CO rebinding to protoheme: Investigations of the proximal and distal contributions to the geminate rebinding barrier

Article abstract of DOI:10.1021/ja042365f

The rebinding kinetics of CO to protoheme (FePPIX) in the presence and absence of a proximal imidazole ligand reveals the magnitude of the rebinding barrier associated with proximal histidine ligation. The ligation states of the heme under different solvent conditions are also investigated using both equilibrium and transient spectroscopy. In the absence of imidazole, a weak ligand (probably water) is bound on the proximal side of the FePPIX-CO adduct. When the heme is encapsulated in micelles of cetyltrimethylammonium bromide (CTAB), photolysis of FePPIX-CO induces a complicated set of proximal ligation changes. In contrast, the use of glycerol-water solutions leads to a simple two-state geminate kinetic response with rapid (10-100 ps) CO recombination and a geminate amplitude that can be controlled by adjusting the solvent viscosity. By comparing the rate of CO rebinding to protoheme in glycerol solution with and without a bound proximal imidazole ligand, we find the enthalpic contribution to the proximal rebinding barrier, H_p, to be 11 ± 2 kJ/mol. Further comparison of the CO rebinding rate of the imidazole bound protoheme with the analogous rate in myoglobin (Mb) leads to a determination of the difference in their distal free energy barriers: ΔG_D ≈ 12 ± 1 kJ/mol. Estimates of the entropic contributions, due to the ligand accessible volumes in the distal pocket and the xenon-4 cavity of myoglobin (～3 kJ/mol), then lead to a distal pocket enthalpic barrier of H_D ≈ 9 ± 2 kJ/mol. These results agree well with the predictions of a simple model and with previous independent room-temperature measurements (Tian et al. Phys. Rev. Lett. 1992, 68, 408) of the enthalpic MbCO rebinding barrier (18 ± 2 kJ/mol).

Full text of DOI:10.1021/ja042365f

Published on Web 03/30/2005
CO Rebinding to Protoheme: Investigations of the Proximal
and Distal Contributions to the Geminate Rebinding Barrier
Xiong Ye, Anchi Yu, Georgi Y. Georgiev, Florin Gruia, Dan Ionascu, Wenxiang Cao,
J. Timothy Sage, and Paul M. Champion*
Contribution from the Department of Physics and Center for Interdisciplinary Research on
Complex System, Northeastern UniVersity, Boston 02115
Received December 20, 2004; E-mail: p.champion@neu.edu
Abstract: The rebinding kinetics of CO to protoheme (FePPIX) in the presence and absence of a proximal
imidazole ligand reveals the magnitude of the rebinding barrier associated with proximal histidine ligation.
The ligation states of the heme under different solvent conditions are also investigated using both equilibrium
and transient spectroscopy. In the absence of imidazole, a weak ligand (probably water) is bound on the
proximal side of the FePPIX-CO adduct. When the heme is encapsulated in micelles of cetyltrimethylam-
monium bromide (CTAB), photolysis of FePPIX-CO induces a complicated set of proximal ligation changes.
In contrast, the use of glycerol-water solutions leads to a simple two-state geminate kinetic response with
rapid (10-100 ps) CO recombination and a geminate amplitude that can be controlled by adjusting the
solvent viscosity. By comparing the rate of CO rebinding to protoheme in glycerol solution with and without
p
a bound proximal imidazole ligand, we find the enthalpic contribution to the proximal rebinding barrier, H ,
to be 11 ( 2 kJ/mol. Further comparison of the CO rebinding rate of the imidazole bound protoheme with
the analogous rate in myoglobin (Mb) leads to a determination of the difference in their distal free energy
barriers: ∆G
in the distal pocket and the xenon-4 cavity of myoglobin (∼3 kJ/mol), then lead to a distal pocket enthalpic
barrier of H
D
≈ 12 ( 1 kJ/mol. Estimates of the entropic contributions, due to the ligand accessible volumes
D
≈ 9 ( 2 kJ/mol. These results agree well with the predictions of a simple model and with
previous independent room-temperature measurements (Tian et al. Phys. Rev. Lett. 1992, 68, 408) of the
enthalpic MbCO rebinding barrier (18 ( 2 kJ/mol).
Introduction
involves at least two steps: the diffusion of ligand through the
surrounding solvent or buffer followed by the bond formation
step between the diatomic ligand and the iron. In proteins, the
process is complicated by the protein matrix where various entry
Iron protoporphyrin-IX (FePPIX or heme) is the prosthetic
group of a large class of heme proteins and is intimately
involved with physiological functions associated with the
binding of small diatomic ligands such as CO, O2, and NO.
