©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Heme-Protein Interactions in Horse Heart Myoglobin at Neutral pH and Exposed to Acid Investigated by Time-resolved Fluorescence in the Pico- to Nanosecond Time Range (*)

(Received for publication, March 3, 1995; and in revised form, May 4, 1995)

Zygmunt Gryczynski (1) Jacek Lubkowski (2) Enrico Bucci (1)(§)

From the  (1)Department of Biochemistry, University of Maryland Medical School at Baltimore, Baltimore, Maryland 21201 and the (2)Department of Chemistry, University of Gdansk, Poland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We measured the steady state and time-resolved emission intensity decay of horse heart myoglobin at various pH values from neutral to pH 4.42. The steady state intensity was reversibly increased with the decreasing pH, almost doubling at pH 4.5. Frequency domain data for emission decay were analyzed separately for each pH and simultaneously by global analyses. The results indicated the presence of four lifetime components, conserved throughout the pH titrations at 40, 116, 1363, and 4822 ps, respectively. The titration affected only their fractional intensities. Assignments of the lifetimes were based on the Förster theory of radiationless dipole-dipole interaction and the atomic coordinates of the system. We assigned the two shorter lifetimes to Trp-14 and Trp-7, respectively, in the presence of normal hemes. The 1363-ps lifetime was assigned to Trp-7 with inverted hemes (i.e. rotated 180° around the alpha--meso axis of the porphyrin ring). The 4822-ns lifetime was assigned to reversibly heme-dissociated myoglobin. Lorentzian lifetime distributions were narrow for the lifetimes at 40, 116, and 4822 ps, indicating a homogeneous protein structure. Instead the lifetime at 1363 ns had a broad, pH-independent distribution consistent with small angle wobblings of inverted hemes inside the heme pocket. These analyses revealed the presence of three species originating from heme-protein interactions: the native form of crystalline myoglobin, the conformation with disordered hemes, and the reversibly dissociated heme-free myoglobin. There was increased heme inversion and heme dissociability at lower pH, consistent with the titration of the proximal and distal histidines inside the heme pocket.


INTRODUCTION

Heme-protein interactions in hemoglobin and myoglobin are now closely scrutinized. In fact, they are probably correlated to oxidizability of heme, heme inversion, protein stability, and allosteric properties(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) . The dissociation equilibrium of heme and protein acquires importance because of the potential toxicity of free heme in vivo, when released either from myoglobin or hemoglobin. This is an important field of study, although difficult to approach under equilibrium conditions.

The kinetics of heme release from several hemoproteins perturbed by acid, alcohol, or detergents have been studied for a number of years (1) . Smith et al.(12) evaluated the free energy of activation of heme release from peroxidase and other hemoproteins to be near 95-105 kJ/mol at neutral pH. This implies that the affinity of the apoproteins for heme is very high, and the fraction of reversibly dissociated hemes is very small and difficult to detect. Still, indirect detections by heme transfer to albumin (4, 5) or to recombinant forms of apomyoglobin (2, 3) are possible, proving the existence of a dissociating equilibrium between heme and protein.

Disordered, i.e. inverted, hemes, rotated 180° around the alpha--meso axis of the porphyrin ring, have been first observed in myoglobin and hemoglobin by La Mar and colleagues using NMR spectroscopy(6, 7, 8) . Since then it was shown that this peculiar conformation of the heme can be detected also by CD spectroscopy and has probable implications on the Bohr effect and oxygen affinity of these systems(9, 10, 11) . These studies were complicated by the relative low amounts, 2-8%, of inverted hemes in these proteins, especially in their functionally active ferrous states. Therefore the approachable information was rapidly exhausted.

In myoglobin we can expect to find at least three species produced by heme-protein interactions, namely myoglobin with normally positioned hemes, as in the crystals, myoglobin with disordered hemes, and reversibly heme-dissociated myoglobin. In unperturbed systems the last two species represent a very small fraction of the total and are difficult to detect and investigate. This is why until now it was impossible to measure the equilibrium distribution of these species.

