The effect of ligand dynamics on heme electronic transition band III in myoglobin.

Band III is a near-infrared electronic transition at ~13,000 cm(-1) in heme proteins that has been studied extensively as a marker of protein conformational relaxation after photodissociation of the heme-bound ligand. To examine the influence of the heme pocket structure and ligand dynamics on band III, we have studied carbon monoxide recombination in a variety of myoglobin mutants after photolysis at 3 K using Fourier transform infrared temperature-derivative spectroscopy with monitoring in three spectral ranges, (1) band III, the mid-infrared region of (2) the heme-bound CO, and (3) the photodissociated CO. Here we present data on mutant myoglobins V68F and L29W, which both exhibit pronounced ligand movements at low temperature. From spectral and kinetic analyses in the mid-infrared, a small number of photoproduct populations can be distinguished, differing in their distal heme pocket conformations and/or CO locations. We have decomposed band III into its individual photoproduct contributions. Each photoproduct state exhibits a different "kinetic hole-burning" (KHB) effect, a coupling of the activation enthalpy for rebinding to the position of band III. The analysis reveals that the heme pocket structure and the photodissociated CO markedly affect the band III transition. A strong kinetic hole-burning effect results only when the CO ligand resides in the docking site on top of the heme group. Migration of CO away from the heme group leads to an overall blue shift of band III. Consequently, band III can be used as a sensitive tool to study ligand dynamics after photodissociation in heme proteins.     

Heme protein dynamics revealed by geminate nitric oxide recombination in mutants of iron and cobalt myoglobin

Nitric oxide myoglobin (MbNO) at 300 K was photodissociated with 405 nm pulses. The NO recombination in several mutants of iron and cobalt myoglobins was investigated at a time resolution of ca. 70 fs. The geminate recombination of NO was nonexponential on sub-nanosecond time scales. For both metals, the change of the detailed structure of the heme pocket (position 68 mutations) caused significant changes in the rates of recombination; however, the metal substitution influenced the recombination much less than did amino acid substitution. The results indicate a primary role of the heme pocket structure in the dynamics, and they suggest that proximal protein relaxation is not the limiting factor in the geminate recombination process. Recombination in cobalt derivatives is somewhat more efficient on the sub-nanosecond time scales than in corresponding iron myoglobins, consistent with other results that show a greater intrinsic reactivity toward the NO of cobalt compared with the iron heme. A comparison of results using Soret band excitation with previous Q-state excitation studies demonstrates that the ligand dissociates with a similar kinetic energy in both cases, suggesting fast intramolecular energy redistribution before dissociation.     

Analysis of the kinetic barriers for ligand binding to sperm whale myoglobin using site-directed mutagenesis and laser photolysis techniques

Time courses for NO, O2, CO, methyl and ethyl isocyanide rebinding to native and mutant sperm whale myoglobins were measured at 20 degrees C following 17-ns and 35-ps laser excitation pulses. His64 (E7) was replaced with Gly, Val, Leu, Phe, and Gln, and Val68 (E11) was replaced with Ala, Ile, and Phe. For both NO and O2, the effective picosecond quantum yield of unliganded geminate intermediates was roughly 0.2 and independent of the amino acids at positions 64 and 68. Geminate recombination of NO was very rapid; 90% rebinding occurred within 0.5-1.0 ns for all of the myoglobins examined; and except for the Gly64 and Ile68 mutants, the fitted recombination rate parameters were little influenced by the size and polarity of the amino acid at position 64 and the size of the residue at position 68. The rates of NO recombination and ligand movement away from the iron atom in the Gly64 mutant increased 3-4-fold relative to native myoglobin. For Ile68 myoglobin, the first geminate rate constant for NO rebinding decreased approximately 6-fold, from 2.3 x 10(10) s-1 for native myoglobin to 3.8 x 10(9) s-1 for the mutant. No picosecond rebinding processes were observed for O2, CO, and isocyanide rebinding to native and mutant myoglobins; all of the observed geminate rate constants were less than or equal to 3 x 10(8) s-1. The rebinding time courses for these ligands were analyzed in terms of a two-step consecutive reaction scheme, with an outer kinetic barrier representing ligand movement into and out of the protein and an inner barrier representing binding to the heme iron atom by ligand occupying the distal portion of the heme pocket. Substitution of apolar amino acids for His64 decreased the absolute free energies of the outer and inner kinetic barriers and the well for non-covalently bound O2 and CO by 1 to 1.5 kcal/mol, regardless of size. In contrast, the His64 to Gln mutation caused little change in the barrier heights for all ligands, showing that the polar nature of His64 inhibits both the bimolecular rate of ligand entry into myoglobin and the unimolecular rate of binding to the iron atom from within the protein. Increasing the size of the position 68(E11) residue in the series Ala to Val (native) to Ile caused little change in the rate of O2 migration into myoglobin or the equilibrium constant for noncovalent binding but did decrease the unimolecular rate for iron-O2 bond formation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structure-dynamics-function relationships in Asian elephant (Elephas maximus) myoglobin. An optical spectroscopy and flash photolysis study on functionally important motions.