The binding of these diatomic ligands is crucial to the varied
(
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catalytic, signaling, and oxygen storage functions of this
important class of proteins.1-5 The diatomic ligand binds to the
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heme iron from the distal side, while an amino acid residue
from the protein matrix (often histidine or cysteine) binds from
the other (proximal) side. The dynamics associated with ligand
binding to the heme group has been studied for many years in
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both native and mutant proteins
and in heme model
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2004, 43, 11109-11117.
compounds.1
6-24
The binding process for model compounds
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J. AM. CHEM. SOC. 2005, 127, 5854-5861
10.1021/ja042365f CCC: $30.25 © 2005 American Chemical Society

CO Rebinding to Protoheme
A R T I C L E S
and exit channels, as well as docking sites and cavities within
the protein, lead to a complexity in the overall reaction process
that is still not fully understood.1
mediated intermediate states and the distal pocket contribution,
is to study the CO rebinding kinetics of heme model compounds
with and without the proximal imidazole ligand. Surprisingly,
a complete set of such kinetic measurements has not been
reported and analyzed. If Fe-PPIX is simply dissolved in
aqueous solution at high pH, then dimerization takes place,
2-14,24-30
The specific interactions that control the activation barrier
for the Fe-diatomic bond formation in terms of the heme, the
proximal ligand, and the distal pocket protein architecture have
been studied extensively. For simplicity, the enthalpic part of
the ligand rebinding barrier can be separated into two contribu-
tions: the proximal term, HP, which is controlled by the
proximal ligand and the heme geometry (e.g., the Fe out-of-
plane displacement) and the distal term, HD, which is determined
by the distal pocket environment around the dissociated ligand.
It remains unclear if one or the other of these two terms
dominates the overall enthalpic barrier, or if both are equally
39
which significantly perturbs the rebinding kinetics. To prevent
heme dimerization, it is possible to use micelles formed from
materials such as CTAB (cetyltrimethylammonium bromide),
which stabilize the hemes in a monodispersed solution.4
Model compound kinetic studies have been performed in
CTAB under conditions where an imidazole ligand is bound to
0,41
1
8,23
the heme.
However, for such compounds, the CO ligand
escape rate is much larger than its geminate rebinding rate, so
the latter process is difficult or impossible to detect.23 Moreover,
in the absence of an added imidazole ligand, the CTAB
encapsulated heme samples again display complex kinetic
evolution, probably due to a base elimination mechanism1
(i.e., upon CO photolysis, there is a delayed dissociation of the
weak proximal base before CO rebinds). The problems men-
tioned above have made clear kinetic comparisons between the
imidazole ligated and unligated species problematic.
important, as suggested by a prior analysis of the nonexponential
rebinding kinetics of MbCO at low temperatures.3
1,32
On the
other hand, when the entropic part of the free energy rebinding
barrier is considered, the distal contribution (associated with
the ligand accessible volumes33 of the distal pocket and the Xe4
cavity) is more important than the proximal contribution.
Several studies of Mb under conditions (e.g., low pH, H93G
mutation) that remove the proximal histidine (His93) ligand have
been carried out in order to determine its contribution to the
8,42
In contrast to FePPIX-CO kinetics in CTAB solutions, early
10,15,34,35
16,17,43-45
rebinding barrier.
Unfortunately, the situation becomes
studies
of CO binding to iron PPIX in highly concen-
complex because of the increased acidity of the iron atom when
CO binds. For example, the complex ligation kinetics observed
for Mb at low pH required a ligand switching model involving
both His93 and H2O as possible ligands.3 The ligand switch
arises because of the presence of the Fe-His bond when CO is
trated glycerol solutions (with no added proximal ligand)
revealed a relatively simple kinetic response, suggesting that
heme aggregation and spurious proximal ligation was not a
problem under these highly viscous solution conditions. Kinetic
measurements of diatomic ligand binding to FePPIX in the
presence of a proximal imidazole ligand are also feasible under
identical conditions of highly concentrated glycerol. For ex-
ample, Traylor and Magde used 1-methlyimidazole (1-MeIm)
as a ligand to form a 1-MeImFePPIX-CO complex in 98%
glycerol and observed a geminate yield of 0.4 with a ∼300 ps
time constant. However, there have been no systematic attempts
made to isolate and assess the effects of proximal imidazole
ligation on the CO geminate rebinding kinetics using a self-
consistent set of solution conditions.
5
bound to the partially unfolded3 protein at low pH. In contrast,
5,36
a transient proximal Fe-OH2 bond is formed3
5,36
when CO is
19
photolyzed. In the case of the H93G mutant of Mb, where the
native proximal His93 ligand is replaced by glycine and no
exogenous ligand is added, another histidine (His97) is recruited
as the proximal ligand when CO binds and acidifies the heme
3
7,38
iron.
As a result of these complexities, the kinetic effects
of removing the proximal histidine ligand have not been isolated
in Mb using either pH induced unfolding or the H93G mutant
system.
One obvious way to probe the proximal contribution to the
rebinding barrier, free of complications from the various protein-
The measurements reported here present the first such
controlled comparisons of the CO geminate rebinding kinetics,
with and without a proximal imidazole ligand. We study heme
in its bare form, FePPIX, without the protein matrix and we
focus on the effect of the proximal ligand by adding exogenous
(
25) Lim, M.; Jackson, T. A.; Anfinrud, P. A. Nat. Struct. Biol. 1997, 4, 209-
14.
2
2-methylimidazole (2-MeIm) to the heme in highly concentrated
(
26) Brunori, M.; Vallone, B.; Cutruzzol a` , F.; Travaglini-Allocatelli, C.;
Berendzen, J.; Chu, K.; Sweet, R. M.; Schlichting, I. Proc. Natl. Acad.
Sci. U.S.A. 2000, 97, 2058-2063.
