(Received for publication, March 3, 1995; and in revised form, May 4, 1995)
From the
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
-
-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.
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 -
-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.
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 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°.
where is the lifetime of donor in the absence
of acceptor and R is the donor-acceptor separation. R
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
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; is the angle
between Dand A,
,
are the angles between Aand T, and Dand T, respectively(23) .
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.
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
-
-meso axis of the
porphyrin ring.
Using the atomic coordinates of horse heart
myoglobin we computed the dependence of 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
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
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 on the angle
between the orientation of the transition moment of the heme
absorption band responsible for energy transfer from tryptophan, and
the
-
-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) .
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 curvatures
shown in Fig. 4.
Figure 4:
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.
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.
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).
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 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 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 distribution of the
three molecular species was described by the lifetime amplitudes in Table 2. The sum of the amplitudes +
-
=
was the fraction of myoglobin with normal
hemes, Mb
. Twice the amplitude of the 1.3 ns
lifetime, 2
=
, was
the fraction of myoglobin with disordered hemes,
Mb
. The amplitude of the lifetime at 4.8 ns,
=
, was the fraction
of RDMb, Mb
. After doubling the amplitude
, all fractions were normalized to 1.0. (
)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
C/(
+
). 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 K
= K
. 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. (
)
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.
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.
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