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J. Biol. Chem., Vol. 280, Issue 46, 38740-38755, November 18, 2005

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The Position 68(E11) Side Chain in Myoglobin Regulates Ligand Capture, Bond Formation with Heme Iron, and Internal Movement into the Xenon Cavities^*

David Dantsker, Camille Roche, Uri Samuni1, George Blouin, John S. Olson, and Joel M. Friedman2

From the Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461 and the Department of Biochemistry and Cell Biology and the W. M. Keck Center for Computational Biology, Rice University, Houston, Texas 77005-1892

Received for publication, June 10, 2005 , and in revised form, August 29, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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 °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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Proteins are inherently complex materials, and even their simplest reactions exhibit layers of complexity that were not anticipated a few decades ago (1-3). A case in point is carbon monoxide (CO) binding to the heme iron atom in myoglobin (Mb)³ (4). Much of the work on Mb has been directed toward understanding the biophysical principles associated with both bimolecular and internal ligand rebinding after photolysis of the Fe-CO bond. Key issues include the following: (i) the roles of distal and proximal heme pocket amino acids; (ii) the roles of internal water molecules near the active site; (iii) the roles of local and global conformational relaxations that are modulated by solvent; and (iv) the roles of pre-existing internal cavities associated with xenon binding. Time-resolved spectroscopic and x-ray crystallographic studies have demonstrated that dissociated ligands can access internal cavities that arise from packing defects in the globin tertiary structure (5-26). A remaining challenge is to establish quantitatively how all these factors contribute to the multiple kinetic phases associated with internal ligand binding at cryogenic temperatures and high viscosity and to those observed at ambient temperatures and physiologically relevant viscosities (21, 27-37).

Much of the cryogenic work and the time-resolved x-ray crystallography have been carried out with MbCO derivatives. At ambient temperatures, most vertebrate MbCOs exhibit very small amplitudes for the internal rebinding after laser photolysis. Consequently, geminate CO rebinding is usually examined at low temperatures or high viscosity where bimolecular rebinding from the solvent is effectively zero, and ligand escape from the protein is slowed greatly. O₂ and NO complexes are typically used to examine recombination phases in low viscosity solvents at room temperature because of their much higher intrinsic reactivity with the heme iron atom, which enhances the rate of iron-ligand bond formation 10-1,000-fold (10, 14, 38-40). However, in contrast to the stable CO complex, the NO and O₂ derivatives pose difficulties because of autoxidation of the equilibrium ligand-bound A state. Over the past several years, we have developed the use of sol-gels and glycerol solutions that enhance geminate recombination by blocking ligand escape to the solvent. These conditions provide a method to follow the evolution of the multiple kinetic phases for MbCO complexes and other hemeproteins by viscosity tuning at room temperatures in nonfrozen states (28, 30, 33).

In this study, we have focused on a set of Mb double mutants that allow direct determination of the role of the position 68(E11) side chain in partitioning photodissociated CO between the distal heme pocket and the xenon cavities. In each protein the distal histidine (E7) was replaced with leucine (H64L) to prevent water from entering the distal heme pocket and hydrogen bonding to the imidazole side chain after photodissociation of bound CO (41). The Leu(E7) side chain in H64L Mb does not shift position after ligand dissociation (42) (Fig. 1, right panel). After ligand dissociation in wild-type Mb, the imidazole side chain of His⁶⁴ swings inward, occupies a position closer to the iron atom, inhibits internal rebinding, and eventually binds an internal water molecule in the equilibrium unliganded state, markedly inhibiting bimolecular rebinding from the solvent phase (Fig. 1, left panel). In contrast, the Leu⁶⁴ side chain does not inhibit geminate rebinding and increases the overall second-order rate of association, k'_CO, 60-fold (TABLE ONE). Thus, when the H64L mutation is coupled to mutations at position 68(E11), all of the observed differences can be assigned to the size and stereochemistry of the E11 side chain.

View larger version (15K): [in this window] [in a new window]   FIGURE 1. Side view of the distal pocket structures of the deoxygenated and CO forms of wild-type (Protein Data Bank codes 2MGL, 2MGK) and H64L (2MGD, 2MGC) recombinant sperm whale myoglobin. The deoxy-Mb amino acids, heme group, iron atom, and internal water are shown in CPK, silver, orange, and transparent blue, respectively. The MbCO amino acids, heme group, iron atom, and bound CO are shown in silver, blue, orange, and CPK, respectively. The structures are described in Quillin et al. (42). CO binding to wild-type deoxy-Mb causes displacement of internal water and a large outward movement of His(E7), whereas CO binding to the H64L mutant causes no change in conformation of any of the distal pocket side chains.

All of the myoglobin mutants were encapsulated in porous sol-gels, and glycerol was added as a bathing medium. The observed ligand and protein conformational dynamics were followed over a wide range of viscosities (1 10³ cP) by varying the temperature of the samples from 257 to 337 K (-15 to 65 °C). This approach both eliminates complications associated with "glass transitions" that occur at more cryogenic temperatures and avoids the limitations of trehalose matrices that act like glasses precluding smooth transitions from intermediate to low viscosity at room temperature. In the sol-gel/glycerol matrix, solvent-coupled phenomena can be selectively enhanced or suppressed, allowing more direct determination of solvent-decoupled kinetic processes.