In this work we report the thermal behavior (10-300 K) of the Soret band lineshape of deoxy and carbonmonoxy derivatives of Asian elephant (Elephas maximus) and horse myoglobins together with their carbon monoxide recombination kinetics after flash photolysis; the results are compared to analogous data relative to sperm whale myoglobin. The Soret band profile is modeled as a Voigt function that accounts for the coupling with high and low frequency vibrational modes, while inhomogeneous broadening is taken into account with suitable distributions of purely electronic transition frequencies. This analysis makes it possible to isolate the various contributions to the overall lineshape that; in turn, give information on structural and dynamic properties of the systems studied. The optical spectroscopy data point out sizable differences between elephant myoglobin on one hand and horse and sperm whale myoglobins on the other. These differences, more pronounced in deoxy derivatives, involve both the structure and dynamics of the heme pocket; in particular, elephant myoglobin appears to be characterized by larger anharmonic contributions to soft modes than the other two proteins. Flash photolysis data are analyzed as sums of kinetic processes with temperature-dependent fractional amplitudes, characterized by discrete pre-exponentials and either discrete or distributed activation enthalpies. In the whole temperature range investigated the behavior of elephant myoglobin appears to be more complex than that of horse and sperm whale myoglobins, which is in agreement with the increased anharmonic contributions to soft modes found in the former protein. Thus, to satisfactorily fit the time courses for CO recombination to elephant myoglobin five distinct processes are needed, only one of which is populated over the whole temperature range investigated. The remarkable convergence and complementarity between optical spectroscopy and flash photolysis data confirms the utility of combining these two experimental techniques in order to gain new and deeper insights into the functional relevance of protein fluctuations.     

The position 68(E11) side chain in myoglobin regulates ligand capture, bond formation with heme iron, and internal movement into the xenon cavities

After photodissociation, ligand rebinding to myoglobin exhibits complex kinetic patterns associated with multiple first-order geminate recombination processes occurring within the protein and a simpler bimolecular phase representing second-order ligand rebinding from the solvent. A smooth transition from cryogenic-like to solution phase properties can be obtained by using a combination of sol-gel encapsulation, addition of glycerol as a bathing medium, and temperature tuning (-15 --> 65 degrees C). This approach was applied to a series of double mutants, myoglobin CO (H64L/V68X, where X = Ala, Val, Leu, Asn, and Phe), which were designed to examine the contributions of the position 68(E11) side chain to the appearance and disappearance of internal rebinding phases in the absence of steric and polar interactions with the distal histidine. Based on the effects of viscosity, temperature, and the stereochemistry of the E11 side chain, the three major phases, B --> A, C --> A, and D --> A, can be assigned, respectively, to ligand rebinding from the following: (i) the distal heme pocket, (ii) the xenon cavities prior to large amplitude side chain conformational relaxation, and (iii) the xenon cavities after significant conformational relaxation of the position 68(E11) side chain. The relative amplitudes of the B --> A and C --> A phases depend markedly on the size and shape of the E11 side chain, which regulates sterically both ligand return to the heme iron atom and ligand migration to the xenon cavities. The internal xenon cavities provide a transient docking site that allows side chain relaxations and the entry of water into the vacated distal pocket, which in turn slows ligand recombination markedly.     