Time-resolved fluorescence spectroscopy overcomes these difficulties. Inverted hemes and heme-dissociated myoglobin produce long lifetimes of 1 ns or more, much longer and easily resolved from the main lifetime components of myoglobin emission (originating from the protein fraction with normally oriented hemes), which are in the picosecond range. Because of the longer lifetime, the minor components contribute a large fraction of the total emission intensity. Frequency domain fluorescence spectroscopy, by measuring the phase and modulation of the emission intensity, is well suited for monitoring the presence and changes of fluorescence intensities for the small fractions of long lifetimes components produced by disordered and heme-dissociated myoglobin. It should be stressed that in fluorescence spectroscopy fractional intensity and relative amplitudes of the lifetime components are readily interconvertible. In this way the very small relative amplitudes of the various components can be obtained, so as to investigate the distribution of the molecular species present in the system.

In this report we used the length of the lifetimes for the identification of the emitting molecular species. Their amplitudes, as obtained from the fractional intensities, were used for estimating their distribution. This allowed a detailed quantization of the relative proportions of heme-protein interaction species in horse heart myoglobin. As anticipated, three species were detected with normal, inverted, and reversibly dissociated hemes. We monitored the pH dependence of their distribution between pH 7 and 4.5.


MATERIALS AND METHODS

Crystalline Ferric Horse Heart Myoglobin

Myoglobin was purchased from Sigma. The protein was purified by preparative free-phase electrofocusing using the Rotofor equipment marketed by Bio-Rad.

Fluorescence Steady State Intensities

Fluorescence was measured using an 8000 SLM photon counting instrument. Purified solutions of myoglobin were washed repeatedly by ultrafiltration with the desired buffers during a period of 2-3 h. After this treatment the pH of the samples and their fluorescence response were stable for several hours. We used 0.05 M phosphate buffers, at neutral pH, and 0.05 M acetate buffers below pH 5.0. Comparable intensities were obtained by calibrating the samples to the same optical density at 294 nm. The absorption spectrum of the samples was independent of pH within the pH range investigated. Protein concentration was near 0.2 mg/ml.

Absorption Spectra

Absorption spectra were measured using a double beam Aviv 14DS spectrophotometer.

Fluorescence Lifetimes

Fluorescence lifetimes were measured using a frequency domain 10 GHz fluorometer equipped with a Hamamtsu 6-µm microchannel plate detector (MCP-PMT) as described previously (14) . The instrument covered a wide frequency range between 15 and 5000 MHz, which allowed detection of lifetimes ranging from several nanoseconds to a few picoseconds. Samples were placed in a 1-cm path ``shielded cuvette,'' previously described(15) , which eliminated stray lights from the emission. The exciting light was tuned at 294 nm. Sample emission was filtered through an Oriel interference filter centered at 340 nm and a Corning 7-60 broad band filter. For reference, we used the scatter of the sample solution filtered through an Oriel interference filter at 289 nm together with neutral density filters. The filters used for the emission and the reference were calibrated, so as to obtain identical optical length at 294 and 340 nm(15) .

The governing equations for the time-resolved intensity decay data were assumed to be either a sum of discrete exponentials as in Beechem et al.(16) and Lakowicz et al.(17)

where alpha is the amplitude (preexponential factor) and the lifetime of the ith discrete component; or a sum of Lorentzian distribution functions as in the the equation

and

where = center value of the lifetime distribution and = full width at half-maximum.

In frequency domain the measured quantities, at each frequency , are the phase shift () and the demodulation factors (m) of the emitted light versus the reference light. Fractional intensity, amplitude, and lifetime parameters were recovered by nonlinear least squares procedures using either the Globals Unlimited software (16) or software developed at the Center of Fluorescence Spectroscopy(17) . The measured data were compared with calculated values (, m) and the goodness of fit was characterized by

where is the number of degrees of freedom, and and are the experimental uncertainties in the phase and modulation values, respectively. The respective values were = 0.2° over a span of 0-90° and = 0.002 over a span of 0-1°.