(80-95%) glycerol solution. We quantitatively compare the
kinetics of the imidazole ligated species to that of the unligated
bare heme in the same highly viscous glycerol solution, where
(
27) Tian, W. D.; Sage, J. T.; Champion, P. M.; Chien, E.; Sligar, S. G.
Biochemistry 1996, 35, 3487-3502.
(
(
28) Elber, R.; Karplus, M. J. Am. Chem. Soc. 1990, 112, 9161-9175.
29) Cao, W.; Christian, J. F.; Champion, P. M.; Rosca, F.; Sage, J. T.
Biochemistry 2001, 40, 5728-5737.
it is known to undergo very rapid and large amplitude CO
20,44
geminate recombination.
Such measurements directly probe
(
30) Schotte, F.; Lim, M.; Jackson, T. A.; Smirnov, A. V.; Soman, J.; Olson, J.
Hp, the contribution of the proximal imidazole ligation to the
CO rebinding barrier. In addition, we are also able to compare
S.; Phillips, G. N., Jr.; Wulff, M.; Anfinrud, P. A. Science 2003, 300, 1944-
1
947.
(
(
31) Srajer, V.; Champion, P. M. Biochemistry 1991, 30, 7390-7402.
32) Srajer, V.; Reinisch, L.; Champion, P. M. J. Am. Chem. Soc. 1988, 110,
6
656-6670.
(39) White, W. I. In The Porphyrins; Dolphin, D., Ed. Academic: NewYork:
1979.
(40) Simplicio, J.; Schwenzer, K. Biochemistry 1973, 12, 1923-1929.
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3248.
(42) Larsen, R. W. Inorg. Chim. Acta 1999, 288, 74-81.
(43) Beece, D.; Eisenstein, L.; Frauenfelder, H.; Good, D.; Marden, M. C.;
Reinisch, L.; Reynolds, A. H.; Sorensen, L. B.; Yue, K. T. Biochemistry
1980, 19, 5147-5157.
(44) Miers, J. B.; Postlewaite, J. C.; Zyung, T.; Chen, S.; Roemig, G. R.; Wen,
X.; Dlott, D. D.; Szabo, A. J. Chem. Phys. 1990, 93, 8771-8776.
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U.S.A. 1988, 85, 5062-5066.
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(
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33) Tilton, R. F., Jr.; Kuntz, I. D. Jr.; Petsko, G. A. Biochemistry 1984, 23,
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932.
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2
3
1
1
J. AM. CHEM. SOC.
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VOL. 127, NO. 16, 2005 5855

A R T I C L E S
Ye et al.
the kinetics of the imidazole ligated FePPIX-CO model com-
pound in glycerol with that of native Mb, to assess the
contribution of the distal pocket protein architecture to the
rebinding barrier.
and spins at a rate of over 7000 rpm to ensure that every pump-probe
pulse pair detects fresh re-equilibrated sample. Care is also taken to
make the pump beam completely overlap the probe beam at the sample.
The time resolution of 200 fs is determined by pump-probe cross-
correlation.
Experimental Methods
The kinetics measurements employ synchronous detection, where
the pump beam is modulated by a mechanical chopper (3 kHz), and
the pump-induced change in the probe signal is detected at each time
delay using a PMT and a lock-in amplifier. Transient absorption spectra
are taken with a diode array detector (TBY1024 Princeton Instruments,
Inc.) by using a shutter (Uniblitz, Inc.) to sequentially block and unblock
the pump beam for an equal amount of time (about 0.5 s) so that pump-
off and pump-on spectra can be accumulated. The logarithm of the
ratio yields the absolute magnitude of the pump induced transient
absorption difference spectrum.
Hemin (ferric protoporphyrin IX chloride) is purchased from
Porphyrin Products Inc., dissolved in 1N NaOH, and then diluted into
glycerol or CTAB solution. For typical 80% (v/v) glycerol solutions
(glycerol obtained from Acros Organics), the final sample pH is 12.
CTAB solutions (1% (w/v) cetyltrimethylammonium bromide obtained
from Sigma, Inc.) are adjusted to pH 7 with 0.1 M phosphate buffer.
The 2-methylimidazole concentration is 0.5 M when used. The hemin
concentrations for kinetics measurements are typically 50-100 µM.
At higher heme concentration in glycerol solution aggregation in deoxy
state is observed, as is evidenced by the blue-shift of Soret and red-
shift of band III in the deoxy sample spectra. After flushing with argon,
the sample is reduced by addition of a small amount of degassed sodium
dithionite solution. The CO adduct is formed from the reduced sample
by flushing with CO for at least 30 min. The equilibrium absorption
spectra of the samples are obtained using a Hitachi U-3410 spectro-
photometer.
