Evidence for Damped Hemoglobin Dynamics in a Room Temperature

Jul 18, 1996 - Conformational Dependence of Hemoglobin Reactivity under High Viscosity Conditions: The Role of Solvent Slaved Dynamics. Uri Samuni, Ca...
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J. Phys. Chem. 1996, 100, 12034-12042

Evidence for Damped Hemoglobin Dynamics in a Room Temperature Trehalose Glass David S. Gottfried, Eric S. Peterson, Asim G. Sheikh, Jiaqian Wang, Ming Yang, and Joel M. Friedman* Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morrris Park AVenue, Bronx, New York 10461 ReceiVed: March 29, 1996; In Final Form: May 24, 1996X

Upon photodissociation of its ligand, COHbA exhibits a wide range of nonequilibrium relaxation phenomena that start within a fraction of a picosecond and extend out to tens of microseconds. In addition, equilibrium fluctuations of the protein result in conformational averaging. All of these dynamics can have an impact on ligand rebinding. In an effort to better understand the relationship between conformational dynamics and ligand-binding reactivity, COHbA was embedded in a room temperature trehalose sugar glass (Hagen et al. Science 1995, 269, 959) in order to uncouple solvent motions from protein dynamics as well as reduce the amplitude of large-scale protein conformational fluctuations. Time-resolved resonance Raman spectroscopy and ligand-rebinding kinetics show that the trehalose glass does not impede the initial fast relaxation of the iron-histidine linkage, but does dramatically impede conformational averaging and completely eliminates ligand escape at all temperatures from 140 K to room temperature. Fluorescence measurements indicate that in the trehalose glass the picosecond tryptophan lifetimes are nearly unchanged, but there is a complete absence of the nanosecond fluorescence decay (observed in aqueous solutions), which is replaced by a decay of ∼700 ps. This change in the fluorescence decay is ascribed to a significant decrease in the structural dynamics that normally allow transient opening of the distal heme pocket.

Introduction The nature of protein dynamics and the role dynamics plays in the control of protein reactivity remain areas of intense biophysical study. One approach to the study of protein dynamics is to reduce protein motions by increasing the solvent viscosity and monitor both the alteration in dynamics and the impact on protein function. In this study, carbon monoxy human adult hemoglobin (COHbA) is embedded in a room temperature glassy matrix. This matrix provides a means of both trapping the equilibrium population and reducing or eliminating largescale protein motions. COHbA is used in this study primarily because of its stability relative to other Hb derivatives as well as the ease of readily probing both its conformational and dynamical properties. Trehalose is a diglucose sugar that confers to certain plant and animal cells the ability to survive dehydration.1,2 Solutions of trehalose are known to form glasses (as opposed to crystals) when dried under appropriate conditions at biological temperatures.3 There is current interest in using trehalose as a means of preserving proteins and peptides at ambient temperatures without the need for lyophilization.4 The recent study by Hagen et al.5 on photodissociated COMb in a trehalose glass indicates that trehalose inhibits some protein dynamics. In the present study, COHbA was embedded in a trehalose glass and compared to COHbA in solution using several experimental techniques. We focus on how this matrix influences several categories of protein dynamics as manifest in differences observed in the tryptophan fluorescence lifetimes and the resonance Raman spectrum (Soret excitation) of the photoproduct 10 ns after photodissociation. In addition, the impact on hemoglobin reactivity is examined as reflected in the time course of geminate recombination and the probability of CO escape from the protein after flash photolysis. The fluorescence signal from tryptophans in proteins and peptides is used extensively as a probe of structure, dynamics, X

Abstract published in AdVance ACS Abstracts, July 15, 1996.