Distances and Angular Relationships

Relationships between tryptophans and hemes were determined using the atomic coordinates of horse heart myoglobin(18) . The Förster parameter kappa^2(19, 20, 21, 22, 23) and its dependence on the orientation of the heme transition moment were computed using software developed in our laboratory.

Lifetimes Computation

The value of donor lifetime in the presence of energy transfer can be computed according to the Förster theory as (19, 20, 21, 22, 23)

where (0) is the lifetime of donor in the absence of acceptor and R is the donor-acceptor separation. R(0) is a characteristic distance (19, 20, 21, 22, 23) defined by:

where n is the refractive index of the solvent, is the quantum yield of the donor in the absence of acceptor, J is the overlap integral between the normalized emission spectrum of the donor and the absorption spectrum of the acceptor, and kappa^2 is the orientation factor for dipole-dipole interaction (19, 20, 21, 22, 23) described by:

The vectors D, A, and T are unit vectors along the transition moment directions of the donor and acceptor, and the translation vector respectively; alphais the angle between Dand A, alpha, alphaare the angles between Aand T, and Dand T, respectively(23) .


RESULTS

Steady State Measurements

As shown in Fig. 1, exposure to acid of ferric horse heart myoglobin more than doubled the intensity of the emission and produced a red shift of the emission spectrum. It also shows that this phenomenon was entirely reversible from pH 4.5.


Figure 1: Emission spectrum of horse heart myoglobin at pH 7.2 (lower curve) and at pH 4.5 (upper curve). The dashed curve was obtained upon neutralization of the sample previously at pH 4.5. The inset shows the pH dependence of the intensity.



Computed Lifetimes

The critical parameter for dipole-dipole interactions is the orientation factor kappa^2, which depends on the conformation of the protein, and can be determined using atomic coordinates. For the donor tryptophan we assumed a fixed orientation of its ^1L(a) transition moment at 38° from the pseudosymmetry axis of the indole rings(24, 25) .

As we reported(23, 26) , energy transfer from tryptophan to heme is regulated by a single absorption band of the heme between 300 and 380 nm, whose orientation was estimated to be within the range of 50-70° (angle ) from the alpha--meso axis of the porphyrin ring.

Using the atomic coordinates of horse heart myoglobin we computed the dependence of kappa^2 on the orientation of the transition moment of the heme by changing the angle from 0 to 90° for normal hemes, and from 0 to -90° for inverted hemes. Fig. 2shows the dependence of kappa^2 on the angle for normal (N) and inverted, i.e. disordered (D), heme positions for Trp-7 and -14, respectively. The shaded area indicates the region between 50 and 70° for the orientation of the heme transition moment. Within that area, as later discussed, we have chosen an orientation centered at 55°. Considering the possible effects of conformational fluctuations, the lifetimes were computed using an average kappa^2 obtained in the interval = 50-60°. Table 1shows the computed lifetimes for Trp-7 and -14, respectively, in the presence of either normal (N) or inverted (D) hemes.


Figure 2: Dependence of the Förster parameter kappa^2 on the angle between the orientation of the transition moment of the heme absorption band responsible for energy transfer from tryptophan, and the alpha--meso axis of the porphyrin ring. N refers to hemes in normal position, D refers to hemes in inverted position. The shaded area is the region previously estimated for the orientation of the transition moment of the heme(26) .





Time Resolved Measurements

The pH dependence of the frequency domain data is presented in Fig. 3and the respective recovered parameters for discrete lifetime distribution are shown in Table 2. Time-resolved analyses showed the presence of four lifetime components in the emission, justified by a 2-4-fold decrease of the ^2 value going from three- to four-component analyses.


