Kinetic Modulation in Carbonmonoxy Derivatives of Truncated Hemoglobins

THE ROLE OF DISTAL HEME POCKET RESIDUES AND EXTENDED APOLAR TUNNEL*

Uri Samuni {ddagger}, David Dantsker {ddagger}, Anandhi Ray {ddagger}, Jonathan B. Wittenberg {ddagger}, Beatrice A. Wittenberg {ddagger}, Sylvia Dewilde §, Luc Moens §, Yannick Ouellet ¶, Michel Guertin ¶ and Joel M. Friedman {ddagger} ||

From the {ddagger}Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, the Department of Biochemistry and Microbiology, Faculty of Sciences and Engineering, Laval University, Quebec G1K 7P4, Canada, and the §Department of Biomedical Sciences, University of Antwerp (UIA), Universitreitsplein 12610 Wilrijk (Antwerpen), Belgium

Received for publication, December 11, 2002 , and in revised form, April 11, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Truncated hemoglobins (trHbs), are a distinct and newly characterized class of small myoglobin-like proteins that are widely distributed in bacteria, unicellular eukaryotes, and higher plants. Notable and distinctive features associated with trHbs include a hydrogen-bonding network within the distal heme pocket and a long apolar tunnel linking the external solvent to the distal heme pocket. The present work compares the geminate and solvent phase rebinding kinetics from two trHbs, one from the ciliated protozoan Paramecium caudatum (P-trHb) and the other from the green alga Chlamydomonas eugametos (C-trHb). Unusual kinetic patterns are observed including indications of ultrafast (picosecond) geminate rebinding of CO to C-trHb, very fast solvent phase rebinding of CO for both trHbs, time-dependent biphasic CO rebinding kinetics for P-trHb at low CO partial pressures, and for P-trHb, an increase in the geminate yield from a few percent to nearly 100% under high viscosity conditions. Species-specific differences in both the 8-ns photodissociation quantum yield and the rebinding kinetics, point to a pivotal functional role for the E11 residue. The response of the rebinding kinetics to temperature, ligand concentration, and viscosity (glycerol, trehalose) and the viscosity-dependent changes in the resonance Raman spectrum of the liganded photoproduct, together implicate both the apolar tunnel and the static and dynamic properties of the hydrogen-bonding network within the distal heme pocket in generating the unusual kinetic patterns observed for these trHbs.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Hemoglobin- and myoglobin-like proteins have been found in virtually all categories of living organisms. Recently, a distinct new class of hemoglobin-like proteins, termed truncated hemoglobins (trHbs),1 has been identified (19). Truncated hemoglobins are small heme proteins that are widely distributed in bacteria, unicellular eukaryotes, and higher plants (10, 11). They share very little in the way of sequence identity with members of other hemoglobin families. Many are found in pathogenic organisms. Most but not all of the examined truncated Hbs exhibit very high oxygen affinities that would tend to rule out oxygen transport as a function (5, 12, 13). Although their function(s) is (are) as yet uncertain, it has been proposed (3, 14, 15) and demonstrated (16, 17) that in some instances they may play a key role in protecting the pathogenic organisms from the toxic effects of NO.

A comparison of amino acid sequences within this family of proteins reveals several common features including a proximal histidine. Most notable from the perspective of functionality are the residues found in the distal heme pocket. Nearly all trHbs have a tyrosine at a position analogous to the leucine at the B10 site in vertebrate myoglobins. The B10 tyrosine motif appears to be very wide spread among myoglobins and hemoglobins from nonvertebrate organisms and may well represent the primitive condition associated with Hb/Mb evolution. For trHbs and many other nonvertebrate Hbs, the distal histidine at position E7 found in most vertebrate Hbs and Mbs is often replaced with a glutamine and in some instances with nonpolar residues such as alanine or leucine. Other distal heme pocket residue sites such as E11 and G8 that have been shown to be of functional importance in vertebrate Hbs and Mbs display considerable variability (1820). As will be further discussed below, the myriad combinations associated with these sites, appear to contribute to a ligation-sensitive hydrogen-bonding network that plays a significant role not only in stabilizing the bound ligand but also in the rebinding kinetics of dissociated ligands.

An initial crystallographic study (6) on two representative trHbs, one from the protozoan Paramecium caudatum (P-trHb) and the other from the alga Chlamydomonas eugamentos (C-trHb) and a subsequent study on trHbN (14), a trHb from Mycobacterium tuberculosis, reveal distinct structural features associated with this group of proteins. Although there is resemblance to the myoglobin fold, there are dramatic differences. A "two-over-two" {alpha}-helical sandwich replaces the classic "three-over-three" myoglobin {alpha}-helical fold. The A helix and the CD-D region are reduced or eliminated, and the F-helix is almost completely replaced by an extended pre-F-helix polypeptide loop. The B10 tyrosine hydrogen also bonds both to hemebound ligands and to other residues within the distal heme pocket as observed in nematode Hb (21), trematode Hb (22), and mutant sperm whale Mbs designed to mimic Ascaris Hb (23, 24). Both the crystallography studies referred to above and the series of spectroscopic and kinetic studies (4, 5, 9, 13, 15, 2528) on these and related proteins reveal considerable conformational plasticity associated with this hydrogen-bonding network.

A truly striking feature observed in the crystal structures of the three studied trHbs, is the large extended apolar tunnel linking the distal heme pocket to the external solvent. Sequence analysis indicates that this tunnel may be a common feature in many members of the trHb family (14). An analogous, but distinctly different (in terms of residues involved), tunnel has also been identified in the structure of a small Mb-like neural heme protein from Cererabratulus lacteus (29). This protein does not group with any of the existing subfamilies of Hbs and Mbs including trHbs. Much smaller discontinuous hydrophobic cavities, referred to as Xe cavities, have been identified in Mbs (30, 31) and other heme proteins (32). Recent studies indicate that these Xe cavities, acting as docking sites for potential heme ligands, can have a profound effect on ligand binding kinetics (23, 3341). In particular multiple phases for the geminate recombination of CO to Mb have been attributed to ligands rebinding from different Xe cavities. It has been proposed that these Xe cavities play a functional role in modulating both ligand binding kinetics and multiligand reactions such as NO-mediated oxidation reactions of O2Mb (36, 39). Similar functional roles have been proposed for the large continuous tunnel in truncated Hbs that covers and links the spatial domains of Xe cavities seen in Mb (14).