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and disorder.6-9 In the absence of a strong quencher, the fluorescence lifetime of tryptophan is typically on the order of a few nanoseconds. In a protein that contains a chromophore to which an excited tryptophan can efficiently and rapidly transfer energy, the lifetime can be dramatically reduced. Under such conditions, the steady-state fluorescence intensity is correspondingly decreased. A change in lifetime in such a system can be attributed to a change in the interaction between the fluorophore and its quencher, since it is the energy transfer between the two that affects the observed decay. The intrinsic tryptophan fluorescence from hemoglobins (Hb’s) and myoglobins (Mb’s) falls into the above category. Energy transfer from an excited tryptophan (Trp) to a nearby heme is very efficient, resulting in highly quenched (approximately 100-fold) fluorescence.10-13 Although of considerable potential value as a probe of both static and dynamic conformational properties in these proteins, the fluorescence has not been extensively utilized for such purposes because of the low signal intensity and the difficulties in characterizing such weak fluorescence. Front-face fluorescence techniques14,15 have made it possible to routinely observe the emission, although the weakness of the signal always raises the possibility that trace impurities may be a major contributor to the detected fluorescence signal. The systematic behavior of the steady-state signal in response to well-defined conformational and compositional changes (induced ligand binding and allosteric effectors) makes it clear that a large fraction of this signal is intrinsic and responsive to functionally relevant conformational changes in the protein.15 Lifetime studies of myoglobins and hemoglobins using lasers and detection systems with picosecond time resolution reveal that nearly all of the fluorescent population (>98%) decays with an excited state lifetime of tens of picoseconds or less.10,12,13,16 This short-lived emission can be accounted for very precisely using the known spatial separations and orientations between the different tryptophan residues and the hemes as observed © 1996 American Chemical Society

Damped Hemoglobin Dynamics in Trehalose from X-ray crystallographically determined structures.13,17 Both distance and orientation play a role in determining the lifetime. In addition to the short-lived fluorescence, intermediate and long-lived components are also reported, and these have been the subject of speculation as well as analysis. These longer lived populations may represent a very small fraction of the total fluorescing population, but they comprise a substantial fraction of the time-integrated or steady-state signal. In addition, if this signal originates from some highly improbable conformation or process, it provides a direct optical probe of these minority or transient conformations. Several explanations have been put forth to account for the intermediate and long-lived fluorescence components ranging from dismissing the long-lived components as being due to impurities10,11,18 to providing a comprehensive detailed mechanism based on disordered heme insertion and transient heme loss.17,19 Data are presented in this report that suggest that the longest lived fluorescence component in COHbA is not due to a minority steady-state population (including impurities) but is instead likely due to a dynamical process that transiently creates unfavorable quenching conditions. This idea was alluded to in a report of molecular dynamics simulations of Mb by Henry and Hochstrasser.20 The resonance Raman spectrum provides information on whether the trehalose induces any major conformational changes in the protein that affect the heme pocket. The Soret band enhanced resonance Raman spectrum of Hb is highly reflective of the interaction of the heme with the surrounding globin; consequently, changes in heme orientation within its pocket will be manifest as frequency and relative intensity changes within the spectrum. The photoproduct spectrum at 10 ns contains spectral bands of the five-coordinate heme, which are both better characterized and more conformationally sensitive than those of the six-coordinate species. In this study, we focus on Raman bands reflective of the proximal heme environment and of the interactions of the heme propionate groups with the protein and solvent environment. Geminate recombination refers to a ligand, such as CO, rebinding to its original heme partner within the protein subsequent to dissociation. This process competes with ligand escape into the solvent. The probability for ligand-iron recombination is related both to the kinetic barrier controlling bond formation and the trajectory of the dissociated ligand in the distal pocket. The former is influenced by both the proximal21-24 and distal heme pocket environments,25-27 and the latter is determined largely by the conformation of the distal pocket.25-27 The ability of a photodissociated ligand to escape from the protein is directly related to the ability of the protein to undergo large-amplitude fluctuations that allow for the transient opening of escape channels. For example, in 75% glycerol escape of the ligand from the protein essentially ceases for photodissociated COMb below the glass transition at ∼180 K and the geminate yield becomes 100%.28 Thus, preventing these fluctuations can prevent ligand escape. Materials and Methods Sample Preparation. Blood samples were freshly drawn and standard procedures used to obtain a highly purified sample of hemoglobin A (HbA).29 D(+)-Trehalose (R-D-glucopyranosyl R-D-glucopyranoside) was obtained from Sigma. A concentrated solution of HbA in 50 mM Tris (pH ) 7.0) was combined with 0.6 g/mL of trehalose, and the mixture was stirred under a CO atmosphere for 30-60 min. The sample was filtered by centrifugation in Centrex tubes (pore diameter of 0.2 µm) for 15-20 min followed by freezing in liquid N2. This cycle of