This viscosity "tuning" approach complements the cryogenic temperature derivative spectroscopy methods utilized by the Nienhaus group (8, 13, 22, 37, 52), which track both positional changes of dissociated CO and the barrier height changes with temperature. Eaton and co-workers (35, 53) have argued that alterations in viscosity, and not temperature per se, are responsible for the large changes in the geminate rebinding patterns seen for MbCO when going from cryogenic to ambient conditions. In the sol-gel work with glycerol as the bathing medium, high viscosity kinetic patterns are seen at temperatures in the range from -15 to 0 °C and change smoothly to the low viscosity pattern upon elevation to 45 and 65 °C.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Horse heart Mb was commercially obtained from Sigma and used without further purification other than centrifugation to eliminate particulates. Mutant Mbs were produced using an expression system originally developed by Springer and Sligar (54). Construction of the different mutant genes purification of the corresponding proteins followed protocols described previously (44, 45, 49, 54).

Overall association time courses for CO binding to the H64L mutants were measured after photolysis of MbCO samples under pseudo first-order conditions, using a 300-ns excitation pulse from a Phase-R model 2100B dye laser. The transmittance signals were collected using a Tektronix TDS 220 100-MHz digital oscilloscope and IGOR data collection software (Fig. 3B) (14, 43). Time courses for geminate recombination at 20 °C in simple phosphate buffer (Fig. 3A) were observed after excitation with a 9-ns Lumonics (Northville, MI) YM600 YAG laser. In this case, transmittance signals were obtained using a high speed Hamamatsu photomultiplier and a Tektronix TDS 3052 500-MHz digital oscilloscope (Beaverton, OR). A pulsed xenon arc lamp (Applied Photophysics models 04-122 and 03-412, Leatherhead, Surrey, UK) was used to increase the source light intensity during the time required for geminate rebinding (500 µs). CO dissociation time courses were measured in a conventional Gibson-Dionex stopped flow apparatus by mixing MbCO samples in 100 µM CO with buffer containing 2,000 µM NO (1 atm). Under these conditions, the observed replacement rate is directly equal to the overall rate constant for CO dissociation, k_CO.

Geminate recombination measurements in sol-gel and glycerol solutions were carried out using 8-ns 532-nm pulses at 1 Hz from a Nd:YAG laser (Minilite, Continuum, Santa Clara, CA) as a photodissociation source and a greatly attenuated continuous wave 442-nm probe beam from a He:Cd laser to monitor time-dependent changes in absorption. Details of the apparatus, data collection, and data display can be found in a previous publications and the citations therein (32, 55, 56). The kinetic traces are displayed on a log-log plot of normalized absorbance (proportional to fraction of deoxy-Mb photoproduct remaining) versus time. The use of the log-log plot helps expose multiple phases. Exponential rebinding originating from a single barrier and rate constant is seen as a near vertical line intersecting the time axis at a point that is roughly the inverse of the first-order rate constant. Kinetic phases originating from a continuous distribution of rates appear as a linear curve on the log-log plot, with a much smaller slope.

View larger version (52K): [in this window] [in a new window]   FIGURE 2. Top view of the distal pocket structures of the deoxygenated and CO forms of wild-type, V68A (Protein Data Bank codes 1MLG, 1MLF), V68L (1MLR, 1MLQ), and V68F (1MLK, 1MLJ) recombinant sperm whale myoglobin. The structures were taken from Quillin et al. (49). Polar residues are colored blue (His64(E7) and Thr67(E10)); apolar residues are yellow; distal pocket water (W) and initial photodissociated docking site ("B") are transparent blue; CO and heme are CPK; and the iron atom is orange. The position 68 residue is represented by green spheres. In the Ala68 mutant, access to the Xe4 cavity and the iron atom is "open" in both forms, and the occupancy of the distal pocket water in the deoxy-Mb is low. The side chain of Leu68 blocks access to the iron atom from the protein interior in deoxy-Mb but also displaces the distal pocket water. CO binding causes large 90° rotation of the Leu68 side chain about the C--C- bond. This conformational relaxation increases access to the iron atom from the initial docking site. There is a secondary and low occupancy conformation indicated by the transparent green spheres in the V68L MbCO structure. The large phenyl blocks movement into the Xe4 cavity in both forms of V68F myoglobin, but access to the iron atom from the initial docking site, B, enhanced once in the distal pocket water is displaced.

View larger version (24K): [in this window] [in a new window]   FIGURE 3. Geminate and bimolecular CO rebinding to H64L MbCO mutants in 0.1 M Pi, pH 7.0, 20 °C. See text for experimental details. A, photodissociation achieved using a 9-ns excitation pulse. B, photodissociation achieved using a 300-ns excitation pulse. WT, wild type.