The usual distal E7 gate for the entering and exiting of ligands to and from the distal heme pocket (18, 42, 43) is not evident in trHbs (14). Thus the tunnel may be a key pathway for ligand diffusion to the heme. By virtue of its large volume, the tunnel may represent a means both of concentrating and storing nonpolar ligands within the protein.

The primary focus of the present study is to compare the CO rebinding patterns of two trHbs: one from the protozoan P. caudatum (P-trHb) and the other from the alga C. eugametos (C-trHb). Their three-dimensional structures indicate that they share all but one of the key distal residues (henceforth designated and ordered as follows: E7/B10/E11/G8/CD1) as shown in Fig. 1 and Table I. For C-trHb and P-trHb these residues are Q/Y/Q/V/F and Q/Y/T/V/F, respectively. There is also an H-bond network between the distal residues B10Y, E7Q, and E11Q/T and between the bound ligands and the B10Y and E7Q. In addition, they both exhibit an open tunnel. Despite these similarities, they display O2 dissociation rates that differ by 2,000-fold. Questions being explored in the present study include: (i) Is there a kinetic pattern common to these trHbs and if so is it distinct from that of vertebrate myoglobins? (ii) Are there indications that the observed kinetic patterns are influenced by the architecture of either the distal heme pocket or the apolar tunnel? (iii) Do protein dynamics play an important role in modulating the kinetic patterns observed for these trHbs?



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FIG. 1.
Structures of P-trHb and C-trHb. These are derived from the x-ray data reported by Bolognesi and co-workers (6) illustrating both the tunnel-like network of hydrophobic cavities linking the solvent to the distal heme pocket and the distal heme pocket residues that are positioned to influence ligand binding dynamics.

 

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TABLE I
Summary of results

 

These questions are addressed by monitoring geminate and solution phase CO rebinding as a function of ligand concentration and solvent viscosity/temperature. A limited set of Raman measurements on some of these samples is used to assess conformational plasticity in response to ligation and deligation. These studies reveal kinetic patterns that are distinctly different from those observed in other Hbs and Mbs.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Previously described methods were used to generate and prepare samples of P-trHb (5) and C-trHb (2). Horse heart Mb was commercially obtained from Sigma and used without further purification other than centrifugation to eliminate particulate matter. Samples of Mb(L29W) and Mb(H64Q/L29Y) were a generous gift from Professor John Olson of Rice University.

Geminate and solvent phase recombination measurements 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 to monitor time-dependent changes in absorption. Details of the apparatus, data collection, and data display can be found in a previous publication and citations therein (41). The Soret enhanced resonance Raman spectra of the photoproducts of several of the samples was generated using 8 ns 435.8-nm excitation pulses to both photodissociate the CO derivatives and generate the Raman spectrum of the resulting photoproduct. Details of the apparatus, data collection, and data analysis are reported elsewhere (44, 45).

Kinetic measurements were typically carried out on solution samples contained in standard 10-mm or 1-mm capped cuvettes placed in a custom-built variable temperature cuvette holder. Sol-gel-encapsulated samples were prepared as a thin layer lining the bottom portion of either 5- or 10-mm diameter NMR tubes as previously described (41, 4547). The glycerol-bathed samples were prepared by replacing the aqueous bathing buffer covering the sol-gel sample with an excess volume of either CO- or N2-purged glycerol. With the exception of encapsulated P-trHb, sol-gel samples, stored at 4 °C, were stable for months or longer (see below). The Raman measurements were carried out on cooled (~4 °C) samples contained in spinning NMR tubes using a front face scattering configuration.

It is important to note that the absorption and resonance Raman spectrum of the deoxy derivative of these trHbs are typical of a five coordinate high spin ferrous heme species. Spectra consistent with a six coordinate deoxy derivative, as seen for some trHbs, were never seen for P-trHb or C-trHb under the solution conditions employed in the current study. It should be noted that C-trHb can adopt a six coordinate heme configuration with B10Y as the likely sixth ligand but under very different pH conditions (2, 27, 28).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The Quantum Yield (QY) for Photodissociation Is Protein and Temperature-dependent—In an aqueous solution, the CO derivatives of Mb and P-trHb are easily and comparably photodissociated using an 8 ns 532-nm excitation. Under similar solution conditions, the CO derivatives of C-trHb is exceedingly difficult to photodissociate. At 3.5 °C, under conditions that yielded a large photoproduct population for the CO derivatives of Mb(horse heart) and P-trHb the corresponding yield of photoproduct for C-trHb was nearly undetectable. Whereas the yield of photoproduct changed minimally with increasing temperature for Mb and P-trHb, and even decreased, the yield substantially increases for C-trHb. Fig. 2 shows the percentage change of an arbitrary measure of the 8 ns quantum yield using the values at 3.5 °C as a reference starting point. It can be seen how for C-trHb the near-zero yield at 3.5 °C substantially and steadily increased with increasing temperature whereas no such increase was seen for Mb and P-trHb, both of which have a substantial quantum yield at 3.5 °C. The very low 8-ns photolysis quantum yield for C-trHb required that the sample be maintained at high temperatures (45–65 °C) in order to generate a photoproduct population sufficient to produce a measurable kinetic trace of the CO recombination.



