Molecular dynamics simulations of heme reorientational motions in myoglobin.

Molecular dynamics simulations of 2-ns duration were performed on carbonmonoxymyoglobin and deoxymyoglobin in vacuo to study the reorientational dynamics of the heme group. The heme in both simulations undergoes reorientations of approximately 5 degrees amplitude on a subpicosecond time scale, which produce a rapid initial decay in the reorientational correlation function to about 0.99. The heme also experiences infrequent changes in average orientation of approximately 10 degrees amplitude, which lead to a larger slow decay of the reorientational correlation function over a period of hundreds of picoseconds. The simulations have not converged with respect to these infrequent transitions. However, an estimate of the order parameter for rapid internal motions of the heme from those orientations which are sampled by the simulations suggests that the subnanosecond orientational dynamics of the heme accounts for at least 30% of the unresolved initial anisotropy decay observed in the nanosecond time-resolved optical absorption experiments on myoglobin reported by Ansari et al. in a companion paper (Ansari, A., C.M. Jones, E.R. Henry, J. Hofrichter, and W.A. Eaton. 1992. Biophys. J. 64:852-868.). A more complete sampling of the accessible heme orientations would most likely increase this fraction further. The simulation of the liganded molecule also suggests that the conformational dynamics of the CO ligand may contribute significantly to discrepancies between the ligand conformation as probed by x-ray diffraction and by infrared-optical photoselection experiments. The protein back-bone explores multiple conformations during the simulations, with the largest structural changes appearing in the E and F helices, which are in contact with the heme. The variations in the heme orientation correlate with the conformational dynamics of the protein on a time scale of hundreds of picoseconds, suggesting that the heme orientation may provide a useful probe of dynamical processes in the protein.     

A photolysis-triggered heme ligand switch in H93G myoglobin

Resonance Raman spectroscopy and step-scan Fourier transform infrared (FTIR) spectroscopy have been used to identify the ligation state of ferrous heme iron for the H93G proximal cavity mutant of myoglobin in the absence of exogenous ligand on the proximal side. Preparation of the H93G mutant of myoglobin has been previously reported for a variety of axial ligands to the heme iron (e.g., substituted pyridines and imidazoles) [DePillis, G., Decatur, S. M., Barrick, D., and Boxer, S. G. (1994) J. Am. Chem. Soc. 116, 6981-6982]. The present study examines the ligation states of heme in preparations of the H93G myoglobin with no exogenous ligand. In the deoxy form of H93G, resonance Raman spectroscopic evidence shows water to be the axial (fifth) ligand to the deoxy heme iron. Analysis of the infrared C-O and Raman Fe-C stretching frequencies for the CO adduct indicates that it is six-coordinate with a histidine trans ligand. Following photolysis of CO, a time-dependent change in ligation is evident in both step-scan FTIR and saturation resonance Raman spectra, leading to the conclusion that a conformationally driven ligand switch exists in the H93G protein. In the absence of exogenous nitrogenous ligands, the CO trans effect stabilizes endogenous histidine ligation, while conformational strain favors the dissociation of histidine following photolysis of CO. The replacement of histidine by water in the five-coordinate complex is estimated to occur in < 5 micros. The results demonstrate that the H93G myoglobin cavity mutant has potential utility as a model system for studying the conformational energetics of ligand switching in heme proteins such as those observed in nitrite reductase, guanylyl cyclase, and possibly cytochrome c oxidase.     

Structural dynamics of myoglobin: effect of internal cavities on ligand migration and binding

Using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) at cryogenic temperatures, we have studied CO binding to the heme and CO migration among cavities in the interior of sperm whale carbonmonoxy myoglobin (MbCO) after photodissociation. Photoproduct intermediates, characterized by CO in different locations, were selectively enhanced by laser illumination at specific temperatures. Measurements were performed on the wild-type protein and a series of mutants (L104W, I107W, I28F, and I28W) in which bulky amino acid side chains were introduced to block passageways between cavities or to fill these sites. Binding of xenon was also employed as an alternative means of filling cavities. In all samples, photolyzed CO ligands were observed to initially bind at primary docking site B in the vicinity of the heme iron, from where they migrate to the secondary docking sites, the Xe4 and/or Xe1 cavities. To examine the relevance of these internal docking sites for physiological ligand binding, we have performed room-temperature flash photolysis on the entire set of proteins in the CO- and O(2)-bound form. Together with the cryospectroscopic results, these data provide a clear picture of the role of the internal sites for ligand escape from and binding to myoglobin.     