Kinetic traces were deconvoluted using the maximum entropy method (MEM) analysis as a visual means of distinguishing kinetic phases and following their evolution as a function of viscosity/temperature. The MEM analysis was performed using an algorithm described previously (57, 58) that is now part of a commercially available package contained within the analysis module in Felix 3.2 software (Photon Technology International, Lawrenceville, NJ). Kinetic data were usually analyzed using a maximum number of anticipated lifetimes and limiting the ² value to 1. Center points of the reported distributions are mean values for the Gaussian peaks. Estimates of the noise ranged from 5 to 10%, depending on the range of the original data. The results of the MEM analysis are presented as the reciprocal of the rates in order to have the distribution displayed on the same axis as the kinetic traces. To a first approximation, the MEM peak position corresponds to the reciprocal of the rebinding rate constant for a given phase, and the peak width provides an estimate of the distribution of rate constants, which presumably represent first-order rebinding in closely related protein-ligand conformations. A single exponential process involving only two conformations and one rebinding process would yield a single, very sharp peak, whereas a large distribution of rates within a given kinetic phase would be reflected in a broad band corresponding to a stretched exponential.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Internal and Bimolecular CO Recombination in Solution—Time courses for both geminate and bimolecular rebinding to the H64L MbCO mutants at pH 7, 20 °C in low viscosity phosphate buffer are shown in Fig. 3 as conventional plots of fraction Mb versus time. The time courses for bimolecular rebinding from the solvent are monophasic and fit to simple, single exponential expressions with k_obs = k'_CO [CO] (Fig. 3A). Time courses for complete CO replacement by NO were also monophasic, and the observed rate constants equals k_CO. Complete sets of overall association and dissociation rate constants are given in TABLES ONE and TWO, and the CO association equilibrium constants were calculated from the ratio k'_CO/k_CO.

The internal nanosecond rebinding time courses exhibit more complex behavior, even in low viscosity buffers (Fig. 3A). Both H64L/V68A and H64L/V68F MbCO show large and rapid geminate phases indicating 80 and 93% internal rebinding. Significantly less recombination is observed for H64L mutants containing Val, Leu, and Asn at position 68(E11). The simplest interpretation of these low viscosity, room temperature data is a two-step binding mechanism shown in Scheme 1, where the fraction of geminate rebinding, F_gem, is defined as k_BA/(k_BA + k_BS), and the apparent first-order rate constant, k_gem, is the sum of the rates of internal rebinding and escape, k_BA + k_BS. Because simple monophasic behavior is observed for bimolecular binding and dissociation, a steady state assumption can be made for the B state intermediate, and the overall association and dissociation rate constants can be expressed as shown in Equation 1,

(Eq. 1)

The individual rate constants in Equation 1 are defined experimentally as k'_SB = k'_CO/F_gem, k_BS = k_gem(1 - F_gem), k_BA = k_gemF_gem, and k_AB = k_CO/(1 - Fgem) (59). Sets of these parameters are listed in TABLE TWO and provide an approximate interpretation of the effects of mutagenesis on CO binding to the H64L mutants at room temperature and low viscosity.

With the exception of H64L/V68N Mb, the rate of thermal bond dissociation, k_AB, is roughly the same for all the mutants and wild-type myoglobin. In the case of the H64L/V68N double mutant, k_AB is reduced roughly 3-fold due hydrogen bonding between the amide NH₂ and the bound ligand, as described previously (51). This decrease in k_AB accounts for the decrease in the overall dissociation rate constant, k_CO, for H64L/V68N MbCO. In contrast, the decreases in k_CO for the H64L/V68A and H64L/V68F mutants are due entirely to greater geminate recombination in these proteins, which decreases the frequency of escape from the initial B geminate state 5- and 10-fold, respectively.

View larger version (6K): [in this window] [in a new window]   SCHEME 1

The results for the H64L, H64L/V68L, and H64L/V68N mutants are difficult to interpret in terms of simple two-step scheme. The poor fits to a single exponential expression of the geminate time courses to the H64L/V68A and H64L/V68N mutants indicate that more complex analyses and interpretations are needed. As has been recognized by many workers in the field, ligand movement into the internal xenon binding cavities must be considered. To examine the competition between escape, iron-ligand bond formation, and migration into these remote sites, we have examined systematically the effects of viscosity on the amplitudes and rates of CO rebinding for the set of myoglobins listed in TABLES ONE and TWO and then used the more general MEM of analysis to obtain distributions of rate constants for the various transitions (TABLE THREE).

Fig. 5 shows the dramatic effects of so-gel encapsulation and high viscosity on the rebinding traces for wild-type, H64L/V68A, H64L/V68L, and H64L/V68F MbCO at -15 °C. Except for samples embedded in trehalose-glass matrices, this condition represents the high viscosity/low temperature limit, in which rebinding from the solvent (k'_BS) is likely to be effectively zero (k'_CO 0.0005 µM, s. Under these conditions, CO recombination in H64L/V68F and H64L/V68A MbCO is extremely fast and nearly monophasic as judged by the large slope of the nearly linear log-log plots (Fig. 4, LA and LF curves). The traces for H64L/V68L sperm whale and native horse heart MbCO, both of which display minimal geminate recombination in solution (Figs. 3 and 4), now show extended initial geminate phases that smoothly evolve into a final slow internal phase that shows either a single rate constant or a narrow distribution of constants and resembles a simple exponential. Wild-type sperm whale Mb exhibit a trace similar to that of the horse heart protein but with slightly different amplitudes for the individual phases. In the trace for horse heart MbCO, the three distinct internal phases are much more clearly delineated in time and are designated B A (10-1000 ns), C A (10-1000 µs), and D A(10 ms)

Figs. 4 and 5 show the extremes in kinetic behavior associated with the viscosity dependence of CO recombination in myoglobin. There is wide range of patterns and multiple internal rebinding phases. To dissect out the structural origin and kinetic parameters for the individual processes, we focused initially on mutants exhibiting the least complicated patterns (H64L/V68F and H64L/V68A) and then moved progressively to those proteins with more complex recombination patterns (H64L/V68L, H64L/V68N, H64L, and wild-type). In each case we started at the low temperature/high viscosity limit and then systematically tuned the system to a temperature/viscosity that yielded kinetic traces resembling more closely those seen in simple solution. TABLE THREE contains the MEM determined rate constants for the different recombination phases obtained for the encapsulated samples bathed in 100% glycerol at 25 °C, except where noted. Higher temperature (65 °C) was needed to generate a measurable population of the slowest phase for two samples (H64L/V68F and H64L/V68A Mb; TABLE THREE).