J. Phys. Chem., Vol. 100, No. 29, 1996 12035 CO saturation, filtering, and freezing was repeated twice more. A 15 mL aliquot of sample was placed on the surface of a fused silica disc or sapphire window, which was then transferred into a gastight chamber with a positive pressure of CO at room temperature. Complete drying occurred after several days. Ligand Recombination. The flash photolysis apparatus used for rebinding rate studies used the doubled output of a Nd:YAG laser at 532 nm to photolyze the samples and the continuous output of a HeCd laser at 441.6 nm to monitor the recombination via changes in sample absorbance. The photolysis pulse was 8 ns in duration and was focused to a diameter of approximately 2 mm at the sample. To achieve 100% photolysis within the illuminated sample volume, the pulse energy was increased until the amplitude of the signal saturated, typically requiring approximately 2 mJ/pulse. The unfocused probe beam passed through the sample collinear with the photolysis beam, was spectrally separated from the 532 nm light using colored glass filters and a single monochromator, and was measured with a photomultiplier tube (Hamamatsu R928). For each laser shot, the detector output was digitized with a 500 MHz oscilloscope, resulting in a trace with 10 000 points with 5 ns separation. Traces for 100 laser shots were averaged. Identical results were obtained with 10 Hz excitation and slow spinning of the sample or 1 Hz excitation without sample spinning. For low-temperature measurements, the sample was mounted between quartz windows on the end of a cold finger in a closed-cycle helium cryostat. The temperature was controlled with a resistive heater in a feedback loop. The data were numerically converted from transmission to absorption change. Resonance Raman Spectroscopy. Visible time-resolved resonance Raman spectra were obtained using an 8 ns, 435.8 nm pulse to both photodissociate the ligand and Raman scatter off the sample. The light source was a Nd:YAG laser producing 450 mJ pulses at 20 Hz in the second-harmonic output at 532 nm. This beam was focused into a homemade 90 cm long cell filled with 120 psi of hydrogen to Raman shift the laser output to 435.8 nm. Neutral density filters were used to reduce the energy of the 435.8 nm pulses to 100 mJ, and these were focused onto the sample at an incidence angle of 45°. The Raman scattering was collected at normal incidence to the sample using a 50 mm Nikon F/1.4 camera lens and focused with an f-matching lens onto the slit of a 0.64 m single monochromator containing an 1800 grooves/mm grating. The Rayleigh line was reduced in intensity with a holographic notch filter, and a depolarizer was used to scramble the polarization of the collected light and thus eliminate intensity artifacts created by polarization-dependent grating reflectivity. The detector was an intensified diode array run in CW mode. The total accumulation time per spectrum was 30-60 min. The spectral bandwidth of the monochromator was approximately 2.5 cm-1, and the discretization on the detector face was approximately 0.9 cm-1 per pixel. Raman spectra were calibrated using solvent spectra with previously determined peak assignments. A least squares fit was used to map pixel number into relative wavenumbers (Raman shift), and the Raman spectra were base-lined using a polynomial fitting routine. Time-Resolved Fluorescence. The time-correlated singlephoton-counting system described below was based substantially on that previously reported.30 Considerable assistance in optimization was obtained from a report by Holtom.31 The light source for the time-correlated single-photon-counting apparatus was a mode-locked, frequency-doubled Nd:YAG laser, which synchronously pumped a dye laser with Rhodamine 6G as the active medium. The excitation source was the output from the cavity-dumped dye laser that was frequency doubled from the

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Gottfried et al.