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FIG. 2.
Percent change in the initial 8-ns photolysis QY at 3.5 °C as a function of increasing temperature for P-trHb(CO), C-trHb(CO), and COMb. This percent change was obtained as follows: At each temperature, the maximum percent change in transmission due to addition of the photolysis pulse was determined. If this maximum percent change in the transmission at temperature T, is symbolized by {varphi}(T), then the ordinate, {Delta}r[%], is [{varphi}(3.5) - {varphi}(T)]/{varphi}(3.5) x 100%. The initial QYs as reflected in the values of {varphi}(3.5), were high and very similar for P-trHb and Mb, whereas for C-trHb the value was close to zero.

 

A Comparison of the CO Recombination Kinetics for the Two trHbs in Solution—Fig. 3 shows CO recombination kinetics in solution at 45 °C for the two trHbs and horse Mb displayed on a log-log plot of normalized absorbance (proportional to survival probability of photoproduct) versus time. On a log-log plot exponential rebinding appears as a horizontal line followed by a precipitous near vertical drop. The intersection of the vertical line with the time axis is approximately equal to the reciprocal of the exponential decay constant. Deviations from this behavior are expected under a variety of conditions including those in which either there are multiple kinetic populations contributing or there are photoproduct population evolving during the time course of the recombination.



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FIG. 3.
A comparison of CO recombination at 45 °C for photodissociated CO derivatives of trHbs and Mb shown as a plot of the normalized absorbance versus time on a log-log plot. Traces a, b, and c are derived from C-trHb, P-trHbm and Mb, respectively.

 

Under the conditions of these measurements the kinetic trace can be divided between geminate and solvent recombination. Geminate recombination under these solution conditions in other hemeproteins occurs on time scales ranging from picoseconds to hundreds of nanoseconds or in some cases to about a microsecond (and is not evident in Fig. 3); whereas, the solvent phase recombination occurs on the many microsecond and longer time scales. For the reasons discussed above relating to the quantum yield, the kinetic comparison was made on samples at elevated temperatures. All the samples were maintained at 45 °C. In all cases, the solutions were fully saturated with CO, and the protein concentrations were ~25–50 µM.

It can be seen that the amplitude for the geminate phase is very low for P-trHb. Despite the similarity to Mb with respect to the low geminate yield, the solvent phase recombination is ~100 times faster for P-trHb. These values are similar to those reported earlier for the combination rate using rapid mix techniques (3, 5).

The small geminate phase seen for C-trHb in Fig. 3 does not exhibit the typical clear cut leveling off seen for most Hbs and Mbs. Instead the highly nonexponential geminate phase smoothly evolves into a very rapid solvent phase recombination process.

Recombination Kinetics as a Function of the [CO]/[Hb] Ratio—Fig. 4 shows the rebinding kinetics of CO at 3.5 °C for photodissociated P-trHb(CO), under a range of conditions where the variable is the ratio of protein concentration to CO concentration. Traces a and e are obtained when the [CO]/[Hb] ratio is low and high, respectively. Trace d is obtained after slightly reducing the [CO]/[Hb] ratio but the sample is still in the pseudo first-order regime. Traces b and c are obtained at intermediate values of the [CO]/[Hb] ratio. When the CO to protein concentration ratio is high there is only the single fast phase that matches the rate reported in an earlier study (5) recorded under similar pseudo first-order condition (CO >> Hb) using rapid mix techniques. For COMb(Horse), a single solvent recombination phase is observed (not shown) under the same full set of concentration conditions. The recombination rate slows in the anticipated fashion as the CO concentration is reduced, e.g. trace d.



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FIG. 4.
The different kinetic patterns observed for the recombination of CO to P-trHb in solution at 3.5 °C as a function of [CO]/[Hb]. The changes in going from trace a to trace e are in response to increasing the value of [CO]/[Hb]. Trace e represents the limiting case when the ratio is high.

 

Several unusual features are apparent in the mid to low [CO]/[Hb] ratio regime. There are the striking biphasic solvent phase traces. Trace a of Fig. 4, obtained when the [CO]/[Hb] ratio is low deviates from the exponential behavior seen for COMb under similar conditions. Another unusual aspect associated with the kinetics from P-trHb(CO) is that the kinetic patterns appear only quasi-stable. The kinetics obtained in the mid to low range for the [CO]/[Hb] ratio exhibit a slow evolution with time (hours to days). It was often observed that an initial trace resembling trace c, generated shortly after a protocol designed to lower the [CO]/[Hb] ratio, would over the course of tens of hours or days evolve into a trace similar to trace b. The most consistent aspect of this phenomenon is that the slow phase becomes slower and the fast phase becomes faster. Amplitude changes are also observed, but a more systematic study is needed before a firm conclusion regarding the amplitudes can be put forth. The data are highly suggestive of the slow phase both slowing and increasing in amplitude over a period of many hours. A similar sequence has been observed when a trace such as trace a is observed. With time, the trace a type pattern slowly evolves into a clear-cut biphasic trace with the fast and slow phases bracketing the initial trace. Acceleration of both the fast and slow phases occurs when successive aliquots (60 µl) of CO-saturated buffer are added to a 25 µM P-trHb(CO) sample (1 ml) in the low [CO]/[Hb] ratio regime. Continued addition of CO results in the progressive loss of the slow phase and eventually a return to the single trace associated with high [CO]/[Hb] ratio regime. The rates and ratio of the two components were not responsive to the addition of up to 0.5 M NaCl.

The flushing with gaseous N2 of a CO-saturated C-trHb(CO) sample at 45 °C yielded decipherable kinetics (not shown) that showed a marked slow down of the fast phase displayed in Fig. 3. There is a suggestion of two phases as seen for P-trHb but the trace is too noisy (due to the low quantum yield) to draw a firm conclusion.

Rebinding Kinetics from Sol-gel-encapsulated P-trHb—Compared with the solution phase samples that exhibit a geminate yield (GY) of at most a few percent, encapsulated samples (designated as [P-trHB(CO)]) show a clearly discernable GY of well over 10% as might be anticipated based on the enhanced internal viscosity associated with the environment surrounding the encapsulated protein (48). Under high [CO]/[protein] values, the solvent phase recombination for encapsulated sample consists of a single phase similar to what is observed for the solution phase samples.