View larger version (33K): [in this window] [in a new window]   FIGURE 4. Kinetic traces following the rebinding of CO to different Mb species in CO-saturated solutions at 3.5 °C subsequent to photodissociation of the initial COMb derivative using 532-nm excitation pulses (7 ns) and a CW 436-nm probe beam. The kinetic traces are displayed as log normalized absorbance (N.A.) versus log time. The traces are derived from the following samples: LF-Mb(H64L/V68F), LA-Mb(H64L/V68A), L-Mb(H64L), LN-Mb(H64L/V68N), LL-Mb(H64L/V68L), and native-Mb(SW). Experimental details can be found in the text.

View larger version (23K): [in this window] [in a new window]   FIGURE 5. CO recombination traces for sol-gel-encapsulated samples of Mb (sol-gel encapsulated samples are designated as the bracketed species in this and subsequent figures) that were initially prepared with an aqueous buffer as the bathing medium for the gel but were subsequently bathed in an excess of glycerol (100%) and then probed at -15 °C. Traces are derived from the following samples: LF-Mb(H64L/V68F), LA-Mb(H64L/V68A), LA-Mb(H64L/V68L), and Native-Mb(Horse). N.A., normalized absorbance.

Fig. 6, a and b, shows time courses for CO recombination in sol-gel-encapsulated H64L/V68F MbCO and the corresponding MEM-derived kinetic populations. At temperatures ranging from -15 to 45 °C, encapsulated H64L/V68F MbCO in 100% glycerol shows essentially a single rapid phase with a major rate constant peak at 40 µs^-1 and little or no secondary geminate recombination processes (TABLE THREE). The only variation over this temperature regime is a slight increase in the peak of the rate constant distribution with increasing temperature. Below 45 °C, this high viscosity sample exhibits neither the solution phase rebinding shown in Fig. 6c (and Fig. 3) nor any clear cut break indicative of the onset of a second well defined phase. The near linear trace seen in Fig. 6a is clearly due to the B A transition, and the MEM analysis resolves this phase into a major and very minor component (small peak at 1.0 µs^-1), as reported previously (21).

X-ray studies (49) have shown that the single replacement of Val⁶⁸ with Phe has two structural consequences. First, there is a loss of steric hindrance with respect to ligand access to the heme iron that arises in the wild-type protein from the C- of Val⁶⁸. Second, the aromatic side chain occupies a fixed position that hinders access from the distal pocket, state B, to the Xe4 cavity (see Fig. 2 of Ref. 49) (13).

Several simulations (23, 24, 26) and time-resolved x-ray crystallography studies (17, 18) have shown that ligand trajectories from the distal pocket to Xe4 are the most probable route of ligand movement into the protein interior after photodissociation. For the H64L/V68F double mutant, the combination of reduced steric hindrance at the iron and partial filling of the Xe4 cavity combine to cause 100% geminate recombination from the initial B state, and the observed kinetic phase can be assigned with certainty to geminate recombination from the initial docking site labeled as "B" in the structure of wild-type MbCO shown in Fig. 2. The internal barrier for this B A transition is low, whereas the barrier to internal ligand movement is large because of the benzyl side chain hindering access to the adjacent Xe4 cavity.

Recombination from Remote Sites; Side Chain-mediated Motion and the Onset of the C A Recombination Phase—Heating of encapsulated H64L/V68F MbCO to 65 °C in glycerol results in the appearance of a second, much slower rebinding phase, which exhibits rate constants on the order of 1000 s^-1 in 100% glycerol (TABLE THREE). Under these high viscosity conditions, the slowest observed phases show no detectable dependence on external [CO] but, as will be discussed below, are comparable with the pseudo first-order rates seen in solution. The distinct second slower phase in the sol-gel experiment is usually designated the C A transition and is associated with ligands that have migrated to and then returned from a more remote site in the protein by analogy to low temperature CO geminate results (28, 60) and room temperature geminate O₂ rebinding studies (7, 14, 61).

In the H64L/V68F MbCO mutant, the onset of this second phase is unlikely to be due to protein side chain relaxations that alter steric hindrance to ligand rebinding at the iron atom. The Leu⁶⁴ and Phe⁶⁸ side chain positions are the same in the equilibrium deoxy-Mb and MbCO structures of the corresponding single mutants (Fig. 2). However, the high temperature required for the onset of the C A transition in the sol-gel-encapsulated sample suggests that there is a requirement for very large side chain fluctuations in the protein, including the Phe⁶⁸ side chain to allow ligand movement into the adjacent Xe4 cavity and eventually to the proximal Xe1 site.

View larger version (34K): [in this window] [in a new window]   FIGURE 6. CO recombination traces (in black) and associated MEM-derived distribution of lifetimes (in gray) for Mb(H64L/V68F) encapsulated in a sol-gel ([L/F]) bathed in glycerol at 3.5 °C (a) and 65 °C (b), and free in aqueous solution at 3.5 °C (c) and Mb(H64L/V68A) encapsulated in a sol-gel ([L/A]) bathed in glycerol at 3.5 °C (d), and 65 °C (e) and free in aqueous solution at 3.5 °C (f). The MEM populations are presented as the reciprocal of the rate constants for the populations and are displayed on a time axis that is approximately the same as for the kinetic traces in order to help visualize the connection between the population and the feature in the kinetic trace.