Figure 1. Log-log plot of the survival probability, N(t), following flash photolysis of CO from COHbA at room temperature in (A) aqueous buffer, (B) 90% glycerol, and (C) a trehalose glass.

visible to the near ultraviolet by focusing the pulse train onto a KDP type I crystal to generate light in the wavelength range 280-315 nm (5-10 ps fwhm). For front-face fluorimetry, an optically thick sample was rotated so that the excitation beam angle of incidence was 34° with respect to the surface normal.14 The fluorescence, collected at right angles to the illuminating light, was collimated and passed through a sheet polarizer (at the magic angle) and a cutoff filter. The emission was then focused onto the entrance slit of a 1/8 m subtractive double monochromator with a pair of 1200 groove/mm gratings. The monochromator output was directed onto the surface of a microchannel plate photomultiplier tube (MCP-PMT). The output of the MCP-PMT was amplified and filtered with a constant-fraction discriminator, the output of which triggered the start of a time-to-amplitude converter (TAC). The residual fundamental beam was incident onto a Si avalanche photodiode whose output was then used to stop the TAC. The TAC output was read by a combination analog-to-digital converter and multichannel analyzer. The instrument response function was typically 35 ps fwhm. Deconvolution of the emission decay curves and fitting to a sum of exponentials were achieved using a Marquardt, nonlinear least squares method. Determination of the quality of fit was judged from the value of the reduced χ2, the RUNS test, the residuals, and the autocorrelation of the residuals.32 There was some difficulty in fitting the very short lifetimes of the quenched Trp in heme proteins due to scattered light, spurious reflections, and the problem of obtaining an appropriate instrument response function. However, in all cases estimates of errors in the recovered parameters were obtained using the support plane method.33,34 Results Ligand Recombination. Figure 1 shows a comparison of the early time recombination behavior subsequent to a nanosecond photolysis pulse of COHbA in an aqueous buffer solution, a 90% glycerol/water mixture, and a trehalose glass. Geminate rebinding is usually demonstrated through a plot of the survival probability of the five-coordinate photodissociated heme as a function of time on a log-log plot. Results in aqueous buffer have been previously reported and show a geminate recombination phase that extends out to ∼100 ns followed by a plateau in which there is no rebinding.35 The plateau phase signals the end of the geminate recombination process, and all subsequent rebinding is derived from ligand molecules within the solvent. As a consequence, the onset of the plateau phase is also associated with the full development of the large-scale motions that allow for ligand escape from the protein.

Figure 2. Temperature dependence of the geminate recombination of CO to HbA in a trehalose glass.

As can be seen from Figure 1, the protein in 90% glycerol has a geminate phase that is both accelerated and extended compared to the sample in aqueous buffer. The rebinding of the trehalose-embedded sample obeys roughly the same kinetics as the 90% glycerol sample, but there is no plateau and the geminate phase persists beyond the 10 ms time scale of our experiment and the limits of signal detectability. All three rebinding curves are nearly linear in the geminate phase on the log-log plot. Figure 2 shows the temperature response of the geminate phase of COHbA in trehalose. It can be seen that the effective rate and yield over the first 500 ns increase as the temperature is decreased from 296 to 180 K (Figure 2A). Between 180 and 140 K, the process displays Arrhenius-type behavior, slowing as the temperature is further decreased (Figure 2B). Resonance Raman Spectra. Figure 3 shows the lowfrequency resonance Raman spectrum of the COHbA photoproduct at 10 ns for samples in aqueous buffer and embedded in a trehalose glass. Three separate regions of the glass were probed to test for homogeneity within the sample and yielded identical spectra. The solution phase and glass phase samples exhibit very similar spectra, indicating that there are no major static structural perturbations at the heme pocket imposed by the glass. The band at ∼230 cm-1 is assigned to the iron-proximalhistidine stretching mode.36 The frequency of this Raman band is very sensitive to stereochemical changes between the proximal histidine and the heme.37-39 In general the frequency is a reflection of proximal strain, the degree to which the iron atom is forced out of the heme plane by steric overlap of the carbon atoms of the proximal histidine and the nitrogen atoms of the heme pyrrole rings. This strain is coupled, via the proximal histidine and neighboring residues, to the F-helix. An increase in the frequency of this mode is associated with a decrease in proximal strain between the F-helix and the heme. The observed band position of 230 cm-1 is at the extreme high-frequency