Figure 3: Frequency dependence of phase shift (lower curves) and demodulation (upper curves) of the intensity decay of horse heart myoglobin at various pH values. (+) pH 7.20; () pH 4.95; () pH 4.72; () pH 4.62; (★) pH 4.42.





Table 2includes data obtained by singular analyses and by simultaneous, global analyses, of all of the data at different pH values, where the four lifetimes were linked across the pH titration. This allowed a precise recovery of the pH-dependent intensities. There was a very good agreement between the parameters recovered from singular and global analyses. The table clearly shows that the increased steady state intensity at acid pH, shown in Fig. 1, was produced solely by the increasing fractions of the lifetimes in the nanosecond range near 1.3 and 4.8 ns, respectively. In other words, they were produced by components whose recalculated amplitudes were a few parts per hundred or parts per thousand. The accuracy and resolution of the recovered lifetimes is best described by the sharpness of the ^2 curvatures shown in Fig. 4.


Figure 4: ^2 curvatures for the global analyses shown in Table 2for the four lifetime components detected in the time resolved emission intensity of horse heart myoglobin. The dotted line goes across the standard deviation of the estimated values.



The data were also analyzed using continuous Lorentzian lifetime distributions. The bandwidth of the distributions is a good indicator of microheterogeneity of the environment of the fluorophore(27, 28) . The recovered Lorentzian parameters are listed in Table 3. The Lorentzian distributions of the longer lifetimes in the nanosecond range are illustrated in Fig. 5.




Figure 5: Lorentzian distributions of the nanosecond lifetimes of horse heart myoglobin at various pH values. From top to bottom at pH 7.20, 4.95, 4.62, and 4.42. All distributions are normalized to the height of the respective peak.



The lifetime of 1.3 ns had a broad Lorentzian distribution, which remained constant at all pH values, indicating microheterogeneity of the environment. In contrast, the lifetime at 4.8 ns had a narrow distribution, consistent with the presence of a homogeneous structure. At pH 4.42 the broadening of the band indicated an initial unfolding of the protein.

The very narrow widths of the Lorentzian distributions of the two shortest lifetimes in the picosecond range were also consistent with a rigid, uniform myoglobin structure. They are not shown in Fig. 5because they were too narrow.


DISCUSSION

Reversibility of the pH Titration and Choice of pH Range

As shown in Fig. 1exposure to acid pH produced a large increase of the emission intensity and a red shift of the emission spectrum. An increase of fluorescence intensity with decreasing pH in myoglobin systems was already reported by Irace et al.(29) . They also noticed that the increase started below pH 5. More recently Posticova and Yumakova (31) reported a similar phenomena in iron-free (PPIX) reconstituted myoglobin, which began at pH 5.5 and was attributed to the release of PPIX from the heme pocket.

The increased emission intensity, upon acid exposure, implied either a significant increase of the lengths of the lifetimes, or a rearrangement of their relative fractions, or both. A correct interpretation of the phenomenon required detail analyses of time resolved emission decays. In order to evaluate the experimental data on the basis of the Förster theory and the atomic coordinates of the system, we limited our observations to the reversible part of the pH titration, where significant distortions of the protein structure were not expected.

In our measurements the intensity increase was entirely reversible between pH 7.0 and 4.5. This implied that all of the modification of the lifetime parameters were also reversible.

Analysis of the pH-dependent Lifetime Components

It was necessary to distinguish whether the intensity increase was due to pH-dependent modifications of either the length or the fractions of the lifetimes. The distinction was important because conformational changes affecting the Trp-heme angular relations would affect the length of the lifetimes, while changes in the fractions would imply a redistribution of molecular species emitting with the respective lifetime.