Encapsulated samples of P-trHb exhibit a slow leakage of protein out of the gel matrix into the buffer that bathes the sol-gel sample. This leakage is not likely due to the size of P-trHb since it is only slightly smaller than vertebrate Mb. It is more likely that it originates from protein surface effects, possibly electrostatic or polarity, that do not allow for strong stabilizing interactions with the sol-gel residues that line the cavities surrounding the encapsulated protein. This effect is not observed for encapsulated HbA, nematode Hb (Ascaris), vertebrate Mbs, and other trHbs (including HbN and HbO from M. tuberculosis). The slow escape out of the sol-gel matrix for P-trHb, can be prevented by adding high concentrations of glycerol as a bathing buffer.

Large Viscosity-dependent Changes in the Rebinding Kinetics for Sol-gel-encapsulated and Trehalose Glass-embedded trHbs—Two strategies (41), one using glycerol added to an encapsulated sample and the other using trehalose to create a glassy matrix, were used both to enhance the amplitude and extend the duration of the geminate phase. The purpose of this approach was to expose possible additional geminate phases that might originate from CO localized within intraprotein sites (such as the apolar tunnel) other than the distal heme pocket.

Earlier studies (44, 45, 47) on sol-gel-encapsulated human adult hemoglobin (HbA) and Mb demonstrated that glycerol freely enters the sol-gel resulting in recombination kinetics that are consistent with the ~103 cP viscosity associated with pure glycerol at near 0 °C. The time-dependent changes in the kinetic traces from a sol-gel-encapsulated sample subsequent to the addition of glycerol are shown in Fig. 5. Two sol-gel-encapsulated samples were prepared. In both instances, the encapsulated sample is P-trHb(CO); however, the CO concentration for the two samples differed. The following protocol was used to prepare the sample whose kinetics are shown in Fig. 5. The P-trHb(CO) sample was encapsulated in a thin sol-gel matrix. The bathing buffer was initially flushed with CO and subsequently flushed with N2. The bathing buffer was then replaced with N2-saturated glycerol. The bathing buffer of the second encapsulated sample (kinetics not shown) was flushed only with CO. Subsequently the bathing buffer was replaced with CO-purged glycerol.



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FIG. 5.
The evolution of the kinetic trace at 3.5 °C for the recombination of CO to sol-gel-encapsulated P-trHb in the low [CO]/[Hb] limit subsequent to the replacement of the bathing buffer with 100% (N2-purged) glycerol (traces a–d) (See text for more detail). Trace a was recorded prior to the addition of glycerol. Trace b was recorded within several minutes of adding glycerol. Trace c was recorded after ~1–2 h and trace d after several hours. Trace e is the kinetic trace for a sample of a carbonmonoxy derivative of P-trHb embedded in a trehalose-glass matrix at 65 °C. The high temperature was necessary to generate a sufficient photoproduct population to allow for monitoring of kinetics.

 

The evolution of the rebinding kinetics over a period of hours subsequent to the addition of the glycerol was very similar for both samples in that there is a progressive increase in the amplitude of a fast geminate phase at the expense of the solvent phase. The solvent phase does not show any significant change in rate only a decrease in amplitude. The two samples differ in that the N2-and CO-purged samples of carbonmonoxy P-trHb exhibit a slow and fast (not shown) solvent phase, respectively.

The resulting kinetic pattern at 3.5 °C after several hours is identical for the two samples, consisting of a single non-exponential geminate phase that is complete within a few microseconds. Also shown in Fig. 5 is a kinetic trace from P-trHb(CO) embedded in a trehalose glass. Unlike the T = 3.5 °C kinetics derived from the glycerol-containing sol-gel samples, the kinetics shown for the trehalose glass sample were obtained at 65 °C. It can be seen that the distribution of rates for the trehalose sample is skewed toward even faster values than for the sol-gel plus glycerol sample. At lower temperatures, there was virtually no detectable photoproduct population at 8 ns for the trehalose sample. Several different trehalose samples all displayed the same effect. Even at the high temperature the QY is relatively low.

Fig. 6 shows the temperature dependence of the rebinding kinetics as a function of temperature for the glycerol (CO-saturated) bathed sample of encapsulated P-trHb (after initially achieving the same glycerol-induced end point kinetics shown in Fig. 5 for the corresponding nitrogen-purged sample). It can be seen that with increasing temperature (decreasing viscosity), there is a reappearance of the solvent phase. Although the solvent phase rebinding is slower than in solution it is still considerably faster than the slow rebinding phases observed for Mb and aromatic B10 mutant Mbs under similar conditions (41) (see below) indicating that these processes are not rate limited by the viscosity of the solvent influencing ligand diffusion in the solvent. Instead it appears that these kinetics are reflective of the internal properties of the protein.



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FIG. 6.
The temperature dependence of the CO rebinding kinetics from a sol-gel-encapsulated sample of P-trHb bathed in 100% CO-saturated glycerol. Trace a shows the kinetics of the sample at 3.5 °C within several minutes of adding the CO-saturated glycerol as the bathing solvent for the sol-gel-encapsulated P-trHb sample. Traces b and c are respectively the kinetics at 25 and 10 °C generated after the glycerol-induced changed in the kinetics fully stabilized (over 12 h after adding the glycerol). The kinetic trace at -15 °C was unchanged from that at 10 °C.

 

The full effect of the added glycerol takes several hours. Despite the large differences in the initial solvent kinetics (due to the high and low concentrations of CO in the glycerol), both encapsulated P-trHb samples (high and low CO) eventually end up with essentially identical traces at 3.5 °C. Under similar conditions horse Mb exhibits a very different final kinetic trace as seen in Fig. 7. Instead of the single rapid distributed kinetic phase seen for P-trHb, there are multiple kinetic phases extending out for many decades in time as reported earlier for Mb in a glassy matrix (41).