Modulated excitation of singly ligated carboxyhemoglobin.

We have extended the method of modulated excitation, a small perturbation kinetic method, to study ligand binding and conformational change of hemoglobin tetramers with a single ligand bound. To restrict the excitation to the first ligand, only 1% of the hemes have bound CO, and the remainder are kept unliganded. A detailed theory is presented which agrees well with the experimental observations. This method of observing ligand recombination also provides a novel and simple method for determination of hemoglobin concentration. Additional relaxation processes are also observed. By fitting independently determined spectra to the spectra associated with the relaxations, these processes are assigned as thermal excitation and thermally driven protonation/deprotonation reactions. These added relaxations arise from the deoxy-Hb portion of the samples, and demonstrate that modulated excitation can be used effectively for temperature perturbation in the absence of photodissociation. The spectra observed are not well described by the spectra of allosteric change, however, and we conclude that there is no significant mixing of quaternary states at the first ligation step. In an appendix we present a derivation of the particular features seen in thermally modulated protonation reactions.     

Dynamics and mechanisms of coupled protein folding and binding reactions

Protein folding coupled to binding of a specific ligand is frequently observed in biological processes. In recent years numerous studies have addressed the structural properties of the unfolded proteins in the absence of their ligands. Surprisingly few time-resolved investigations on coupled folding and binding reactions have been published up to date and the dynamics and kinetic mechanisms of these processes are still only poorly understood. Especially, it is still unsolved for most systems which conformation of the protein is recognized by the ligand (conformational selection vs. folding-after-binding) and whether the ligand influences the folding kinetics. Here we review experimental methods, kinetic models and time-resolved experimental studies of coupled folding and binding reactions.     

Thermodynamics of alligator metmyoglobin unfolding

The conformational free energy of alligator metmyoglobin was examined over a pH range of 4.4-8.0, a temperature range of 18-48 degrees C, and a guanidinium chloride concentration of 0-2.3 M. For isothermal unfolding at 25 degrees C essentially the same value was obtained for the conformational free energy from all the data: 7.0 +/- 0.5 kcal/mol. The cooperativity of the unfolding with respect to denaturant is considerably less than for mammalian myoglobins. On unfolding three to four side chains with a pKa of 6.3 in the unfolded protein are protonated instead of the six expected. The temperature at which delta H (unfolding) is zero is much lower than for previously characterized myoglobins. Alligator metmyoglobin, considerably less stable than other previously characterized myoglobins, may not be as compactly folded.     

Viscosity-dependent relaxation significantly modulates the kinetics of CO recombination in the truncated hemoglobin TrHbN from Mycobacterium tuberculosis

Kinetic traces were generated for the nanosecond and slower rebinding of photodissociated CO to trHbN in solution and in porous sol-gel matrices as a function of viscosity, conformation, and mutation. TrHbN is one of the two truncated hemoglobins from Mycobacterium tuberculosis. The kinetic traces were analyzed in terms of three distinct phases. These three phases are ascribed to rebinding: (i) from the distal heme pocket, (ii) from the adjacent apolar tunnel prior to conformational relaxation, and (iii) from the apolar tunnel subsequent to conformational relaxation. The fractional content of each of these phases was shown to be a function of the viscosity and, in the case of the sol-gel-encapsulated samples, sample preparation history. The observed kinetic patterns support a model consisting of the following elements: (i) the viscosity and conformation-sensitive dynamics of the Tyr(B10) side chain facilitate diffusion of the dissociated ligand from the distal heme pocket into the adjacent tunnel; (ii) the distal heme pocket architecture determines ligand access from the tunnel back to the heme iron; (iii) the distal heme pocket architecture is governed by a ligand-dependent hydrogen bonding network that limits the range of accessible side chain positions; and (iv) the apolar tunnel linking the heme site to the solvent biases the competition between water and ligand for occupancy of the vacated polar distal heme pocket greatly toward the nonpolar ligand. Implications of these finding with respect to biological function are discussed.     