We used global analyses, simultaneously involving all of the data shown in Fig. 3. This technique is best for recovering parameters common to different, independent sets of data, as in our case(16, 17) . When it was assumed that the intensities remained constant at all pH values, the recovered pH-dependent lifetimes were unacceptably long with intensities totally inconsistent with those estimated by individual analyses. Instead, when it was assumed that the lifetimes remained constant across the pH titration, the recovered pH-dependent intensities, and recovered constant lifetimes were in very good agreement with the parameters estimated by singular analyses. This indicated that the global analyses were refining the parameters recovered with individual analyses and that pH affected only the relative fractions of the lifetime components (Table 2).

Accuracy in Detecting Small Fractions of Long Lifetime Components

Both tryptophans in myoglobin are in close proximity to the heme. Our lifetimes computations show that in horse heart myoglobin energy transfer to heme consumed more than 97% of the tryptophan excitation energy. Thus, the observed steady state fluorescence intensity was less than 3% of that expected in the absence of quenching. When in this heavily quenched system a small fraction is present of either significantly less or non quenched tryptophan its contribution to total intensity will be very relevant. Table 2shows that at neutral pH, for the lifetime at 4.8 ns, 25% of total emission intensity of myoglobin corresponded to an amplitude of 7 parts per thousand. At low pH this amplitude increased to 31 parts per thousand and was responsible for 54% of total emission intensity. These intensity variations are largely outside the errors of the experiments. Inspection of Fig. 3clearly shows that the differences between the various curves largely exceed the noise of the experiments.

Absence of Impurities

Reversible changes of lifetime intensities on constant lifetime lengths, upon the pH titration, excluded the presence of non-myoglobin impurities of nonquenched tryptophans. In fact there was no justification for pH-dependent fractions of impurities endowed with long lifetimes. (^1)

Lifetimes Assignment Based on the Atomic Coordinates

As we reported(23, 26) , a single electronic dipole in the heme is the acceptor of radiationless energy transfer from tryptophan in hemoproteins. We estimated its orientation (angle ) at 60 ± 10° from the alpha--meso axis(26) .

In this study orientations between 50 and 60° gave very similar computed lifetimes. Increasing the angle above 60° made the short picosecond lifetimes detected in the system very difficult to explain. For these reasons, and taking into account possible wobblings of the heme, we used an average kappa^2 value obtained in the interval = 50-60°. This produced a very good agreement between computed and measured lifetime parameters.

For the computation we also assumed that the lifetime of nonquenched tryptophan was 4.8 ns. The choice was based on the observation that it was the longest lifetime detectable in the system (Table 2) and was also consistent with the lifetimes of free tryptophan in water environment.

The resulting values shown in Table 1anticipated the presence of three experimentally detectable lifetimes in the system, one near 60 ps due to Trp-14 in the presence of normal hemes, one near 130 ps, due to both Trp-14 in the presence of inverted hemes and Trp-7 in the presence of normal hemes, and one near 1.8 ns due to Trp-7 in the presence of inverted hemes.

These values were very similar to those measured and listed in Table 2. Therefore we assigned the lifetime at 40 ps to Trp-14 in the presence of normal heme, and the lifetime at 116 ps to Trp-7 in the presence of normal hemes and to Trp-14 in the presence of inverted hemes. The lifetime at 1.3 ns was assigned to Trp-7 in the presence of inverted hemes. The lifetime at 4.8 ns could be assigned only to reversibly heme-dissociated myoglobin and its value used as that of nonquenched tryptophan in the system.

It should be noted that the relative amount of inverted hemes in the system was double the amplitude of the 1.3-ns lifetime. In fact, the numerical analysis did not take into account that it originated only from Trp-7. Notably, the presence of inverted hemes in myoglobin systems was detected also by NMR spectroscopy with fractions (4-8%) consistent with the amplitudes of the 1.3-ns lifetime reported here (6, 7, 8) .