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FIG. 7.
The evolution of the kinetic trace at 3.5 °C for the recombination of CO to sol-gel-encapsulated COMb in the high [CO]/[Hb] limit subsequent to the replacement of the bathing buffer with 100% (N2-purged) glycerol. Trace a is the kinetic trace prior to replacing the pH 6.5 Bis-Tris acetate buffer with CO-purged glycerol. Traces b through e show the evolution over many hours (>15) of the kinetic trace subsequent to the replacement of the bathing buffer with CO-purged glycerol. No obvious decrease in the QY is observed.

 

The CO rebinding kinetics under high viscosity conditions for two Mb mutants having an aromatic in the B10 site (GlnE7/TyrB10 and TrpB10) are shown in Fig. 8. As reported previously for a glass embedded Mb(TrpB10) sample, the 10-µs geminate yield is significantly reduced compared with Mb(wt). The overall rebinding is characterized by a long drawn out nonexponential geminate phase followed by very slow exponential phase.



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FIG. 8.
Kinetic traces a, b, and c for the CO recombination to a sol-gel-encapsulated sample COMb(H64Q/L29Y) bathed in CO-saturated glycerol as a function of temperature: 45, 3.5, and -15 °C respectively. Also shown, in trace d, are the kinetics at -15 °C of a similarly prepared glycerol-bathed sample of encapsulated COMb(L29W).

 

Resonance Raman of the 8-ns Photoproduct of the CO Derivative of P-trHb—The frequency of the Fe-proximal histidine stretching mode, {nu}(Fe-His) is a sensitive measure of both proximal strain and conformational change associated with the F-helix (4952). The frequency of {nu}(Fe-His) for the 8-ns photoproduct of carbonmonoxy P-trHb in aqueous buffer, is at most 1 cm-1 higher than the 220 cm-1 value reported for the equilibrium deoxy derivative (5). For the glycerol-bathed sample the frequency is increased to ~227 cm-1. Shown in Fig. 9 are spectra obtained after addition, removal, and re-addition of glycerol to the sol-gel-encapsulated samples. The results demonstrate the reversible nature of the glycerol effect. In the trehalose glass, the {nu}(Fe-His) band occurs at ~230 cm-1 (not shown). The ~230 cm-1 is the maximum attainable value for this frequency for Hbs and Mbs at non-cryogenic temperatures. It is of interest to note that C-trHb manifests this end point 230 cm-1 frequency for the equilibrium deoxy derivative in aqueous solution (27). In contrast, the photoproduct of COMb does not exceed ~224 cm-1 even in trehalose glass; whereas, COHbA yields a frequency of ~230 cm-1 under high and low viscosity conditions (45).



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FIG. 9.
The Soret band enhanced resonance Raman spectrum from the 8-ns photoproduct of the CO derivative of P-trHb at ~4 °C as a function of viscosity. VRR spectra of [HbParameciumCO] as a function of viscosity and time. All traces were normalized to {nu}7. a, blue, P-trHb(CO) in solution; b, purple, [P-trHb(CO)]; c, green, [P-trHb(CO)]+glycerol, dt = 2 days; d, black, [P-trHb(CO)]+glycerol-glycerol dt = 2 h; e, red, [P-trHb(CO)]+glycerol-glycerol+glycerol; just after last glycerol addition; f, green, [P-trHb(CO)]+glycerol-glycerol +glycerol, dt (from last glycerol addition) = 2 days; g, gray, pure glycerol, as a control. The designation dt refers to the delay time between the last modification to the sample and the onset of the Raman measurements. The peak labeled H2 is due to Raman scattering from the hydrogen-filled tube used to convert the initial 8-ns green excitation pulse (second harmonic from a Nd:YAG laser) to a blue excitation at 436 nm.

 

Summary of Results—A summary of the results is presented in Table I. The geminate yield (GY) data refer to the fraction of the initially photodissociated population (at 8 ns) that has undergone rebinding within 10 µs of photodissociation.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
A Kinetic Model for P-trHb and C-trHb—Fig. 10 shows a schematic for a reaction coordinate diagram that is to be used to interpret the CO rebinding data for C-trHb and P-trHb. The nomenclature is similar to that used by others in describing Mb behavior. The potential minima labeled A, B, and C refer to the protein with the ligand: bound to the iron, localized within the distal heme pocket, and localized in the apolar tunnel adjoining the distal heme pocket respectively. Barrier I refers to the innermost barrier controlling bond formation between the ligand and the heme-iron. Barrier II refers to the barrier controlling flow between the distal heme pocket and the adjacent tunnel. The presented CO recombination data are analyzed based on assigning kinetic phases to either B -> A or C -> A.



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FIG. 10.
A hypothetical reaction coordinate diagram to help account for the observed kinetics in trHbs. Entropic contributions have been included as reflected in the proposed changes in well depths as a function of volume and occupancy. In an attempt to explicitly and simplistically include the effect of solvent viscosity, we made the height of Barrier II dependent on solvent viscosity. This action follows because the motions of residue side chains directly mediate the diffusion of ligands within the protein. The solvent viscosity impacts ligand diffusion within the protein by modulating conformational mobility. Thus for each value of the solvent viscosity, there is a different "effective" barrier that represents the ease with which a ligand can access different cavities within the protein.

 

The B -> A process refers to geminate recombination of the dissociated ligand from within the distal heme pocket. In aqueous solution, the B -> A process for Mbs and Hbs can occur on time scales ranging from picoseconds out to hundreds of nanoseconds. The time scale and the geminate yield for this process can be understood in terms of the interplay among the heights of Barriers I and II and the stability of the B state. Studies on Hbs and Mbs have provided much insight into factors that influence both the heights of Barriers I and II and the stability of the B state.