Damped Hemoglobin Dynamics in Trehalose

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Figure 3. Resonance Raman spectra of the 10 ns photoproduct of COHbA in a trehalose glass taken from three locations within the same sample (A, B, and C) and in pH 7.4 phosphate buffer (D). The “h” denotes a hot H2 line from the hydrogen shift cell used to frequency shift the laser, and the “s” denotes a Raman line from the sapphire window.

end of the range of frequencies observed for both equilibrium and transient forms of Hb and Mb and is indicative of a fivecoordinate ferrous heme surrounded by the unrelaxed tertiary structure of the liganded R state form of HbA. It can be seen from Figure 3 that the position of this Raman band in both the solution and glass samples is identical. The only significant solvent-dependent differences observed in the low-frequency Raman spectra (150-800 cm-1) are those associated with the heme Raman bands that are sensitive to the heme propionate groups40 in the region between 320 and 370 cm-1. In the solution sample, the region between the 305 and 365 cm-1 bands contains only weak shoulders; however, in the glass two well-defined bands emerge at 333 and 350 cm-1. These two bands are similar to those observed for the COHbA photoproduct at cryogenic temperatures near 10 K.38,41 Lastly, the band at 365 cm-1 shifts to 367 cm-1 in going from the solution to the glass. The sharp, intense band seen in the solution spectrum (marked with an “s”) originates from the sapphire window that was used with that sample. Control spectra were taken of neat trehalose, both in solution and as a glass, and these indicate that the trehalose bands do not contribute to the heme spectra presented here. Time-Resolved Fluorescence. Both solution samples and trehalose-embedded samples of COHbA have nearly identical absorption and emission spectra for excitation in the tryptophan band (data not shown), indicating that there is no gross perturbation of the protein structure, in agreement with the Raman spectra. The recent work of Hagen et al.,5 which examined the kinetics of ligand rebinding in COMb embedded in trehalose, concluded that the solvent viscosity slows substate interconversion but does not drive the equilibrium to a narrower distribution of substates. In other words, the solution conformations are simply frozen in place when the sample is dried. The tryptophan fluorescence intensity decay of aqueous COHbA is shown in Figure 4A, with the lifetimes and amplitudes from a three-exponential fit given in Table 1. The fitting parameters are similar to those reported by other laboratories.10-13 The majority (>98%) of the population42 has a short lifetime of approximately 30 ps due to quenching of the Trp excited state by the nearby hemes.17 Approximately 1% of the population has an intermediate lifetime near 300 ps and less than 0.1% decays with a long lifetime of 2-3 ns. With regard to this last component, it is important to realize that even though this is a very small population of fluorophores, the

Figure 4. Fluorescence intensity decays, fits, and residuals for COHbA in (A) solution and (B) a trehalose glass at room temperature. λexc ) 290 nm; λem ) 340 nm. Data are the dots, and fits the solid lines. IRF is the instrument response function (fwhm ) 32 ps). A longer decay (20 ns) was collected and fit for part A, but only 4 ns are displayed for ease of comparison.