The experimental system for the longer time (> 10 ns) kinetics
2
9
measurement has also been described in detail previously. Briefly, a
cw beam produced by a universal arc lamp (Oriel Instruments, Model
66021) and a 0.25 m monochromator (Oriel Instruments, Model 77200)
probe the kinetic response of the sample at selected wavelengths. The
transmitted beam is detected by a high-linearity, low noise, photomul-
tiplier (Hamamatsu, H6780) and recorded by a transient digitizer
(LeCroy 9420). A laser pulse (10 ns, 532 nm), produced by a 10 Hz
Both cw and pulsed laser sources were used to excite Raman
scattering in resonance with the heme Soret band. Raman scattering
excited by the 413.1 nm line of a Kr ion laser (Innova 300, Coherent,
Inc.) was collected at right angles to the excitation beam and focused
into a single grating monochromator (1870B, Spex Industries). A 76
MHz train of 3 ps pulses, obtained by second harmonic generation of
the picosecond oscillator (Mira 900P, Coherent, Inc.), excited scattering
at 435 nm, which was collected in a backscattering geometry. In both
cases, we used interferometric notch filters (Kaiser Instruments) to reject
the intense Rayleigh scattered light and a liquid nitrogen cooled CCD
detector (Princeton Instruments) to record the spectra. In additional
experiments, either the 363.8 nm line from an Ar laser (Coherent) or
tunable output from a dye laser, containing stilbene 1 or stilbene 3 and
pumped by the UV lines of the Ar laser, was used in conjunction with
a triple monochromator (SPEX) equipped with an intensified photodiode
array (Princeton Instruments). Infrared data were recorded on a Bio-
rad FTS-60A FTIR spectrometer.
Nd-doped yttrium-aluminum-garnet (YAG) laser (Continuum, Inc.),
is used to photolyze the sample with a typical pulse energy of 25 mJ.
The polarization of the photolysis beam is scrambled using quartz
wedges to eliminate the rotational relaxation artifact.
A 3-state kinetic model49 is used to describe the ligand rebinding
process after photolysis of CO from the Fe-PPIX
kout
FePPIXCO 7kBA FePPIX:CO {kin } FePPIX + CO
(1)
where the FePPIX:CO represents a contact pair state50 that can undergo
geminate recombination and FePPIX + CO represents the state with
CO free in solution. Within this simple 3 state model (under the
condition [FePPIX] , [CO]), the absorption change as a function of
time can be expressed as
-
(kgt)â
-kst
∆A(t) ) ∆A {I e
+ (1 - I )e } ) ∆A N(t)
(2)
0
g
g
0
Two laser systems are used to measure the kinetics over all time
scales. One covers the subpicosecond time scale out to 5 ns, and the
other system covers from 10 ns to 100 ms. The ultrafast kinetics
measurement setup and experimental procedure are described in detail
with 0 e â e 1, accounting for short time scale relaxation processes.
The observed geminate rate k ) kBA + kout is the sum of the intrinsic
rate of bond formation and the ligand escape rate. The geminate yield
is I
g
4
6,47
elsewhere.
An amplified Ti:sapphire laser system (REGA 9050,
g
) {kBA/(kBA + kout)}.
Coherent, Inc.) generates ∼50 fs laser pulses at ∼800 nm with an
average power of ∼ 1 W and repetition rate of 200 kHz. These
fundamental pulses are used to pump an optical parametric amplifier
Results and Analysis
Equilibrium Spectra. The absorption spectra of the reduced
(OPA, Coherent 9400) and to generate a white light continuum. The
(
1
or “deoxy”) state of FePPIX in 80% glycerol is shown in Figure
A. The spectra with and without the proximal ligand (2-
methylimidazole, 2-MeIm), show the characteristic asymmetric
OPA output is tuned at 580 nm and used to photolyze the sample. The
white light continuum, which probes the sample, is created by focusing
the 800 nm fundamental into a spinning fused silica disk, with a
resulting spectrum that extends from 390 to 800 nm. A monochromator
5
1
line shape of a five-coordinate heme. The thick solid line
peaking at 420 nm corresponds to the sample with no added
ligand and the thick dashed line peaking at 432 nm corresponds
to the 2-MeIm adduct. The other lines in Figure 1A correspond
to the samples with (thin dashed) and without (thin solid) 2-
MeIm, following the addition of CO, peaking at 418 and 411
nm, respectively. As can be seen, addition of the 2-methylimi-
dazole shifts the Soret peaks of both the deoxy and CO-bound
(Instruments SA, Triax 320) is used to disperse the probe light after
the sample. The time delay for the probe (white light continuum) with
respect to the pump (OPA pulse) is controlled using a servo-motor
translation stage (Daedal Division, Parker Automation) with a full
translation range of 5.6 ns.
The pump and probe beams are focused into a spinning sample cell
using an achromatic lens. Polarizations of pump and probe beams form
the magic angle to remove rotational relaxation artifacts, which are (in
any case) negligible for the heme group on short time scales in a high
(48) Hansen, P. A.; Moore, J. N.; Hochstrasser, R. M. Chem. Phys. 1989, 131,
49-62.
48
viscosity (glycerol based) solvent. The sample cell has a 2 in. diameter
(
49) Henry, E. R.; Sommer, J. H.; Hofrichter, J.; Eaton, W. A. J. Mol. Biol.
1
983, 166, 443-451.
(
46) Wang, W.; Ye, X.; Demidov, A. A.; Rosca, F.; Sjodin, T.; Cao, W.; Sheeran,
M.; Champion, P. M. J. Phys. Chem. B 2000, 104, 10789-10801.
(50) Jongeward, K. A.; Magde, D.; Taube, D. J.; Marsters, J. C.; Traylor, T.
G.; Sharma, V. S. J. Am. Chem. Soc. 1988, 110, 380-387.
(51) Srajer, V.; Schomacker, K. T.; Champion, P. M. Phys. ReV. Lett. 1986,
57, 1267-1270.