Modification of the heme distal side in myoglobin by cyanogen bromide. Heme environmental structures and ligand binding properties of the modified myoglobin

Met, deoxy, and CO forms of myoglobin (Mb) react with a stoichiometric amount of cyanogen bromide (BrCN) to cause substantial changes in the 1H NMR, optical absorption, and infrared spectra. These spectral changes were interpreted as arising from the substantial alterations in the heme environments, most probably due to the modification of the histidine residue at the heme distal side. It is also revealed that the modified Mb does not combine with some exogenous ligands such as CN-, CH3NH2, and O2, although it does with N-3 or CO. These unique ligand binding properties are also discussed with relevance to a role of the distal histidine in stabilizing the coordinated ligand through a hydrogen bond and to a steric constraint.     

1H NMR study of the effect of variable ligand on heme oxygenase electronic and molecular structure

Heme oxygenase carries out stereospecific catabolism of protohemin to yield iron, CO and biliverdin. Instability of the physiological oxy complex has necessitated the use of model ligands, of which cyanide and azide are amenable to solution NMR characterization. Since cyanide and azide are contrasting models for bound oxygen, it is of interest to characterize differences in their molecular and/or electronic structures. We report on detailed 2D NMR comparison of the azide and cyanide substrate complexes of heme oxygenase from Neisseria meningitidis, which reveals significant and widespread differences in chemical shifts between the two complexes. To differentiate molecular from electronic structural changes between the two complexes, the anisotropy and orientation of the paramagnetic susceptibility tensor were determined for the azide complex for comparison with those for the cyanide complex. Comparison of the predicted and observed dipolar shifts reveals that shift differences are strongly dominated by differences in electronic structure and do not provide any evidence for detectable differences in molecular structure or hydrogen bonding except in the immediate vicinity of the distal ligand. The readily cleaved C-terminus interacts with the active site and saturation-transfer allows difficult heme assignments in the high-spin aquo complex.     

The effect of heterogeneous structural diffusion on ligand binding to heme proteins

The kinetics of ligand binding to heme proteins studied by flash photolysis display an algebraic time dependence at low temperatures in contrast exponential recombination observed under physiological conditions. This result shows that protein structures should be viewed as a time average of interconverting microstates which are frozen in at low temperatures. We propose a quasi-one-dimensional model of heterogeneous structural diffusion coupled to ligand binding which describes freezing transition as an inherent property of protein fluctuations. The structural hopping rates are derived from a temperature invariant spectrum of activation energies. The model predicts power law kinetics of the form t ^- at long times. The exponent is constant (0.5) at high temperatures but decreases below a critical temperature in the frozen regime. These results are compared to experiments performed with myoglobin and -chains of hemoglobin.     

Spectroscopic characterization of heme A reconstituted myoglobin.

The focus of this study was to examine the functional role of the unusual peripheral substitution of heme A. The effects of heme A stereochemistry on the reconstitution of the porphyrin have been examined in the heme A-apo-myoglobin complex using optical absorption and resonance Raman and electron paramagnetic resonance spectroscopies. The addition of one equivalent of heme A to apo-Mb produces a complex which displays spectroscopic signals consistent with a distribution of high- and low-spin heme chromophores. These results indicate that the incorporation of heme A into apo-Mb significantly perturbs the protein refolding.     