integrated intensity from this component (fi ) Riτi/∑Riτi) is nearly 10%, making the lifetime easily distinguishable. The amplitude of this long component is variable depending on solution conditions and sample preparation and has been found to range from 90% glycerol samples, indicating that these samples have the lowest average barrier to rebinding. Fluctuations that lead to ligand diffusion through the protein and into the solvent comprise a fourth tier of Hb dynamics. Frauenfelder and co-workers have argued that the same category of large-amplitude fluctuations that cause interconversion of the different conformational substates associated with the proteinligated CO (the A states) are also responsible for the onset of ligand escape from the heme pocket subsequent to photodissociation.69 In a related study of GR in COHbA in 0%-95% glycerol/water mixtures at room temperature, it has been shown

Gottfried et al. that there is a correlation between the acceleration of the rebinding rate and the increased delay in the appearance of the plateau that indicates the onset of ligand escape to the solvent.35 Note that this is not simply an increase in the geminate yield due to a slowing of the competing process of ligand escape. The rate of GR as well as the yield increases with increasing viscosity. This suggests that the large-scale fluctuations involved in ligand escape may also be coupled to the tertiary relaxations responsible for the increase in the barrier to geminate rebinding. The effective viscosity within the trehalose glass at room temperature is sufficiently high to significantly inhibit the nanosecond to microsecond tertiary relaxation in the COHbA photoproduct involving the movement of the F-helix. As can be seen in Figure 1, in the trehalose sample the plateau never appears and the geminate recombination continues unabated on the time scale of our probe. Thus, within the limits of the dynamics scheme presented above, the trehalose glass entirely prevents both the tertiary relaxation involving the shifting of the F-helix and the large-amplitude fluctuations that allow ligand escape. In summary at this point, the log-log plot of the survival probability of the unrecombined photoproduct of COHbA in trehalose shows several features that are illustrative of the extent to which the dynamical processes are influenced by the trehalose glass. The linearity of the plot is indicative of there being either an inhomogeneous distribution of conformational substates or progressive relaxation of the tertiary structure. The acceleration of the rebinding compared to an aqueous sample is consistent with a reduction in tertiary relaxation and a decrease in the average kinetic barrier for recombination. The absence of a plateau phase indicates the quenching of those motions that allow for ligand escape. Thus, the protein exists as a nonrelaxing, inhomogeneous distribution of conformations. Yet, in spite of all these changes in the dynamics, the Raman and fluorescence results indicate that the equilibrium structure of the COHbA is essentially unperturbed. Evidence for Dynamics within the Glassy State. Despite the evidence that the trehalose glass at ambient temperature is inhibiting protein dynamics, the temperature dependence of the geminate rebinding of COHbA in trehalose indicates that there is still a tier of dynamics that is relatively resistant to the glassy state. As mentioned above, GR in both COMb and COHbA in a glycerol/water solvent exhibits an inverse temperature effect, whereby the rebinding rate decreases from 180 K to room temperature, due to the onset of relaxations that increase the barrier height for GR. Figure 2 shows that the inverse temperature effect persists in COHbA embedded in trehalose, although it is much reduced compared to COHbA in 75% or 90% glycerol (unpublished results). In addition, whereas the turnover point for the onset of Arrhenius behavior occurs sharply at ∼180 K for the glycerol samples, a change is observed between 180 and 140 K for the trehalose sample. Since the large-amplitude motions such as the F-helix relaxation are already damped out at 300 K in the trehalose sample, the motions responsible for this effect must belong to a different tier of dynamics. We have observed time-dependent line broadening in band III that reverses the initial line narrowing due to KHB at temperatures greater than 140 K. The fluctuations responsible for this thermal averaging process do not show the same extreme viscosity dependence as the tertiary relaxation and ligand escape facilitating motions, and as such, they must be small motions within the globin. It is likely that the residual inverse temperature effect seen for COHbA in trehalose arises from the damping of these low-amplitude internal fluctuations.