(
47) Ye, X.; Demidov, A. A.; Champion, P. M. J. Am. Chem. Soc. 2002, 124,
5
914-5924.
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CO Rebinding to Protoheme
A R T I C L E S
when the imidazole ligand is absent and sharpens at low
temperature (not shown). Several studies have identified cor-
relations between the band III frequency and diatomic ligand
9
,31,55,56
binding rates.
More specifically, the iron displacement
from the mean plane of the heme has been proposed as a
common underlying structural parameter that influences both
the band III frequency, the Fe-His frequency, and ligand
3
1,56,57,62,64
binding rates.
On the other hand, it should be
recognized that local electric fields can also influence the band
5
9,60
III frequency.
Recently, a strong correlation between the
Fe displacement and the oscillator strength of band III has been
6
1
used to explain the unusually strong temperature dependence
3
1
observed for band III in myoglobin. The proposed correla-
61
tions suggest that the blue shift and intensity decrease of band
III, which are observed when 2-MeIm binds and displaces the
water ligand to Fe(PPIX), reflect an increased Fe displacement
from the heme plane and thus a higher barrier to CO binding.3
Measurements of geminate CO rebinding to these compounds,
described below, allow a direct test of this hypothesized
correlation.
1,32
Transient Spectra. The upper part of Figure 2 shows the
transient absorption difference spectra following CO photolysis
from the bare heme in glycerol buffer. The transient spectra
show the characteristics of a simple two state rebinding
dynamics, with fixed isosbestic points and with a photoproduct
spectrum that is nearly identical to corresponding equilibrium
deoxy species. In contrast, the transient spectra of heme in
CTAB solution display a dynamic evolution of the photoproduct
in the 420-450 nm region. The initial photoproduct is close to
that in glycerol solution, but is different from the equilibrium
deoxy Mb species in CTAB. Within the first five ns, the initial
photoproduct in CTAB does not rebind with CO, as is shown
by the increase in the bleach signal. It instead relaxes (with a
time constant of about 1 ns) to a four-coordinate and/or weakly
bound five-coordinate equilibrium deoxy state, as is shown by
the equilibrium difference spectrum (dashed line) and the
characteristic multiple Soret peaks.
Figure 1. Equilibrium spectra of protoheme in 80% (v/v) glycerol (A)
and 1% (w/v) CTAB (B). Dashed and solid lines are samples with and
without the 2-MeIm ligand. The thin and thick lines are for CO-bound and
deoxy protoheme, respectively. Soret peak positions for heme in glycerol
without 2-MeIm are 420 nm (deoxy) and 411 nm (CO bound). When
2
-MeIm is bound the Soret peaks are 432 nm (deoxy) and 418 nm (CO
Vibrational Spectra. Figure 3A and 3B show the low and
high-frequency Raman spectra of the deoxy FePPIX excited at
435 nm. For heme in glycerol, the core marker bands show
bound). For CO bound heme in CTAB in the absence of 2-MeIm, the Soret
peak is found at 412 nm. When 2-MeIm is bound to heme in CTAB, the
Soret peaks are at 434 nm (deoxy) and 420 nm for CO bound. The multiple
peaks seen in the Soret region for FePPIX in CTAB in the absence of
65
features characteristic of a high-spin five-coordinated species
2
-MeIm and CO are located at 392, 415, 430, and 444 nm.
with or without the addition of 2-methylimidazole. The 2-MeIm
-
1
adduct shows a strong υ(Fe-NIm) mode at 212 cm , demon-
FePPIX species to the red, very near the analogous Soret peaks
found in native Mb. Figure 1B shows the corresponding spectra
in CTAB solution. The multiple Soret peaks of the deoxy form
strating the ligation of the imidazole ligand. When excited at
4
35 nm, as shown in Figure 3B(c), the resonance Raman
5
2
(53) Iizuka, T.; Yamamoto, H.; Kotani, M.; Yonetani, T. Biochim. Biophys. Acta
without external ligand have also been observed in benzene,
1
974, 371, 126-139.
and assigned to a four-coordinated heme. However, the four-
coordinate heme in partially unfolded deoxy Mb has a single
(54) Eaton, W. A.; Hanson, L. K.; Stephens, P. J.; Sutherland, J. C.; Dunn, J.
B. R. J. Am. Chem. Soc. 1978, 100, 4991-5003.
(
55) Agmon, N. Biochemistry 1988, 27, 3507-3511.
35
(56) Ahmed, A. M.; Campbell, B. F.; Caruso, D.; Chance, M. R.; Chavez, M.
D.; Courtney, S. H.; Friedman, J. M.; Iben, I. E. T.; Ondrias, M. R.; Yang,
M. Chem. Phys. 1991, 158, 329-351.
Soret peak at 383 nm . Raman vibrational markers provide a
more definite test for heme coordination and suggest (see below)
a mixture of four- and five-coordinate species for FePPIX in
CTAB solution in the absence of imidazole.
(
(
(
57) Rich, P. R.; Moody, A. J.; Ingledew, W. J. FEBS Lett. 1992, 305, 171-
173.
58) Christian, J. F.; Unno, M.; Sage, J. T.; Champion, P. M.; Chien, E.; Sligar,
S. G. Biochemistry 1997, 36, 11198-11204.
59) Franzen, S.; Moore, L. J.; Woodruff, W. H.; Boxer, S. G. J. Phys. Chem.
