoxycoumarin, in the putative distal mutants but is not the same in the wild type and proximal mutants. From these findings together with other spectral data, it was suggested that the region from Glu318 to Thr322 is located at the distal region of the heme in membrane-bound P-450d as suggested from the X-ray crystal structure of water-soluble P-450cam and amino acid alignments of P-450s.