The kinetic traces for H64L/V68A MbCO in Fig. 6, d and e, also show only two recombination phases. The rate associated with the first B A transition in both sol-gel and solution samples is 2-3-fold slower than that for the H64L/V68F mutant (Figs. 3 and 6), whereas the rate for the second C A transition seen at 65 °C in the sol-gel is 100-fold larger (0.1 µs^-1).

Replacement of Val⁶⁸ with Ala eliminates the steric contribution of the naturally occurring isopropyl side chain to the recombination barrier as in the case of the V68F substitution. In both mutants, the amplitude of the B A transition dominates the observed time courses, whereas in wild-type Mb, the amplitude of these initial phases is very small, even in the glycerol. However, the alanine substitution also increases the free volume of the distal pocket by making the Xe4 cavity continuous with the B site (Fig. 2). Thus, the ligand in H64L/V68A Mb has more space to occupy in an enlarged B state space, which raises the activation entropy for the B A process. In the H64L/V68F mutant, the ligand is sequestered in a smaller volume closer to the iron atom, which enhances k_BA by lowering in the entropy barrier to iron-ligand bond formation. These effects are seen as faster recombination in the initial nanosecond recombination phases for H64L/V68F MbCO compared with those for the H64L/V68A mutant (see Figs. 3, 4, 5, 6). In aqueous solutions, the H64L/V68A mutant also shows a larger distribution of B A rates. This heterogeneity is manifest as a broader MEM peak in Fig. 6f and a poorer fit to single exponential function in Fig. 3A. The distribution of rates is probably a result of ligand rebinding from a variety of positions in the space associated with the distal pocket and the Xe4 cavity, which are now contiguous. State B in this mutant represents CO delocalized in a significantly larger distal cavity. A much sharper distribution and better fit to a single exponential expression is observed for the H64L/V68F mutant in both sol-gel/glycerol samples and aqueous solution, due presumably to the ligand being constrained in smaller volume adjacent to the iron atom (Figs. 3 and 6).

The larger amplitude and rate of the C A transition in gel-encapsulated H64L/V68A MbCO at 65 °C is because of the open channel between the distal pocket and the interior Xe4 cavity allowing greater access to the protein interior and the proximal Xe1 cavity. As a result, the C A transition in this mutant starts to appear at temperatures well below 45 °C. The amplitude for this second geminate phase increases at the expense of B A as the temperature of the sol-gel/glycerol sample increases. The absence of the C A phase under high viscosity conditions (100% glycerol, sol-gel, 3.5 °C; Fig. 6d), indicates that, even though the B and Xe4 sites are continuous, side chain fluctuations are still needed for the ligand to migrate further into the protein interior and into the Xe1 site, from which ligand rebinding is then seen as a separate recombination phase. However, rebinding from these internal positions is significantly more rapid in the H64L/V68A mutant compared with H64L/V68F because of the lack of a steric barrier between the Xe4 cavity and the distal pocket. As can be seen from TABLE THREE, the C A process for H64L/V68A is comparable with that of the wild type, consistent with the early time wild-type photoproduct not as yet having water in the distal pocket and retaining a conformation allowing for an unrestricted trajectory between Xe4 and the distal heme pocket.

The time courses for CO rebinding to the H64L/V68F and H64L/V68A mutants suggest strongly that the onset of the C A process occurs with the diffusion of the CO out of the distal pocket into remote sites in the protein interior, presumably through a pathway that includes the Xe4 cavity. The observed rapid transit of the ligand among the xenon cavities at ambient temperatures precludes associating the C A transition in the sol-gels with recombination originating from the ligand being completely localized in any one specific xenon cavity (17, 18). This conclusion is in contrast to cryogenic measurements below 200 K where CO occupancy within specific xenon cavities can be monitored spectroscopically and crystallographically (8, 11, 15, 20, 22, 37, 62-64). Under these cryogenic conditions, a clear progression of CO occupancy from the adjacent Xe4 to remote Xe1 sites can be followed as a function of temperature. As a result, specific kinetic phases can be associated with rebinding from the individual cavities (8, 11, 13, 15, 20, 22, 37, 63, 64). The major difference between these cryogenic experiments and the high viscosity, ambient temperature measurements described here is that at the higher temperatures, low amplitude-solvent decoupled fluctuations are considerably less dampened. As a result, the C A transition represents CO rebinding from a distribution of remote sites that in some cases may reflect the equilibrium distribution among the accessible xenon cavities (65).

Recombination from Remote Sites after E-helix Side Chain Relaxations; the D A Transition—In contrast to the V68A and V68F Mb mutants, significant differences are observed between the position of the E11 side chain in the deoxy-Mb and MbCO structures of V68L sperm whale myoglobin (Fig. 2) (49, 51). Greater steric hindrance of access to the iron atom occurs in the deoxy conformation. Under high viscosity, sol-gel conditions, it is probable that rotation of the isobutyl side chains is dampened to greater degree by the sol-gel and glycerol, because of its close proximity to the solvent phase, than the side chain dynamics that regulate diffusion of the CO between the interior of the distal heme pocket and the xenon cavities. Under such conditions, the following two geminate phases from the remote sites occur in the H64L/V68L mutant: one prior to the onset of the large amplitude relaxations of the E11 side chain; and a second, even slower internal rebinding phase occurring after relaxations that place one of the Leu C- atoms directly over the iron atom, inhibiting the B A transition. A similar large relaxation of the His⁶⁴ side chain to a position over the iron atom occurs in wild-type Mb after photodissociation of bound CO (Fig. 1, left panel).