B. 1999, 103, 3070-3072.
Band III, shown in the insert, is found near 760 nm in the
presence of 2-MeIm for both glycerol and CTAB solutions and
(60) Nienhaus, K.; Lamb, D. C.; Deng, P.; Nienhaus, G. U. Biophys. J. 2002,
82, 1059-1067.
9,31,53-62
is typical of a five-coordinated heme species.
This band
(
61) Stavrov, S. S. Biopolymers 2004, 74, 37-40.
shifts to 770 nm in the absence of 2-MeIm as indicative of four-
(62) Kiger, L.; Stetzkowski-Marden, F.; Poyart, C.; Marden, M. C. Eur. J.
Biochem. 1995, 228, 665-668.
3
5,63
coordinate hemes.
Another band near 840 nm also appears
(
(
(
63) Brault, D.; Rougee, M. Biochemistry 1974, 13, 4598-4602.
64) Champion, P. M. J. Raman Spectrosc. 1992, 23, 557-567.
65) Parthasarathi, N.; Hansen, C.; Yamaguchi, S.; Spiro, T. G. J. Am. Chem.
Soc. 1987, 109, 3865-3871.
(52) Brault, D.; Rougee, M. Biochemistry 1974, 13, 4591-4597.
J. AM. CHEM. SOC.
9
VOL. 127, NO. 16, 2005 5857

A R T I C L E S
Ye et al.
Figure 3. Panels A and B show the resonance Raman spectra of deoxy
FePPIX in glycerol with (a) and without (b) 2-MeIm. Deoxy FePPIX in
CTAB without 2-MeIm is shown in (c) of panel B and as a function of
excitation wavelength in panel C.
Figure 2. Transient absorption spectra of photolyzed FePPIX-CO. Panel
A shows a sample in 80% (v/v) glycerol solution. Panel B shows data for
a sample in 1% (w/v) CTAB solution, the dashed line in B is the scaled
equilibrium difference spectra between the deoxy and CO bound sample
curve probably cannot effectively separate a five coordinate CO
bound state from a six coordinate CO species with a weak
proximal ligand (e.g., water).
(from Figure 1B without 2-MeIm).
spectrum of deoxy FePPIX in CTAB solution without the
addition of 2-methylimidazole shows mainly features of inter-
mediate spin, which is characteristic of a four coordinated heme
species. The spectra also show minor features of a high spin
species, most notably the doublet in the ν4 region indicates a
mixture of both four and five coordinate species. A more
detailed Raman study of deoxy FePPIX in CTAB solution,
which involved varying the excitation wavelength, is shown in
Figure 3C. A minority species of five coordinate heme manifests
its presence when the laser excitation is tuned to 415 nm and
CO Rebinding Kinetics. As can be seen from Figure 5 and
Table 1, the high viscosity of the glycerol samples mainly affects
the CO escape rate, kout, (see eq 1) and only weakly affects the
bond formation rate, kBA (see eq 1). This same conclusion was
6
8
also arrived at in earlier studies. Addition of 2-MeIm as a
proximal ligand significantly reduces the geminate rebinding
rate and the amplitude of the geminate process. For heme
without a strong proximal ligand, kBA is about 2 orders of
magnitude larger than for the heme with 2-methylimidazole.
The CO rebinding barrier difference between the proximally
ligated (with 2-MeIm) and unligated (no 2-MeIm added) states
can be found from the rates in Table 1:
-1
the high-spin (ν3) marker band at 1474 cm is clearly resolved.
As the excitation moves further to the blue near 364 nm, the
intermediate spin species again dominates the spectra. This
behavior is consistent with the multiple peaked nature of the
absorption spectrum shown in Figure 1B and suggests that a
For 80% glycerol
8
kBA(+ Im)
kBA(- Im)
∆HP
3.3 × 10
5
-coordinate heme minority species, absorbing near 420 nm
)
)
) exp -
(3a)
(3b)
(
)
1
0
kT
(e.g., see Figure 1A), underlies the multiple peaked 4-coordinate
1.5 × 10
heme absorption spectrum observed in CTAB solution.
In Figure 4, we show the Raman and infrared data for the
CO bound heme species in glycerol (without adding 2-MeIm).
The correlation of υ(C-O) and υ(Fe-CO) indicates the
proximal ligand is either weak66 or absent. The correlation
For 95% glycerol
8
kBA(+ Im)
kBA(- Im)
∆HP
1.7 × 10
) exp -
(
)
1
0
67
kT
1.9 × 10
5858 J. AM. CHEM. SOC.
9
VOL. 127, NO. 16, 2005

CO Rebinding to Protoheme
A R T I C L E S
Figure 5. FePPIX-CO recombination kinetics: in 80% (v/v) glycerol
solution, with (open circle) and without (open rectangle) 2-MeIm; in 95%
(
2
v/v) glycerol solution, with (solid circle) and without (solid rectangle)
-MeIm. The results of fitting the data are given in Table 1.
Mb). In the latter case, barriers associated with the distal pocket
will also retard the CO rebinding process. The ratio of the
rebinding rates with and without the protein matrix can be used
to find the free energy difference in the rebinding barrier due
to the presence of the distal pocket and protein matrix.