The height of Barrier I is ligand-dependent. It decreases in the order of CO>O2>NO. As a consequence of this ordering, a B -> A process on the picosecond time scale is often seen for NO, occasionally seen for O2 and rarely seen for CO. For a given ligand, the height of Barrier I is conformation dependent. There are two broad categories of conformational influence: proximal and distal.

Proximal control of Barrier I arises from the energetic cost of moving the iron sufficiently toward the heme plane so as to generate a suitable transition state for ligand-iron bond formation. This movement of the iron is made protein conformation dependent through its covalent attachment to the proximal histidine on the F-helix. The photoproduct frequency of {nu}(Fe-His) appears to be a good indicator of this energetic cost. The maximum observed frequencies at or near 230 cm-1 are associated with lower values for Barrier I.

Distal control of Barrier I is typically associated with steric factors arising from the side chains of distal heme pocket residues that influence access of the dissociated ligand to the iron. Distal heme pocket residues also influence the B -> A recombination by influencing both the stability of the B state through volume effects and the height of Barrier II by either constraining the ligand within the distal heme pocket or facilitating escape.

Diffusion of the ligand through the protein is coupled to conformational fluctuations of residue side chains within the protein (23, 5356). Thus Barrier II, which controls ligand diffusion between B and C, increases and decreases as protein fluctuations increase and decrease respectively. Since conformational fluctuations are viscosity dependent, the viscosity dependence of ligand diffusion between B and C is symbolically represented in Fig. 10 through the viscosity-dependent variation in the height of Barrier II.

Ligand rebinding arising from ligands localized within the apolar tunnel (C -> A) is analogous to the Xe cavity recombination in Mb but with an important distinction. In this case, B -> C and C -> B are part of the linear pathway linking A to the solvent. In Mb, the evidence is most consistent with B -> C being a dead end offshoot of the A -> B -> solvent pathway. Nevertheless, it appears that in many cases associated with Mb and Mb mutants, ligand access to the Xe cavities is sufficiently rapid that under high ligand to protein concentration ratios, the C -> A rebinding is also the rate-limiting process for the solvent phase recombination (solvent -> C -> A).

A Low Nanosecond Quantum Yield for the CO Derivative of C-trHb Implies a Picosecond B -> A Process—Despite the similar sequence of distal heme pocket residues, C-trHb and P-trHb exhibit drastically different photodissociation quantum yields for the carbonmonoxy derivatives at 8 ns in solution. The low 8-ns quantum yield for the CO derivative of C-trHb is unusual. Most CO derivatives of Mbs and Hbs are easily photodissociated. Since the actual instantaneous quantum yield for CO derivatives is known to be unity, the low quantum yield implies an efficient picosecond B -> A process with an anomalously low Barrier I.

Picosecond CO recombination has been observed for COMb at low pH under cryogenic conditions where the iron-proximal histidine bond is broken (57). By eliminating the energetic cost of moving the iron into the heme plane upon ligand binding, the loss of the iron-proximal histidine bond results in a very substantial decrease in Barrier I. Under the conditions of the present experiments, there is no indication for a loss of the iron-proximal histidine linkage. In fact the high frequency observed for {nu}(Fe-His) is indicative of a stable bond. This high frequency also implies a reduced proximal contribution to Barrier I but not sufficient to account for picosecond recombination.

Picosecond geminate recombination for CO arising from distal effects could arise from a combination of contributions. Appropriate architecture within the distal heme pocket can result in the dissociated ligand being transiently trapped near the iron thus increasing Barrier II (53). Barrier I for the B -> A process could be reduced if distal residues constrain the dissociated CO to retain a transition state orientation in close proximity to the iron. If these residues also reduce the volume of the distal pocket, then the B state becomes destabilized (58) which further contributes to the lowering of Barrier I for the B -> A process. The reported reduced volume (6, 59) for the distal heme pocket in C-trHb as well as the high frequency for {nu}(Fe-His) makes it plausible that the low quantum yield observed for the CO derivative arises from a combination of effects that both decrease Barrier I relative to Barrier II and destabilize the B state. Based on this explanation, the increase in the quantum yield with increasing temperature would arise out of an increase in thermally driven motion of the side chains of distal heme-pocket residues. This increased motion of the side chains is expected to enhance ligand diffusion thus reducing Barrier II relative to Barrier I.

Constraint of the dissociated ligand near the heme accounts for changes observed in the B -> A recombination for several mutants of Mb. These constraint-induced effects include the increase in picosecond rebinding of NO and O2 for the B10(L -> F) mutant (53, 60) and the dramatic increase in the geminate yield for CO in the E11(V -> W) mutant. The situation for C-trHb differs from that observed for these Mb mutants. In those cases the aromatic residues cause localization of the dissociated ligand at the heme; however, rebinding of ligands that have escaped from the distal heme pocket is very slow, indicative of either an enhanced Barrier II for C -> B (for the E11 mutant) or a relaxation-induced enhancement of Barrier I (for the B10 mutant). In contrast, for C-trHb, the rebinding of CO molecules that have escaped from the distal heme pocket is extremely fast, consistent with both a facile C -> B process and a very low Barrier I that persists beyond the ligand escape time from the distal heme pocket.

Origin of the Large Viscosity-dependent Change in the Geminate Yield for the B -> A Process for P-trHb—In aqueous buffer, the B -> A geminate yield for P-trHb(CO) is near zero. Increasing the viscosity to ~1000 centipoise by adding 100% glycerol to the sol-gel-encapsulated samples, results in the 8-ns QY for photodissociation being reduced and in the appearance of a nonexponential B -> A process that extends from nanoseconds out to a few microseconds. This process accounts for the rebinding of all the dissociated CO. Increasing the viscosity still further by using a trehalose-glass matrix, results in a situation similar to that of C-trHb where there is no observable 8-ns QY below ~10 °C. These results show that under these high viscosity conditions, the dissociated CO does not escape from the distal heme pocket of P-trHb.