Damped Hemoglobin Dynamics in Trehalose Origin of the Fluorescence Lifetime Change. Based on the results presented above, it is likely that the elimination of the nanosecond population in the fluorescence decay originates from the damping of a class of motions that generate transient conformations having a low probability of energy transfer. Bucci and co-workers have used separation distances and orientation factors obtained from X-ray structures to show that the fluorescence from β37 Trp has the shortest lifetime of the three Trp residues under all conditions.17 Their analysis indicates that the long-lived components could arise from the emission of the A-helix tryptophans (R14 and β15) if there exists a small population of heme that is disordered within the pocket. The occurrence of disordered heme may result from large coupled motions of the A- and E-helices (connected in part through hydrogen bonds between the A-helix tryptophans and the serine or threonine partners on the E-helix) that could open or enlarge the heme pocket and allow the heme to assume a transient orientation that reduces energy transfer. In a recent study,70 it was shown, using both fluorescence and UV resonance Raman spectroscopy, that a naturally occurring mutation on the A-helix of the β chains (HbC, β6 Glu f Lys) causes a loosening of the A-E helix interaction for the CO-bound derivative with a concomitant increase in the steady-state fluorescence. This result, as well as the UV resonance Raman studies of Spiro and co-workers,54 supports the idea that the A-E helix interaction is responsive to changes within the protein. In the presence of a highly viscous medium, such as the trehalose glass, the fluctuating motions of the Aand E-helices are likely to be damped. Thus, both the enhanced anchoring of the heme through the stabilization of the hydrogenbonding network involving one of the propionate groups (discussed above) and the reduction in the motion of the Aand E-helices could contribute to the loss of the nanosecond fluorescent component by inhibiting the formation of a transient disordered configuration in which the heme has undergone a shift in position and thereby reduced the efficiency of energy transfer from the tryptophans. Motion of the tryptophan residues is also a potential variable in the origin of the long-lived component. A cryogenic nanosecond-gated tryptophan fluorescence study on Hb using pulsed red edge excitation (295-310 nm)71 has shown that site selection and fluorescence line narrowing associated with the tryptophan fluorescence occur below 100 K. This indicates that the tryptophans and the surrounding protein environment of each of the fluorescence components are dynamically disordered, but can be immobilized at cryogenic temperatures and presumably also by high solvent viscosity. However, the altered long-lived fluorescent component cannot result simply from trehaloseinduced perturbations of the Trp alone, as this would be insufficient to explain the observed changes in the CO-rebinding kinetics. The appearance of the new subnanosecond fluorescent component may reflect a modified transient population in which residual low-amplitude versions remain of the protein motions that, in aqueous solution, give rise to the more fully disordered heme states and the longer nanosecond lifetime. Clearly not all motions are completely damped in the trehalose-embedded protein, as revealed both by the time-resolved Raman spectrum that shows that the rapid initial relaxation of the iron-proximalhistidine linkage upon photodissociation is not noticeably affected by the trehalose glass at ambient temperatures and by the residual inverse temperature effect in the rebinding kinetics of the photodissociated CO.