The Lifetimes in the Nanosecond Range

As shown in Table 3and Fig. 5, the lifetime near 1.4-1.6 ns, due to heme inversion, had a Lorentzian distribution characterized by a broad bandwidth, indicating microheterogeneity of the environment of the fluorophore(27, 28) . This lifetime and its Lorentzian bandwidth were not pH-dependent. This is an important observation because it indicated that the heterogeneity was not due to unfolding of the protein which would have been pH-dependent, therefore affecting both parameters. Instead, it was a characteristic of the native system. It suggested a disorder produced by small angle wobblings of the inverted hemes inside the heme pocket resulting from the wrong stereospecificity of the protein-heme interactions. For this reason we refer to this as a disordered heme conformation of myoglobin. The question may be posed whether the presence of inverted hemes alters the tertiary structure of the protein. The consistence between the lifetimes computed on the basis of atomic coordinates and those found experimentally would suggest that heme inversion does not alter significantly the tertiary structure of the protein. However, the broad-band Lorentian distribution of the lifetimes produced by heme inversion would be consistent with tertiary structure modifications involving the heme pocket. In either case heme disorder is recognized by the longer lifetimes that it produces.

The lifetime at 4.8 ns was the expression of reversible heme loss. Its Lorentzian distribution was narrow, confirming a homogeneous structure for the reversibly heme-dissociated protein. The bandwidth increased in size at pH 4.42, indicating an initial unfolding of the dissociated protein. At this pH the reversibility was only 80-90%. The initial unfolding of myoglobin at pH 4.42 increased the size of the bandwidth of the Lorentzian distribution of the 4.8-ns lifetime, only. This indicated that the heme-dissociated protein was the species most sensitive to pH denaturation. It also showed that heme in both normal and disordered positions had a strong stabilizing influence on the folding of myoglobin.

The narrow distribution of the longest lifetime component up to pH 4.5 clearly indicates homogeneity of the heme-dissociated protein. We do not know whether this homogeneity reflects or not a loss of helical content. The conformation of isolated apomyoglobin is known to have a substantially reduced CD signal in the far UV region, indicating a loss of helical content(1) . However, it should be stressed that the reversibly dissociated protein and isolated apomyoglobin are two different systems. Apomyoglobin was exposed to extreme acid pH (near pH = 2) and to organic solvents so to allow heme extraction. Therefore it is a protein refolded after acid denaturation. Moreover, it is in the absence of heme. Reversibly heme-dissociated myoglobin was not denatured at very low pH and was not exposed to organic solvents. Also, because of its high affinity for heme, it may not remain heme-free long enough for relaxing into the conformation of isolated apomyoglobin. We believe that reversibly heme-dissociated myoglobin is still a fully folded protein; however, we do not have enough information to distinguish between the two alternatives.

The Lifetimes in the Picosecond Range

It is important to note that the very similar amplitudes of the lifetimes at 40 and 116 ps implied that the two tryptophans in myoglobin were equally exposed to incident light. They were the expression of the native crystalline protein therefore the sum of their amplitudes (alpha(1) + alpha(2) - alpha(3)) decreased with pH from 0.95 at pH 7.0 to 0.91 at pH 4.62, while the disordered and heme-free fractions were monotonically increasing. The narrow Lorentzian distribution of these lifetimes confirmed their origin from a compact homogeneous structure of the system.

Distribution of Heme-Protein Interaction Species in Myoglobin

The lifetime assignment formulated above allowed the distinction of three heme-protein interaction species in horse heart myoglobin. One was the native conformation of crystalline myoglobin with normally oriented hemes which gave the two lifetimes in the picosecond range. The second conformation was myoglobin with disordered hemes which produced the lifetime near 1.3 ns. The third conformation was the reversibly dissociated heme-free myoglobin (RDMb) (^2)whose lifetime was near 4.8 ns.