Redox-induced structural dynamics of Fe-heme ligand in myoglobin by X-ray absorption spectroscopy

The Fe(III) --> Fe(II) reduction of the heme iron in aquomet-myoglobin, induced by x-rays at cryogenics temperatures, produces a thermally trapped nonequilibrium state in which a water molecule is still bound to the iron. Water dissociates at T > 160 K, when the protein can relax toward its new equilibrium, deoxy form. Synchrotron radiation x-ray absorption spectroscopy provides information on both the redox state and the Fe-heme structure. Owing to the development of a novel method to analyze the low-energy region of x-ray absorption spectroscopy, we obtain structural pictures of this photo-inducible, irreversible process, with 0.02-0.06-A accuracy, on the protein in solution as well as in crystal. After photo-reduction, the iron-proximal histidine bond is shortened by 0.15 A, a reinforcement that should destabilize the iron in-plane position favoring water dissociation. Moreover, we are able to get the distance of the water molecule even after dissociation from the iron, with a 0.16-A statistical error.     

Dynamics of ligand binding to myoglobin.

Myoglobin rebinding of carbon monoxide and dioxygen after photodissociation has been observed in the temperature range between 40 and 350 K. A system was constructed that records the change in optical absorption at 436 nm smoothly and without break between 2 musec and 1 ksec. Four different rebinding processes have been found. Between 40 and 160 K, a single process is observed. It is not exponential in time, but approximately given by N(t) = (1 + t/to)-n, where to and n are temperature-dependent, ligand-concentration independent, parameters. At about 170 K, a second and at 200 K, a third concentration-independent process emerge. At 210 K, a concentration-dependent process sets in. If myoglobin is embedded in a solid, only the first three can be seen, and they are all nonexponential. In a liquid glycerol-water solvent, rebinding is exponential. To interpret the data, a model is proposed in which the ligand molecule, on its way from the solvent to the binding site at the ferrous heme iron, encounters four barriers in succession. The barriers are tentatively identified with known features of myoglobin. By computer-solving the differential equation for the motion of a ligand molecule over four barriers, the rates for all important steps are obtained. The temperature dependences of the rates yield enthalpy, entropy, and free-energy changes at all barriers. The free-energy barriers at 310 K indicate how myoglobin achieves specificity and order. For carbon monoxide, the heights of these barriers increase toward the inside; carbon monoxide consequently is partially rejected at each of the four barriers. Dioxygen, in contrast, sees barriers of about equal height and moves smoothly toward the binding site. The entropy increases over the first two barriers, indicating a rupturing of bonds or displacement of residues, and then smoothly decreases, reaching a minimum at the binding site. The magnitude of the decrease over the innermost barrier implies participation of heme and/or protein. The nonexponential rebinding observed at low temperatures and in solid samples implies that the innermost barrier has a spectrum of activation energies. The shape of the spectrum has been determined; its existence can be explained by assuming the presence of many conformational states for myoglobin. In a liquid at temperatures above about 230 K, relaxation among conformational states occurs and rebinding becomes exponential.     

The structural dynamics of myoglobin

Conformational fluctuations in proteins were initially invoked to explain the observation that diffusion of small ligands through the matrix is a global phenomenon. Small globular proteins contain internal cavities that play a role not only in matrix dynamics but also in controlling function, tracing a pathway for the diffusion of the ligand to and from the active site. This is the main point addressed in this Review, which presents pertinent information obtained on myoglobin (Mb). Mb, a simple globular heme protein which binds reversibly oxygen and other ligands. The bond between the heme Fe(II) and gaseous ligands can be photodissociated by a laser pulse, generating a non-equilibrium population of protein structures that relaxes on a picosecond to millisecond time range. This process is associated with migration of the ligand to internal cavities of the protein, which are known to bind xenon. Some of the results obtained by laser photolysis, molecular dynamics simulations, and X-ray diffraction of intermediate states of wild-type and mutant myoglobins are summarized. The extended relaxation of the globin moiety directly observed by Laue crystallography reflects re-equilibration among conformational substates known to play an essential role in controlling protein function.     

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.     

Controlling ligand binding in myoglobin by mutagenesis.