The temperature/viscosity-dependent geminate traces are shown in Fig. 7 for H64L/V68L MbCO. Two phases, designated B A and C A transitions, are seen even at the highest viscosity sol-gel condition at -15 °C. The overall distributions of rates associated with both the B A and the C A phases are very similar to those seen for H64L/V68A, but the amplitude of the slower processes are much larger at all viscosities and temperatures for the H64L/V68L mutant (Figs. 6 and 7). Based on the similarity of the kinetic patterns, we attributed the B A and C A phases for the H64L/V68L mutant at high viscosity (-15 to 3.5 °C, sol-gel) to rebinding from the distal pocket B site and from internal the xenon cavities, respectively.

View larger version (38K): [in this window] [in a new window]   FIGURE 7. CO recombination traces (in black) and associated MEM-derived distribution of lifetimes (in gray) for Mb(H64L/V68L) in glycerol-bathed sol-gel at -15 and 3.5 °C; 25, 45, and 65 °C (a-e, respectively), and in solution at 3.5 °C (f).

Relaxation of the Leu⁶⁸ side chain not only inhibits rebinding from site B but, at the same time, also appears to "push" the photodissociated ligands either toward the protein interior or out of the protein through the histidine gate (Fig. 2, lower left). The latter effect accounts for the 4-8-fold higher calculated rate of CO escape from this mutant (k_BS 44 µs^-1; TABLES ONE and TWO). Note that a similar increase in the rate of escape of photodissociated O₂ was observed for the single V68L mutant (14). In the V68L MbCO structure, multiple orientations of the C- atoms are seen in the electron density maps, differing in rotation about the C--C- bond (Fig. 2, green circles). The alternative conformations indicate steric pressure on the bound ligand and presumably on the ligand in the initial site B "docking" site. Thus, ligand diffusion into the Xe4 channel and toward the Xe1 site is, like direct ligand escape, "activated" by the motions of the Leu(E11) side chain. Above 15 °C, these rotations reduce the fraction of rapid initial rebinding (B A) to 30% in the sol-gel/glycerol samples and to 10% in solution (Figs. 3A and 6f). In the latter case, no slow secondary geminate phases are observed indicating that CO either rebinds from site B in the first 50 ns after photolysis or escapes to the solvent phase.

Further increases in temperature and decreases in viscosity of the sol-gel H64L/V68L sample (Fig. 7, c-e) lead to the appearance of a third, even slower geminate rebinding phase, which is designated D A. This third phase increases in amplitude with increasing temperature at the expense of the C A transition. The amplitude of the first B A transition remains relatively unchanged from 25 to 65 °C. The simplest interpretation is that the onset of the D A phase corresponds to full relaxation of the E11 leucine side chain, which causes a large increase in the barrier to iron-ligand bond formation and a small rate for internal ligand recombination. The increases in amplitude of the slower B A process shown in the high temperature, low viscosity sol-gel time courses (Fig. 7, d and e) support this interpretation. The observed progression of phases suggest that complete E11 side chain relaxation only occurs after photodissociated CO diffuses out of the B site. The assumption is that the fluctuations required for ligand movement into the xenon sites are less dependent on solvent viscosity than those associated with relaxation of the E11 side chain, which are closer to the solvent phase.

In solution the time course for geminate rebinding to H64L/V68L Mb is remarkably simple. Only a small (10%), rapid B A phase (10-50 µs^-1) is observed at either 20 or 3.5 °C. Relaxation of the E11 side chain occurs on the same time scale, pushing the ligand further into the protein interior or out of the protein and, at the same time, inhibiting rebinding from all remote sites, including the solvent phase. The net result is that no geminate rebinding is observed after 50 ns, and 90% of the photodissociated ligands escape from the protein (Fig. 3A). Similar behavior has been observed for CO, O₂, and NO geminate rebinding to V68I Mb. In this case, the C- atom of the sec-butyl side chain rotates directly over the iron atom after photolysis, pushing the dissociated ligand out of the protein or into the Xe4 site and markedly inhibiting iron-ligand bond re-formation. The net result is the absence of geminate CO rebinding, only 12% O₂ nanosecond recombination, and 5-10-fold slower rates of picosecond NO rebinding in the V68I single mutant (14, 49, 59).

The progressive loss of the C A and appearance of the D A phases for the H64L/V68L double mutant with decreasing viscosity is the result of the rate of relaxation of the E11 side chain becoming competitive with the rate of C A rebinding transition. However, even the D A phase is composed of at least two kinetic components based on MEM analysis, with peaks at 15,000 and 5,000 s^-1. These results indicate that, even at 65 °C, some protein conformational inter-conversions are still slower than the remote rebinding process in this double mutant.