6
kBA(Mb)
∆GD
2.8 × 10
3.3 × 10
)
) exp -
)
(
)
8
kBA(+ Im)
kT
∆HD
∆SD
exp -
exp
. (5)
(
)
(
)
kT
k
Thus, the difference in the free energy barrier at 293 K for CO
rebinding due to the presence of the distal pocket in Mb is
approximately
Figure 4. Upper panel shows measurements of FePPIX-CO in 80% (v/v)
glycerol solution. The υ(Fe-CO) assignment is made using isotopic labeling
and resonance Raman spectroscopy (excitation at 413 nm). The υ(C-O)
mode is detected using infrared absorption (inset). The lower panel is a
correlation plot of υ(Fe-CO) and υ(C-O), with all data points except the
solid star (present work) from Vogel et al. The open squares are data for
imidazole (or histidine) ligated heme systems and the dashed line is a fit to
the data. The open triangles represent thiolate ligated heme systems and
the open circles and the solid star (this work) represent heme systems with
weak or absent proximal ligands.
∆G
≈ 12 ( 1 kJ/mol
(6)
D
6
7
where we explicitly acknowledge (through ∆SD in eq 5) that
the entropic contribution to the distal barrier may differ between
the glycerol solution and the protein matrix.
Discussion
where we have assumed that the entropic contribution to the
free energy barrier is independent of the proximal ligation state
Ligation States of Fe-PPIX in Glycerol. Protoheme in
glycerol solution without the addition of 2-MeIm has a weak
proximal ligand, derived either from water or glycerol.5
For example, the Soret band in Figure 1 displays an asymmetric
line shape, characteristic of a five-coordinate heme,51 but blue
shifted to 420 nm as might be expected for a weak fifth ligand.
In addition, the Raman core-size marker bands of PPIX in
glycerol are indicative of a high-spin, five coordinated heme
and are very similar to the marker bands we observe when
(i.e., the spin entropy changes are common to both reactions as
2,63,69,70
seen by the characteristic spin-sensitive spectroscopic indicators
in Figures 1A, 3, and 4). Thus, the proximal contribution to the
difference in the enthalpic rebinding barriers at 293 K is found
to be ∆HP ) 9.5 kJ/mol (80% glycerol) and ∆HP ) 11.2 kJ/
mol (95% glycerol), leading us to conclude
7
1
∆
H ≈ 10 ( 1 kJ/mol
(4)
P
2
-MeIm is added (see Figure 3). In contrast, the Raman spectra
FePPIX complexed with 2-MeIm in glycerol, in the absence of
a distal pocket protein matrix, also shows a faster rebinding
rate than when the heme is embedded in the protein matrix (i.e.,
of CTAB-encapsulated Fe(PPIX) show marker bands charac-
teristic of both 4- and 5-coordinate Fe(II) hemes, indicating that
(
69) Brault, D.; Rougee, M. Biochem. Biophys. Res. Commun. 1974, 57, 654-
(
(
(
66) Ray, G. B.; Li, X. Y.; Ibers, J. A.; Sessler, J. L.; Spiro, T. G. J. Am. Chem.
659.
Soc. 1994, 116, 162-176.
(70) Rougee, M.; Brault, D. Biochemistry 1975, 14, 4100-4106.
(71) Spiro, T. G.; Li, H. Y. Resonance Raman Spectroscopy of Metallopor-
phyrins; In Biological Applications of Raman Spectroscopy; Spiro, T. G.,
Ed.; Wiley-Interscience Publication: New York, Chichester, Brisbane,
Toronto, Singapore, 1988; pp 1-37.
67) Vogel, K. M.; Kozlowski, P. M.; Zgierski, M. Z.; Spiro, T. G. Inorg. Chim.
Acta 2000, 297, 11-17.
68) Marden, M. C.; Hazard, E. S. I.; Gibson, Q. H. Biochemistry 1986, 25,
2
786-2792.
J. AM. CHEM. SOC.
9
VOL. 127, NO. 16, 2005 5859

A R T I C L E S
Ye et al.
Table 1. Kinetics of CO Rebinding in Glycerol Solution
k
BA (s-1)
k
out (s-1)
k
g
(s-1)
I
g
â
k
s
(s-1)
1
8
1
8
1
6
0
0
0
9
10
9
3
Heme-CO
Mb-CO
80% glycerol
- 2-MeIm
2-MeIm
- 2-MeIm
2-MeIm
- 2-MeIm
1.5 × 10
3.3 × 10
1.9 × 10
1.7 × 10
1.5 × 10
2.8 × 10
5 × 10
4.4 × 10
1 × 10
2 × 10
4.7 × 10
2 × 10
0.75
0.07
0.95
0.2
0.38
0.12
0.33
1
0.36
1
0.37
0.5
3.3 × 10
5 × 10
9
2
2
2
+
9
10
8
9
5% glycerol
1.9 × 10
1.1 × 10
8
+
6.7 × 10
2.5 × 10
2 × 10
8.3 × 10
4 × 10
10
7
10
7
9
9% EG
2
75% glycerol
2.3 × 10
6.5 × 10
the weak ligand is probably water, which is destabilized in the
hydrophobic interior of the CTAB micelle.
displays more complicated behavior, probably involving a ligand
switching mechanism.