The distribution of the three molecular species was described by the lifetime amplitudes in Table 2. The sum of the amplitudes alpha(1) + alpha(2) - alpha(3) = alphawas the fraction of myoglobin with normal hemes, Mb. Twice the amplitude of the 1.3 ns lifetime, 2alpha(3) = alpha, was the fraction of myoglobin with disordered hemes, Mb. The amplitude of the lifetime at 4.8 ns, alpha = alpha(4), was the fraction of RDMb, Mb. After doubling the amplitude alpha(3), all fractions were normalized to 1.0. (^3)Also, it could be safely assumed that in the system there were equimolar amounts of Mb and free heme.

On these assumptions, the equilibrium between the three conformations could be described by the Fig. S1, where


Figure S1: Scheme 1



where C is the molar protein concentration.

The computed constants are listed in Table 4. The overall heme-protein dissociation constant is alpha^2C/(alpha + alpha). This is practically equivalent to K. It is the first time that this constant is directly measured. Early estimations (1) were inferred from kinetic data obtained with CN- or CO-heme on sperm whale myoglobin and human hemoglobin.



As expected, at all pH values disordered hemes were more dissociable than those of normal hemes. It is to be noted that in this model KK= K, and Kalpha= Kalpha. This implies that it was not possible to distinguish which fraction of RDMb originated either from Mb or from Mb. This was consistent with the observation that dissociated hemes were in random rotational positions when recombining with the protein. The selection between the two conformations, normal and disordered, was done by the stereospecificity of the recombination. (^4)

The increasing amounts of both Mb and Mb with decreasing pH very probably reflected the protonation of the proximal and distal histidines(35, 36) , resulting in increased heme loss and decreased stereospecificity of the recombination.

Conclusion

We believe that this is the first time that heme-protein interactions in myoglobin have been investigated in such quantitative detail. The observations regarding the structure of heme-dissociated protein and its initial unfolding are novel and challenging.

The experimental flexibility of time resolved fluorescence spectroscopy promises a wealth of information regarding heme-protein interactions in myoglobin and hemoglobin under a variety of conditions, i.e. pH, temperature, ligands, and high spin and low spin status of the iron. This will shed light on the functional and biological relevance of heme-protein interactions in these systems.


FOOTNOTES

*
This work was supported in part by United States Public Health Service Grants R01-HL-13164, P01-HL-48517 and by National Institutes of Health Grant RR-08119 to the Center for Fluorescence Spectroscopy at the University of Maryland at Baltimore. Computer time and facilities were supported by the computer network of the University of Maryland at Baltimore, and at College Park, MD. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, University of Maryland Medical School, 108 N. Greene St., Baltimore, MD 21201. Tel. 410-706-6380; Fax: 410-706-7390; ebucci{at}umabnet.ab.umd.edu.

(^1)
Hochstrasser and Negus(32) , Janes et al.(33) , and Bismuto et al.(28) also report long nanosecond lifetimes for sperm whale myoglobin. As discussed elsewhere (23) , they may originate from heme-protein interaction species, as for horse heart myoglobin, Willis et al.(34) did not find nanosecond lifetimes in sperm whale myoglobin and claimed that they originate from impurities. The discrepancy between these data are difficult to explain. A few speculations have already been proposed (23) .

(^2)
The abbreviations used are: RDMb, reversibly dissociated heme-free myoglobin; Mb, fraction of disordered myoglobin; Mb, fraction of native myoglobin; Mb, fraction of heme-free myoglobin.

(^3)
The sum of the fractions were (alpha(1) + alpha(2) - alpha(3)) + 2alpha(3) + alpha(4) = 1.0.

(^4)
This points out another difference between isolated apomyoglobin and reversibly heme-dissociated myoglobin. When apomyoglobin is reconstituted with heme, the initial ratio between normal and disordered heme is 50/50(6, 8) . Reorientation of the heme after reconstitution is very slow, while the protein refolds into native myoglobin.


ACKNOWLEDGEMENTS

We are indebted to Dr. Gary D. Brayer of the University of British Columbia, Vancouver, CA, who generously released the atomic coordinates of horse heart myoglobin before publication.


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