A quadruple mutant of sperm whale myoglobin was constructed to mimic the structure found in Ascaris suum hemoglobin. The replacements include His(E7)-->Gln, Leu(B10)-->Tyr, Thr(E10)--> Arg, and Ile(G8)-->Phe. Single, double, and triple mutants were characterized to dissect out the effects of the individual substitutions. The crystal structures of the deoxy and oxy forms of the quadruple mutant were determined and compared with that of native Ascaris hemoglobin. Tyr(B10) myoglobin displays low O(2) affinity, high dissociation rate constants, and heterogeneous kinetic behavior, suggesting unfavorable steric interactions between the B10 phenol side chain and His(E7). In contrast, all mutants containing the Tyr(B10)/Gln(E7) pair show high O(2) affinity, low dissociation rate constants, and simple, monophasic kinetic behavior. Replacement of Ile(107) with Phe enhances nanosecond geminate recombination singly and in combination with the Tyr(B10)/Gln(E7)/Arg(E10) mutation by limiting access to the Xe4 site. These kinetic results and comparisons with native Ascaris hemoglobin demonstrate the importance of distal pocket cavities in governing the kinetics of ligand binding. The approximately 150-fold higher O(2) affinity of Ascaris hemoglobin compared with that for Tyr(B10)/Gln(E7)-containing myoglobin mutants appears to be the result of favorable proximal effects in the Ascaris protein, due to a staggered orientation of His(F8), the lack of a hydrogen bonding lattice between the F4, F7, and F8 residues, and the presence of a large polar Trp(G5) residue in the interior portion of the proximal heme pocket.     

Effects of formylation of vinyl side chains of heme on optical and ligand binding properties of horse heart ferric myoglobin.

Effects of substitution of vinyl groups of hemin with formyl groups on the optical and ligand binding properties of horse heart ferric myoglobin were investigated. The peak positions as well as the line shapes of the absorption spectra of the ferric derivatives of three kinds of formylmyoglobin, 2-vinyl-4-formyl-, 2-formyl-4-vinyl-, and 2,4-diformylmyoglobins depend on the number and the position of the formyl groups. Absorption maxima in the Soret region of the acid forms of these ferric formylmyoglobins in 0.1 M potassium phosphate buffer, pH 6.0, at 20 degrees were 415.2, 422, and 429 nm, respectively. The acid forms of these formylmyoglobins exhibit absorption spectra of the mixture of high- and low spin states at ambient temperature. Since proto-, deutero- and mesomyoglobins have a high spin state under the same condition, the increase of the low spin iron in these formylmyoglobins may be due to the strong electron withdrawal by the formyl groups toward the periphery of the porphyrin ring. The affinities of these ferric formylmyoglobins and protomyoglobin for N3-, F-, OCN-, and SCN- increased in the order of proto-, monoformyl-monovinyl-, 2,4-diformyl-myoglobin, which corresponds to the increasing order of electron-withdrawing power of the porphyrin side chains. The pKa values of the acid-alkaline transition decreased in the same order. Although the ferric forms of the two isomeric monoformyl-monovinylmyoglobins exhibited different optical spectra, the dissociation constants of the complexes of these isomers for various ligands were similar to each other. The pKa values of the acid-alkaline transition were also similar. These results indicate that affinities of ferric myoglobin for ligands, in contrast to those of the ferrous form for oxygen and carbon monoxide (Sono, M., and Asakura, T. (1975) J. Biol. Chem. 250, 5527-5232 and Sono, M., Smith, P.D., McCray, J.A., and Asakura, T. (1976) J. Biol. Chem 251, 1418-1426), are not affected by the position of modifications at the two vinyl groups, but are determinedby the number of the formyl groups and that two vinyl groups at position 2 and 4 are equivalent in the binding of various ligands by ferric myoglobin. The electron density of the ferric iron appears to be similar for the two isomeric monoformyl-monovinylmyoglobins.     