Structural dynamics of myoglobin: ligand migration among protein cavities studied by Fourier transform infrared/temperature derivative spectroscopy.

Fourier transform infrared (FTIR) spectroscopy in the CO stretch bands combined with temperature derivative spectroscopy (TDS) was used to characterize intermediate states obtained by photolysis of two sperm whale mutant myoglobins, YQR (L29(B10)Y, H64(E7)Q, T67(E10)R) and YQRF (with an additional I107(G8)F replacement). Both mutants assume two different bound-state conformations, A(0) and A(3), which can be distinguished by their different CO bands near 1965 and 1933 cm(-1). They most likely originate from different conformations of the Gln-64 side chain. Within each A substate, a number of photoproduct states have been characterized on the basis of the temperature dependence of recombination in TDS experiments. Different locations and orientations of the ligand within the protein can be distinguished by the infrared spectra of the photolyzed CO. Recombination from the primary docking site, B, near the heme dominates below 50 K. Above 60 K, ligand rebinding occurs predominantly from a secondary docking site, C', in which the CO is trapped in the Xe4 cavity on the distal side, as shown by crystallography of photolyzed YQR and L29W myoglobin CO. Another kinetic state (C") has been identified from which rebinding occurs around 130 K. Moreover, a population appearing above the solvent glass transition at approximately 180 K (D state) is assigned to rebinding from the Xe1 cavity, as suggested by the photoproduct structure of the L29W sperm whale myoglobin mutant. For both the YQR and YQRF mutants, rebinding from the B sites near the heme differs for the two A substates, supporting the view that the return of the ligand from the C', C", and D states is not governed by the recombination barrier at the heme iron but rather by migration to the active site. Comparison of YQR and YQRF shows that access to the Xe4 site (C') is severely restricted by introduction of the bulky Phe side chain at position 107.     

Contributions of residue 45(CD3) and heme-6-propionate to the biomolecular and geminate recombination reactions of myoglobin

Overall association and dissociation rate constants were measured at 20 degrees C for O2, CO, and alkyl isocyanide binding to position 45 (CD3) mutants of pig and sperm whale myoglobins and to sperm whale myoglobin reconstituted with protoheme IX dimethyl ester. In pig myoglobin, Lys45(CD3) was replaced with Arg, His, Ser, and Glu; in sperm whale myoglobin, Arg45(CD3) was replaced with Ser and Gly. Intramolecular rebinding of NO, O2, and methyl isocyanide to Arg45, Ser45, Glu45, and Lys45(native) pig myoglobins was measured following 35-ps and 17-ns excitation pulses. The shorter, picosecond laser flash was used to examine ligand recombination from photochemically produced contact pairs, and the longer, nanosecond flash was used to measure the rebinding of ligands farther removed from the iron atom. Mutations at position 45 or esterification of the heme did not change significantly (less than or equal to 2-fold) the overall association rate constants for NO, CO, and O2 binding at room temperature. These data demonstrate unequivocally that Lys(Arg)45 makes little contribution to the outer kinetic barrier for the entry of diatomic gases into the distal pocket of myoglobin, a result that contradicts a variety of previous structural and theoretical interpretations. However, the rates of geminate recombination of NO and O2 and the affinity of myoglobin for O2 were dependent upon the basicity of residue 45. The series of substitutions Arg45, Lys45, Ser45, and Glu45 in pig myoglobin led to a 3-fold decrease in the initial rate for the intramolecular, picosecond rebinding of NO and 4-fold decrease in the geminate rate constant for the nanosecond rebinding of O2. (ABSTRACT TRUNCATED AT 250 WORDS)     

Structural relaxation and nonexponential kinetics of CO-binding to horse myoglobin. Multiple flash photolysis experiments.