Observations on distal heme pocket mutants of Mb (41) indicate that the viscosity induced increase in amplitude of B -> A yields a series of amplitudes that scales with the initial geminate yield (at ~10 µs). The behavior observed for P-trHb where the geminate yield at 10 µs increases from 0 to 100% with viscosity is just not seen for Mbs that have comparably low geminate yields in the solution phase.

An explanation of these P-trHb associated viscosity effects follows from an extension of the analysis used to account for the recombination in C-trHb. The observation of a near zero geminate yield for B -> A and a moderately fast solution phase recombination (C -> A) for P-trHb(CO) in solution indicates that Barrier I is not especially high and that escape from the distal heme pocket is very efficient. This combination is consistent with Barrier I being higher than Barrier II and the B state being destabilized (presumably due to residue crowding in the distal heme-pocket).

Increasing the viscosity both increases the yield of B -> A (from near zero to nearly 100%) and shortens the temporal window over which the B -> A process occurs. This effect requires a decrease in Barrier I and an increase in Barrier II. The increase in Barrier II is attributed to a viscosity-induced slowdown in the motion of distal residue side chains that drives the activated diffusion of the dissociated CO. The viscosity-induced decrease in protein motion is also invoked to account for the reduction in Barrier I. Barrier I can decrease due to an increased localization of the dissociated CO near the iron in a transition state orientation (as invoked for C-trHb) and a substantial slow down of a subnanosecond photodissociation-induced relaxation process.

Evidence that the increased viscosity limits a fast occurring relaxation is seen in the Raman results presented in this work. The 8-ns photoproduct frequency for {nu}(Fe-His) from P-trHb-(CO) in solution at 220 cm-1 is consistent with full relaxation of the proximal environment within 8 ns, a result similar to what is observed for Mb (61, 62). Under high viscosity conditions the frequency for the 8 ns photoproduct of P-trHb(CO) attains a value close to 230 cm-1, characteristic of a proximal environment where the F-helix is in much closer proximity to the heme. The frequency observed for the photoproduct of P-trHb(CO) in trehalose or 100% glycerol is much higher than the corresponding value from MbCO photoproducts under similar conditions (230 versus 224 cm-1) (45). The large dynamic range (220–230 cm-1) for {nu}(Fe-His) likely arises from the high degree of flexibility seen in the greatly reduced F-helix and the adjacent pre-F-helix non-ordered loop (63). It is conceivable that an enhanced solvent effect may arise from this pre-F-helix loop through solvent induced coil-helix transitions associated with this solvent-exposed segment of protein.

Solvent Phase Recombination: the Role of Relaxation—The fast solvent phase rates seen for P-trHb and C-trHb are comparable to the fast rates observed in some other trHbs (7). These fast solvent phase recombination rates are faster than those observed for Mb mutants having an aromatic residue at the B10 position (18, 23, 24). The side chains of aromatic B10 residues in mutant Mbs have been shown to undergo ligation-induced shifting away from the iron (23, 24, 37, 64). In these mutants, the relaxation of B10 subsequent to photodissociation results in the side chain blocking access to the heme-iron. This relaxation of the B10 side chain subsequent to ligand dissociation can have a profound effect on the fate of the dissociated ligand. By limiting the accessible volume available to the ligand near the iron and by blocking access to the iron, it causes a "squeezing" of the ligand away from the binding site toward adjacent Xe cavities (18). As a result of the increase in Barrier I, the C -> A recombination in these Mb mutants is very slow. Clearly the fast C -> A process observed for C-trHb and P-trHb reveals that relaxation of the B10 side chain, to the extent that it occurs in these two trHbs, does not progress to the same extent as in the above noted Mb mutants. The presence of a polar E11 residues in P-trHb and C-trHb, probably prevents the degree of relaxation of the B10 phenolic side chain that would occur if E11 were a comparably sized or smaller nonpolar residue.

Solvent Phase Recombination: the Role of the Apolar Tunnel—Volume analysis of the apolar tunnel suggests that it can accommodate as many as 6 CO molecules.2 The apolar interior of the tunnel should increase the solubility of nonpolar diatomics such as CO in the tunnel relative to the aqueous phase. As a result the tunnel should function as a concentrator of ligands enhancing the local effective concentration of available molecules to bind to the heme. This can be represented symbolically in Fig. 10 by having the stability of the C state dependent on occupancy with multiple ligands. Alternatively at low ligand concentrations, the rates may become unexpectedly slow due to both limited access to the tunnel opening(s) and the long N2 or perhaps water filled narrow pathway that must be traversed in order to access the distal heme pocket.

The biphasic kinetics observed for P-trHb are consistent with tunnel-dictated kinetic patterns for the solvent phase. We propose that the slow phase arises from protein molecules that have a CO-depleted tunnel and the fast kinetic phase from protein molecules that have CO-occupied tunnels. There are several important observations that need to be considered in evaluating this proposed role of the tunnel. This model predicts the observed increase in the rates of both slow and fast rates with the addition of small aliquots of CO-saturated buffer. It also explains why under low [CO]/[trHb] conditions the biphasic rebinding kinetic continue to evolve for many hours. Invariably the pattern evolves such that the slow phase gets slower and the faster phase gets faster. This evolution is consistent with a CO partition function favoring tunnel occupancy. The tunnel becomes a CO trap scavenging free CO. Thus when a sample that starts out with a high [CO]/[trHb] ratio is initially flushed with N2 to the extent that [CO] is reduced but still non-negligible, with time, the free CO will tend to partition into the apolar tunnel thereby further reducing the free CO in solution. As a result there is a progressive increase in tunnel occupancy by CO with a concomitant reduction in aqueous CO. Those P-trHb molecules lacking CO in their tunnel will experience a slow down in the solvent phase rebinding due to the decrease in the concentration of CO in the solvent. This explanation also accounts for the observation that the single slow recombination phase observed after extensive N2 flushing of a CO-saturated P-trHb(CO) sample will, with time, partition into a biphasic pattern with the slower phase being even slower than the average rate seen for the initial single phase.