Less well defined multiple phases are seen for geminate CO recombination in H64L/V68N Mb (Fig. 8). At the high viscosity limit (-15 to 3.5 °C, Fig. 8, a and b), a broad and slow B A process dominates the rebinding time course. MEM analysis indicates multiple barriers with two rate distribution peaks, the first of which is still significantly smaller than that for the H64L/V68A, H64L/V68F, and H64L/V68L mutants. Similar large barriers to internal rebinding were observed for the single V68N sperm whale and pig Mb mutants (51) and are attributed to direct hindrance from the relatively inflexible planar amide side chain, which sterically hinders both access to the iron atom and to the Xe4 pocket. Log-log plots for the sol-gel/100% glycerol samples are roughly linear with a small slope in the temperature range from -15 to 25 °C. There is no clear "plateau" separating the B A and C A transition. Only at elevated temperatures, 45 °C, are two phases clearly seen: the first being the broad initial B A transition, and the second being a simple exponential but slow process resembling the D A transition seen for the H64L/V68L mutant (Fig. 8e).

The correspondence of the slower geminate phases for the Leu⁶⁸ and Asn⁶⁸ containing double mutants reflects the similarity of the conformations seen in the equilibrium deoxy-Mb structures of the single mutants (49, 51). In both cases, the side chain adopts a conformation where the terminal atoms, C-1 and C-2 in Leu⁶⁸ and O- and N- in Asn⁶⁸, lie almost parallel to the heme plane. One atom blocks access to Xe4, and the other hinders binding to the heme iron from the B site (Fig. 2, V68L deoxy-Mb structure). The marked differences between the V68L and V68N mutants at shorter times and higher viscosities reflect the flexibility of the isobutyl Leu side chain, which can rotate out of the way for bound ligands and adopt a conformation that allows easy access to the iron atom (Fig. 2, V68L MbCO structure). It is the complete rotation of the Leu⁶⁸ side chain from the MbCO to the deoxy-Mb conformation that accounts for relaxation from the C A to the slower D A transition in the H64L/V68L mutant. However, once rotation to the deoxy conformation has occurred, the H64L/V68L mutant shows a rate constant for slow internal rebinding from remote sites that are very similar to that for the V68N double mutant, 3,000 s^-1 (TABLE THREE and Figs. 7 and 8). The similarity of the equilibrium deoxy-Mb Asn⁶⁸ and Leu⁶⁸ conformations also accounts for why the bimolecular rate constants for the H64L/V68L and H64L/V68N double mutants are almost the same, 15-22 µM^-1s^-1 (TABLE ONE).

The Role of His⁶⁴ ; a Comparison between H64L and Wild Type—The temperature/viscosity dependence of CO rebinding in the single H64L mutant is compared with that in wild-type sperm whale Mb in Fig. 9. In sol-gels at the high viscosity limit (100% glycerol, -15 to 3.5 °C), log-log plots are linear with the slope for the wild-type protein being much smaller than that for the faster reacting H64L mutant. The peak positions and distribution of the B A rate parameters for the single H64L mutant are similar to those for the H64L/V68N double mutant, implying that the naturally occurring Val⁶⁸ side chain confers almost as much resistance to iron-ligand bond re-formation as Asn⁶⁸.

In contrast, the rate of initial CO rebinding in wild-type sperm whale Mb is much smaller both in solution and in sol-gels in 100% glycerol at low temperature because of a marked decrease in the amplitude of the B A transitions (Fig. 3A; TABLES ONE and TWO; and Fig. 9d). This effect is clearly due to the His⁶⁴ side chain in wild-type Mb. In the bound A state, the imidazole ring is donating a proton to the bound ligand, and this electrostatic interaction places the N- atom within 3.0 Å of the ligand O atom. After photolysis, the His⁶⁴ side chain actively pushes the dissociated ligand away from the iron as it swings inward to occupy a position over the iron atom. The final conformation of the imidazole ring restricts CO rebinding from any position in the protein (Fig. 1). This inward movement of His⁶⁴ is seen when comparing equilibrium MbCO to deoxy-Mb structures, in recent time-resolved, x-ray crystallographic studies at room temperature with wild-type sperm whale MbCO (18, 66), and accounts for the Stark splitting of the IR bands of photodissociated CO (B states) (67).

View larger version (35K): [in this window] [in a new window]   FIGURE 8. CO recombination traces (in black) and associated MEM-derived distribution of lifetimes (in gray) for Mb(H64L/V68N) in glycerol-bathed sol-gel at -15 and 3.5 °C; 25, 45, and 65 °C (a-e), and in solution at 3.5 °C (f).

View larger version (37K): [in this window] [in a new window]   FIGURE 9. CO recombination traces (in black) and associated MEM-derived distribution of lifetimes (in gray) for Mb(H64L) in a glycerol-bathed sol-gel at -15, 25, and 65 °C (a-c, respectively), and wild-type Mb(sperm whale) in glycerol-bathed sol-gel at 3.5, 25, and 65 °C (d-f, respectively).