The geminate recombination kinetics of CO-myoglobin strongly deviates from single exponential behavior in contrast to what is expected for unimolecular reactions (1). At low temperatures, this result was attributed to slowly exchanging conformational states which differ substantially in barrier height for ligand binding. Above 160 K the kinetics apparently slow down with temperature increase. Agmon and Hopfield (2) explain this result in terms of structural relaxation perpendicular to the reaction coordinate, which enhances the activation energy. In their model, structural relaxation homogenizes the kinetic response. Recently, Steinbach et al. (3) proposed a relaxation model which conserves the kinetic inhomogeneity. Below we test these conjectures by single and multiple excitation experiments. This method allows for discrimination between parallel (inhomogeneous) and sequential (homogeneous) kinetic schemes. The kinetic anomaly above 160 K is shown to result from a homogeneous, structurally relaxed intermediate. However a second anomaly is found above 210 K concerning the inhomogeneous phase which may indicate either a shift in activation energy or entropy.     

Magnetic resonance studies of the binding of 13C-labeled carbon monoxide to myoglobins and hemoglobins containing modified hemes.

The effects of changes in the groups attached to the periphery of the porphyrin ring of the heme of various hemoglobin and myoglobins on the environment experienced by the ligand, carbon monoxide, have been studied by observation of the chemical shift of the bound 13CO. The results indicate that the major interaction between bound ligands and substituents around the porphyrin is that transmitted electronically from substituent to ligand. The nature of the protein environment around the ligand and the interaction between the proximal histidine (F8) and the ligand (through the iron atom) impose differences between subunits of hemoglobin and between myoglobins and hemoglobins which are largely, but not entirely, independent of these substituent effects. To assess the influence of protein structure on the chemical shifts of bound ligand, the shifts of 13CO bound to myoglobin and hemoglobins from a wide range of species have also been measured.     

Nitric oxide binding to the cardiolipin complex of ferric cytochrome C

Cardiolipin, a phospholipid specific to the mitochondrion, interacts with the small electron transfer heme protein cytochrome c through both electrostatic and hydrophobic interactions. Once in a complex with cardiolipin, cytochrome c has been shown to undergo a conformational change that leads to the rupture of the bond between the heme iron and the intrinsic sulfur ligand of a methionine residue and to enhance the peroxidatic properties of the protein considered important to its apoptotic activity. Here we report that the ferric cytochrome c/cardiolipin complex binds nitric oxide tightly through a multistep process in which the first step is the relatively slow displacement (5 s(-1)) from heme coordination of an intrinsic ligand that replaces methionine in the complex. Nanosecond photolysis of the nitrosyl adduct demonstrated that a fraction of the nitric oxide escapes from the heme pocket and subsequently recombines to the heme in second-order processes (k = 1.8 × 10(6) and 5.5 × 10(5) M(-1) s(-1)) that, under these conditions, were much faster than recombination of the intrinsic ligand with which they compete. Ultrafast (femtosecond) laser photolysis showed that the geminate recombination of nitric oxide to the heme occurred with time constants (τ = 22 and 72 ps) and that ～23% of the photolyzed nitric oxide escaped into the bulk phase. This high value for the escape fraction relative to other heme proteins indicates the open nature of the heme pocket in this complex. These results are summarized in a scheme and are discussed in terms of the possible modulation of the apoptotic activity of cytochrome c by nitric oxide.     

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.     