Biphasic recombination kinetics, observed in several other instances have been attributed to multiple conformations (13, 20, 65, 66). Several of these proteins exhibit a six coordinate ferrous heme when ligand-free, the sixth ligand being an internal residue from the distal heme pocket. The biphasic rebinding kinetics can arise from rebinding to photoproduct populations that are initially five coordinate but with time evolve into a six coordinate species (with the sixth ligand being an internal residue) (7, 13, 65). Similarly biphasic rebinding kinetics could arise from the relaxation of side chains of "gate keeper" residues such as B10 or E11 causing a time-dependent change in either Barrier I or II. In both of these cases the time scale for the photodissociation-triggered relaxation of the distal heme pocket is the determining factor giving rise to the multiple phases. If this effect were operative in the P-trHb(CO) case, one would anticipate that the combination rate observed in a stopped flow setting would reflect the slow limit (fully relaxed deoxy conformation) whereas the photodissociation-triggered measurement at high CO concentrations would reflect the fast recombining conformation. The comparable rates in both cases are not consistent with there being a significant relaxation effect that could account for the two phases. There is also no spectroscopic evidence for any six coordinate deliganded ferrous form of P-trHb. The pattern of changes seen for the two phases as a function of CO concentration and N2 flushing, is much more consistent with the tunnel-based explanation than any conformation-based description.


    CONCLUSIONS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The kinetic patterns observed for the recombination of CO to trHbs are varied and atypical when compared with many other Hbs and Mbs. The observed patterns for the two trHbs studied in the present work can largely be explained based on residue-specific variations in the B10Y-based hydrogen-bonding network within the distal heme pocket and the presence of a long apolar tunnel linking the distal heme pocket and the solvent.

The large difference in the rebinding kinetics between these trHbs and Mb mutants also having a TyrB10 (or Trp) is attributed to differences in the E11-governed relaxation properties of the B10 side chain subsequent to ligand dissociation. Both C-trHb and P-trHb have a ligand-accessible heme presumably due in part to the "spacer" role of either Gln or Thr at the E11 site. Their interaction with B10 likely prevents the B10 side chain from occupying a site directly blocking the iron as occurs for the TyrB10 (or Trp) Mb mutants that have a valine at the E11 site.

C-trHb and P-trHb share all but one (E11) of functionally key residues that comprise the distal heme pocket. Seemingly, small difference in positioning of the distal heme pocket residues is sufficient to create dramatically different kinetics. The convergence of the CO rebinding kinetics and Raman spectra for P-trHb and C-trHb when the P-trHb is subjected to high viscosity media, indicates that the kinetic differences at low viscosity arise in part from protein specific differences in the thermal mobility of residue side in the distal heme pocket and in the relaxation properties of proximal heme environment. It is likely that a difference in the stability of the hydrogen-bonding network due to GlnE11 versus ThrE11 is the factor influencing the dynamical behavior of the distal heme pocket residues.

The ability of the apolar tunnel to concentrate CO with respect to the surrounding solvent is proposed to be a contributing factor to both the enhanced solvent phase recombination rates and the biphasic kinetics observed for P-trHb(CO). This ability to concentrate ligands could in principle result in anomalous oxygen titration curves that have the appearance of cooperativity but are not the result of any ligation-dependent change in conformation. The present results support the claim made with regard to a hydrophobic tunnel observed in a large bifunctional enzyme that the tunnel can act to concentrate nonpolar diatomics such as CO (67).

The observed kinetic patterns for these trHbs and the suggestive indications of their responsiveness to environmental as well as molecular factors raise interesting possibilities with respect to functionality. Those ligands such as dioxygen that can actively participate in the hydrogen-bonding network will contribute to the stability of a conformation of the distal heme pocket that greatly limits ligand dissociation. In effect the dissociation process now requires sufficient thermal energy to not only break the hydrogen bonds to the bond ligand but also the other hydrogen bonds that contribute to the overall network. Recent calculations indicate that it is this type of effect that contributes to the exceptionally low dissociation rate of oxyHb(Ascaris) rather than the strength of the hydrogen bonds directed at the bound ligand (68). This added stability for hydrogen bonding ligands may be a mechanism to greatly enhance the ligand selectivity typically seen in Mbs and Hbs and perhaps plays a role in biasing multiligand reactions (e.g. O2 and NO) in direction not anticipated based on solution concentrations.

The complete shut down by glycerol or trehalose of the seemingly facile ligand escape pathway in P-trHb may be an adaptive mechanism that allows for the long term storage of bound oxygen. By further stabilizing the tight hydrogen-bonding network, glycerol and trehalose will likely stop thermally driven oxygen dissociation and thus prevent autoxidation. Many trHb containing microorganisms including M. tuberculosis, generate osmoprotectants such as trehalose in response to various stress situations (6971). The ability of such organisms to survive severe osmotic stress for extended periods has been directly correlated with the sufficient production of trehalose to generate intracellular glassy matrices. It is possible that some trHbs are specifically generated and utilized to respond to these conditions in parallel with the enhanced production of trehalose or other osmolytes and cryoprotectants.


    FOOTNOTES
 
* This work was supported by National Institutes of Health Grants R01 EB00296 and P01 GM58890 (to J. M. F.) and Natural Sciences and Engineering Research Council Grant of Canada 46306–01 (to M. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed. Tel.: 718-430-3591; Fax: 718-430-8819; E-mail: jfriedma{at}aecom.yu.edu.

1 The abbreviations used are: trHb, truncated hemoglobin; Hb, hemoglobin; Mb, myoglobin; UVRR, UV resonance Raman; VRR, visible resonance Raman; P-trHb, truncated hemoglobin from P. caudatum; C-trHb, truncated hemoglobin from C. eugametos; QY, quantum yield; GY, geminate yield. Back

2 M. Bolognesi, private communication. Back



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