Structural dynamics of myoglobin: ligand migration and binding in valine 68 mutants

We have combined Fourier transform infrared/temperature derivative (FTIR-TDS) spectroscopy at cryogenic temperatures and flash photolysis at ambient temperature to examine the effects of polar and bulky amino acid replacements of the highly conserved distal valine 68 in sperm whale myoglobin. In FTIR-TDS experiments, the CO ligand can serve as an internal voltmeter that monitors the local electrostatic field not only at the active site but also at intermediate ligand docking sites. Mutations of residue 68 alter size, shape, and electric field of the distal pocket, especially in the vicinity of the primary docking site (state B). As a consequence, the infrared bands associated with the ligand at site B are shifted. The effect is most pronounced in mutants with large aromatic side chains. Polar side chains (threonine or serine) have only little effect on the peak frequencies. Ligands that migrate toward more remote sites C and D give rise to IR bands with altered frequencies. TDS experiments separate the photoproducts according to their recombination temperatures. The rates and extent of ligand migration among internal cavities at cryogenic temperatures can be used to interpret geminate and bimolecular O2 and CO recombination at room temperature. The kinetics of geminate recombination can be explained by steric arguments alone, whereas both the polarity and size of the position 68 side chain play major roles in regulating bimolecular ligand binding from the solvent.     

Structural dynamics of myoglobin: spectroscopic and structural characterization of ligand docking sites in myoglobin mutant L29W

We have studied CO binding to the heme and CO migration among protein internal cavities after photodissociation in sperm whale carbonmonoxy myoglobin (MbCO) mutant L29W using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) and kinetic experiments at cryogenic temperatures. Photoproduct intermediates, characterized by CO at particular locations in the protein, were selectively enhanced by applying special laser illumination protocols. These studies were performed on the L29W mutant protein and a series of double mutants constructed so that bulky amino acid side chains block passageways between cavities or fill these sites. Binding of xenon was also employed as an alternative means of occluding cavities. All mutants exhibit two conformations, A(I) and A(II), with distinctly different photoproduct states and ligand binding properties. These differences arise mainly from different positions of the W29 and H64 side chains in the distal heme pocket [Ostermann, A., et al. (2000) Nature 404, 205-208]. The detailed knowledge of the interplay between protein structure, protein dynamics, and ligand migration at cryogenic temperatures allowed us to develop a dynamic model that explains the slow CO and O(2) bimolecular association observed after flash photolysis at ambient temperature.     

Structural dynamics of liganded myoglobin.

X-ray crystallography can reveal the magnitudes and principal directions of the mean-square displacements of every atom in a protein. This structural information is complementary to the temporal information obtainable by spectroscopic techniques such as nuclear magnetic resonance. Determination of the temperature dependence of the mean-square displacements makes it possible to separate large conformational motions from simple thermal vibrations. The contribution of crystal lattice disorder to the overall apparent displacement can be estimated by Mössbauer spectroscopy. This technique has been applied to high resolution x-ray diffraction data from sperm whale myoglobin in its Met iron and oxy cobalt forms. Both crystal structures display regions of large conformational motions, particularly at the chain termini and in the region of the proximal histidine. Overall, the mean-square displacement increases with increasing distance from the center of gravity of the molecule. Some regions of the heme pocket in oxy cobalt myoglobin are more rigid than the corresponding regions in Met myoglobin.     

Proximal ligand motions in H93G myoglobin.

Resonance Raman spectroscopy has been used to observe changes in the iron-ligand stretching frequency in photoproduct spectra of the proximal cavity mutant of myoglobin H93G. The measurements compare the deoxy ferrous state of the heme iron in H93G(L), where L is an exogenous imidazole ligand bound in the proximal cavity, to the photolyzed intermediate of H93G(L)*CO at 8 ns. There are significant differences in the frequencies of the iron-ligand axial out-of-plane mode nu(Fe-L) in the photoproduct spectra depending on the nature of L for a series of methyl-substituted imidazoles. Further comparison was made with the proximal cavity mutant of myoglobin in the absence of exogenous ligand (H93G) and the photoproduct of the carbonmonoxy adduct of H93G (H93G-*CO). For this case, it has been shown that H2O is the axial (fifth) ligand to the heme iron in the deoxy form of H93G. The photoproduct of H93G-*CO is consistent with a transiently bound ligand proposed to be a histidine. The data presented here further substantiate the conclusion that a conformationally driven ligand switch exists in photolyzed H93G-*CO. The results suggest that ligand conformational changes in response to dynamic motions of the globin on the nanosecond and longer time scales are a general feature of the H93G proximal cavity mutant.