Conformational free energies of myoglobins of small mammals

Myoglobins from three small placental mammals and one small marsupial were isolated from cardiac or skeletal muscle. The conformational free energies of these four myoglobins were estimated from guanidinium chloride unfolding data at pH 8 and 25 degrees. The myoglobins from rat and rabbit are more stable than that of the most stable myoglobin previously studied, that of the sperm whale. In addition, these two myoglobins unfold with greater cooperativity than previously characterized myoglobins. The data obtained herein demonstrate unequivocally for the first time that the stability of homeotherm myoglobins correlates with neither the size of the organism nor its metabolic rate.     

Picosecond geminate recombination of nitrosylmyoglobins

The kinetics of NO geminate recombination to sperm whale and elephant myoglobins has been studied on the picosecond time scale using an amplified colliding-pulse mode-locked ring dye laser. The dynamics of ligand rebinding are shown to be affected by the distal structure of the protein surrounding the heme pocket.     

Ligand and proton exchange dynamics in recombinant human myoglobin mutants

Site-specific mutants of human myoglobin have been prepared in which lysine 45 is replaced by arginine (K45R) and aspartate 60 by glutamate (D60E), in order to examine the influence of these residues and their interaction on the dynamics of the protein. These proteins were studied by a variety of methods, including one and two-dimensional proton nuclear magnetic resonance spectroscopy, exchange kinetics for the distal and proximal histidine NH protons as a function of pH in the met cyano forms, flash photolysis of the CO forms, and ligand replacement kinetics. The electronic absorption and proton nuclear magnetic resonance spectra of the CO forms of these proteins are virtually identical, indicating that the structure of the heme pocket is unaltered by these mutations. There are, however, substantial changes in the dynamics of both CO binding and proton exchange for the mutant K45R, whereas the mutant D60E exhibits behavior indistinguishable from the reference human myoglobin. K45R has a faster CO bimolecular recombination rate and slower CO off-rate relative to the reference. The kinetics for CO binding are independent of pH (6.5 to 10) as well as ionic strength (0 to 1 M-NaCl). The exchange rate for the distal histidine NH is substantially lower for K45R than the reference, whereas the proximal histidine NH exchange rate is unaltered. The exchange behavior of the human proteins is similar to that reported for a comparison of the exchange rates for myoglobins having lysine at position 45 with sperm whale myoglobin, which has arginine at this position. This indicates that the differences in exchange rates reflects largely the Lys----Arg substitution. The lack of a simple correlation for the CO kinetics with this substitution means that these are sensitive to other factors as well. Specific kinetic models, whereby substitution of arginine for lysine at position 45 can affect ligand binding dynamics, are outlined. These experiments demonstrate that a relatively conservative change of a surface residue can substantially perturb ligand and proton exchange dynamics in a manner that is not readily predicted from the static structures.     

Conformational selection in protein binding and function

Protein binding and function often involves conformational changes. Advanced nuclear magnetic resonance (NMR) experiments indicate that these conformational changes can occur in the absence of ligand molecules (or with bound ligands), and that the ligands may “select” protein conformations for binding (or unbinding). In this review, we argue that this conformational selection requires transition times for ligand binding and unbinding that are small compared to the dwell times of proteins in different conformations, which is plausible for small ligand molecules. Such a separation of timescales leads to a decoupling and temporal ordering of binding/unbinding events and conformational changes. We propose that conformational‐selection and induced‐change processes (such as induced fit) are two sides of the same coin, because the temporal ordering is reversed in binding and unbinding direction. Conformational‐selection processes can be characterized by a conformational excitation that occurs prior to a binding or unbinding event, while induced‐change processes exhibit a characteristic conformational relaxation that occurs after a binding or unbinding event. We discuss how the ordering of events can be determined from relaxation rates and effective on‐ and off‐rates determined in mixing experiments, and from the conformational exchange rates measured in advanced NMR or single‐molecule fluorescence resonance energy transfer experiments. For larger ligand molecules such as peptides, conformational changes and binding events can be intricately coupled and exhibit aspects of conformational‐selection and induced‐change processes in both binding and unbinding direction.