J. Phys. Chem., Vol. 100, No. 29, 1996 12041 Conclusions The results obtained in the present study of COHbA embedded in a trehalose glass at ambient temperatures show that the long-lived nanosecond component of the tryptophan fluorescence of COHbA does not originate from either an impurity or a stable conformation of COHbA, but must instead arise from a transiently disordered conformation. In addition, it can be seen that the trehalose greatly reduces many of the dynamical processes in COHbA, from tertiary relaxation of the F-helix to those that give rise to ligand escape from the protein. However, it does not appear to constrain either the initial rapid elastic recoil of the iron and proximal histidine upon ligand dissociation or a class of low-amplitude internal fluctuations. The resonance Raman spectrum of the COHbA photoproduct suggests that a trehalose-enhanced interaction involving the hydrogen bond network to the heme propionates may contribute to the rigidity of the heme within its pocket in COHbA in the glassy matrix. The loss of the 2-3 ns fluorescence component of the tryptophan in COHbA in trehalose and its replacement by a sub-nanosecond component are explained in terms of glass (high viscosity)-imposed constraints on the magnitude of thermally induced fluctuations in the structure of the protein. Although fluctuations still occur at 300 K, the trehalose glass prevents the large excursions of structure necessary for the formation of a transient conformation with the extremely unfavorable energy transfer parameters that result in a nanosecond component in the fluorescence decay. This explanation can be further tested by measuring the fluorescence lifetime below 140 K, where the inverse temperature effect terminates. At these temperatures the residual fluctuations should cease and the 0.6 ns component should also vanish. Acknowledgment. The authors wish to thank Ellen Chien, Steve Sligar, Leonid Levin, and Jerry Huang for allowing their unpublished results to be mentioned. Funding was provided by the NIH (GM-44343-04) and the W. M. Keck Foundation. References and Notes (1) Womersley, C. Comp. Biochem. Physiol. 1981, 70B, 669. (2) Crowe, J. H.; Crowe, L. M.; Carpenter, J. F.; Wistrom, C. A. Biochem. J. 1987, 242, 1. (3) Green, J. L.; Angell, C. A. J. Phys. Chem. 1989, 93, 2880. (4) Colac¸ o, C.; Sen, S.; Thangavelu, M.; Pinder, S.; Roser, B. Bio/ Technology 1992, 10, 1007. (5) Hagen, S. J.; Hofrichter, J.; Eaton, W. A. Science 1995, 269, 959. (6) Ross, J. B. A.; Eftink, M. R. In Time-ResolVed Laser Spectroscopy in Biochemistry III; Lakowicz, J. R., Ed.; The Society of Photo-optical Instrumentation Engineers: Bellingham, WA, 1992; pp 2-9. (7) Grinvald, A.; Steinberg, I. Z. Biochim. Biophys. Acta 1976, 427, 663. (8) Beechem, J. M.; Brand, L. Annu. ReV. Biochem. 1985, 54, 43. (9) Eftink, M. R. In Methods of Biochemical Analysis, Vol. 35: Protein Structure Determination; Suelter, C. H., Ed.; John Wiley & Sons: New York, 1991; pp 127-205. (10) Szabo, A. G.; Willis, K. J.; Krajcarski, D. T. Chem. Phys. Lett. 1989, 163, 565. (11) Bucci, E.; Malak, H.; Fronticelli, C.; Gryczynski, I.; Laczko, G.; Lakowicz, J. R. Biophys. Chem. 1988, 32, 187. (12) Bucci, E.; Gryczynski, Z.; Fronticelli, C.; Gryczynski, I.; Lakowicz, J. R. J. Fluoresc. 1992, 2, 29. (13) Hochstrasser, R. M.; Negus, D. K. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 4399. (14) Eisinger, J.; Flores, J. Anal. Biochem. 1979, 94, 15. (15) Hirsch, R. E. In Hemoglobins, Part C, Biophysical Methods: Methods in Enzymology; Everse, J., Vandegriff, K. D., Winslow, R. M., Eds.; Academic Press: San Diego, CA, 1994; Vol. 232, pp 231-246. (16) Janes, S. M.; Holtom, G.; Ascenzi, P.; Brunori, M.; Hochstrasser, R. M. Biophys. J. 1987, 51, 653. (17) Gryczynski, Z.; Tenenholz, T.; Bucci, E. Biophys. J. 1992, 63, 648. (18) Bucci, E.; Malak, H.; Fronticelli, C.; Gryczynski, I.; Lakowicz, J. R. J. Biol. Chem. 1988, 263, 6972.

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