View larger version (33K): [in this window] [in a new window]   FIGURE 10. CO recombination traces (in black) and associated MEM-derived distribution of lifetimes (in gray) for COMb(horse) in trehalose glass at 25 °C as a function of increasing exposure to a humid environment (a-d). The details of sample preparation and the measurements can be found in an earlier paper (32).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Assignment and Nomenclature for Recombination Phases; Combining Localization Sites and Protein Relaxation—Our results at ambient temperatures, where the distinction among the remote Xe4 and Xe1 sites cannot be easily made, led us to propose a slightly different nomenclature that takes into account both conformational and CO coordinates. In our interpretations, the A-D states now refer to the CO localized in the following: A, the distal heme pocket, covalently bound to the heme;

B, the distal heme pocket (and sometimes the Xe4 site) but dissociated from the iron; C, the remote xenon cavities (primarily Xe1) under conditions when the major relaxations leading to equilibrium deoxy-Mb structure are not complete; and D, the remote xenon cavities under conditions when the relaxations back to the equilibrium deoxy-Mb population have occurred (Fig. 11). The relaxations that result in the D state can include not only conformational relaxations but also water re-entering a polar distal heme pocket. Thus the A to B transition corresponds to dissociation with the ligand remaining in the distal heme pocket environment; the B to C transition corresponds to the diffusion of the ligand from the heme pocket to remote sites before large amplitude relaxations, and C to D transitions correspond to large amplitude relaxations occurring with the ligand in the remote sites. In Fig 11 we differentiate between B and {B}, which designate states where the dissociated CO is still in the distal heme pocket/Xe4 under conditions where the distribution of local sites and rebinding rates either are not being modulated or are being modulated by low amplitude fluctuations of the DHP side chains, respectively. Similarly, Fig. 11, B_relaxed, designates the state in which the ligand is in the DHP under conditions where full relaxation back to the equilibrium deoxy-Mb conformational distribution has occurred.

Factors Influencing Rebinding from within the Distal Heme Pocket, B A—All of the H64L mutants used in this study display what appear to be two distinct B A phases, which we designate as major and minor for the faster and slower B A phases, respectively. The faster B component is assigned to geminate recombination under conditions where there is minimal steric hindrance at the binding site, which occurs immediately after photolysis but before the onset of side chain relaxations (Fig. 11, top tier of the reaction coordinate diagram and top structure). The minor B A transition appears to be due to a slow down of B A due to side chain fluctuations that either mediate movement of the ligand into the interior portion of the distal pocket and/or sterically slow the return of the ligand to the iron atom (Fig. 11, middle tier structure).

View larger version (35K): [in this window] [in a new window]   FIGURE 11. Scheme and proposed structures for key states and intermediates in ligand binding to Mb. This branched model is similar to that proposed by Scott et al. (14), except that multiple relaxations of the active site are considered. Designations for states are given based on both sites of localization for the ligand and the conformational status of the protein. The A states indicate that the ligand is bound to the heme. The B states indicate that a noncovalently bound ligand is localized within the DHP, which can include some of the contiguous space associated with Xe4. The three B states shown, B, {B} and Brelaxed, refer to ligand localization within the extended DHP over a time window where the local conformation is as follows: (i) still characteristic of the initial liganded derivative (B); still characteristic of the initial liganded derivative but with side chains undergoing conformational fluctuations ({B}); and(iii) fully relaxed to the conformational distribution associated with the deoxy derivative (Brelaxed). The C and D states indicate that the ligand is largely localized within the remote sites such as Xe1, and the protein is largely unrelaxed and fully relaxed, respectively. The S state refers to when the ligand is in the bulk solvent. The large letters on the right-hand side of the structural schematics indicate the dominant contributing state associated with each of the ligand rebinding reactions shown adjacent to each conformational schematic. The structural schematics provide a more detailed interpretation of these states with the gray areas representing estimates of the size of the accessible internal cavities based on observed crystal structures (see Figs. 1 and 2).

Recent fitting of the temperature dependence of the kinetic traces of many of the present mutants (69) reveals a low activation energy protein relaxation occurring on the same time scale as the B minor phase. This time scale coincides with the slowing of the B A phase. In cases where there are no side chains that can contribute to steric hindrance at the iron (H64L/V68A and H64L/V68F mutants), there is minimal slowing of the B A process prior to the onset of the plateau that leads to the slower C A phase,. In addition, in these mutants the Xe4 site is either completely blocked (H64L/V68F Mb) or already open and accessible (H64L/V68A Mb) so that small scale protein relaxations have no effect on ligand movement further into the protein interior. However, as described above, those mutants, which have intermediate-sized side chains, display a clear slowing of the B A phase with increasing temperature (decreasing viscosity), just prior to the onset of the C A phase. In these mutants, the side chains can potentially hinder but not completely block access to the Xe4 site. This temperature slowing effect is seen most clearly as increases in the amplitude of the minor B A phase and the appearance of multiple minor B A MEM populations for the single H64L(V68) and the double H64L/V68N mutants. Based on these observations, we attribute the onset of the minor B A phase to low amplitude side chain motions that facilitate ligand movement out of the B site into the Xe4 cavity. In cases where the side chains are large enough and positioned appropriately, these relaxations may also hinder access to the iron, slowing bond formation directly.

Recombination from Remote Sites; the C A and D A Transitions—In our nomenclature, the C A process is associated with recombination of CO from the remote xenon sites before the completion of large amplitude protein relaxations that restrict the size of site B and iron-ligand bond formation. The rates of the C A transition are very similar (50,000-200,000 s^-1; TABLE THREE) for all the H64L mutants except H64L/V68F Mb, presumably because the sizes of the xenon sites are unaltered and movement between them is governed by more general but internal protein fluctuations that are less influenced by external viscosity. In the case of the H64L/V68F mutant, the Xe4 site is blocked and, as a result, rates of movement into and rebinding from Xe1 are very slow, 2,000 s^-1. This latter result strongly supports our assumption that the Xe4 cavity is part of the pathway for ligand movement into the major remote site, Xe1.

The third and slowest recombination phase, D