3
6,37
Entropic Contribution to the Distal Barrier. We can
estimate the differences in the entropic contributions to the distal
barrier between Mb and the 2-MeIm bound FePPIX by assuming
that the entropy at the transition state for CO bond formation
The spectral evidence indicates that the CO-bound PPIX
species in both the CTAB and the glycerol samples is six-
coordinated with a weak proximal ligand from water or OH.
Photolysis of CO does not dissociate the weak proximal ligand
initially. In glycerol solution, the CO has a much smaller escape
rate due to the glycerol viscosity, so that geminate rebinding of
CO takes place before any other proximal ligation changes
occur. On the other hand, for samples in the aqueous CTAB
solution, the viscosity is much lower so that CO can escape
more efficiently. When this happens, a dynamic dissociation
of the weak proximal ligand can take place in the first few ns
because the affinity of the heme iron for the proximal ligand
†
to the heme, S D, is independent of the details of the distal
†
†
architecture for the different samples (i.e., S D(Mb) ≈ S D(+Im)
in eq 5). Thus, the difference in the entropic contribution to the
distal free energy barrier in eq 5 is given by
†
D
†
D
∆S ) [S (Mb) - S (Mb)] - [S (+ Im) - S (+ Im)] ≈
D
D
D
S (+ Im) - S (Mb) (7)
D
D
(
probably water) in the hydrophobic micelle environment is
where SD(Mb) ) k ln ΩMb and SD(+Im) ) k ln Ω+Im with Ω
denoting the number of possible distal pocket states associated
with the CO ligand in its initial configuration prior to geminate
recombination. Thus, eq 7 leads directly to
lowered when the CO bond is broken.
Figure 4 displays the correlation between the bound C-O
and the Fe-CO vibrational frequencies in the absence of
2
-MeIm and demonstrates that, when the CO complex is formed
in glycerol, the proximal ligand bound trans to the CO is either
weak or absent. The deoxy Fe-PPIX sample in glycerol also
has a red-shifted (and more intense) band III (see Figure 1),
which is consistent with a more planar conformation of the
heme, as is expected for a high-spin ferrous heme with a weak
∆
SD
Ω+Im
ΩMb
) ln
.
(8)
(
)
k
We now take the number of accessible initial states for the CO
ligand in Mb, ΩMb, to be proportional to the volume of the
region from which the CO ligand must return during the
geminate rebinding process. Similarly, the number of possible
reactant states for geminate rebinding of the CO ligand to
2-MeImFePPIX in glycerol solvent, Ω+Im, is proportional to
the volume of the glycerol solvent that is displaced by the
photolyzed CO ligand. In the case of Mb, the ligand accessible
volume, relevant for geminate rebinding, can be taken as the
(or absent) proximal ligand. Finally, the Raman spectra show
that the heme is coordinated by a weak fifth ligand, even in the
absence of CO and 2-MeIm. This fact, along with the ca. 100-
fold increase in heme affinity for water observed following CO
70
binding to deuterioheme, suggests that (in the absence of added
imidazole) water is likely bound as a weak proximal ligand in
the CO bound complex as well as in the deoxy state.
When 2-MeIm is added to Fe-PPIX in glycerol solution, the
ligation state remains five coordinate because the steric interac-
tions between the 2-methyl group and the heme prevent the bis
7
2
sum of the distal and Xe4 pocket volumes, which has been
7
3a,b
3
quantified
and is found to be ∼4.6 Å . For the FePPIX in
7
3b
glycerol, we approximate the accessible volume as the cube
(
six coordinate) complex from being formed.69 The Soret bands
3
of the CO bond length, ca. 1.3 Å . Taking the ratio and
combining eqs 5, 6, and 8, we find T∆SD ) kT ln(Ω+Im/ΩMb)
with and without CO (see Figure 1) mimic very closely the
spectra observed for native Mb. The low-frequency Raman
spectra, shown in Figure 3, locate the Fe-NIm mode at 212
)
-3.2 kJ/mol so that
-
1
cm and give definitive evidence that the 2-MeIm has bound
to form the 5 coordinate complex. Band III of the 2-MeIm
FePPIX complex is also located at 760 nm, as observed in deoxy
Mb.
∆
H ) ∆G + T∆S ≈ 8.8 kJ/mol
(9)
D
D
D
Thus, given the various approximations, we conclude
In summary, the ligation properties of the model system
composed of FePPIX in glycerol is remarkably well-behaved.
When 2-MeIm is added, the sample displays optical and Raman
spectra that mimic very closely what is observed for native Mb.
More importantly, when no exogenous 2-MeIm ligand is added,
the spectral evolution observed following CO photolysis remains
characteristic of a simple two state rebinding process (Figure
(
72) Hummer, G.; Schotte, F.; Anfinrud, P. A. Proc. Natl. Acad. Sci. U. S. A.
2
004, 101, 15330-15334.
(
73) (a) Liang, J.; Edelsbrunner, H.; Fu, P.; Sudhakar, P. V.; Subramaniam, S.
Proteins 1998, 33, 18-29. (b) Alternatively, as an estimate of the minimum
total volume explored by the dissociated ligand we can take the 45%
occupancy factor (Srajer, V. et al., Biochemistry 40, 13802-13815) found
3
for CO localized in the distal pocket volume of 1.4 Å . This leads to
3
3
V
min(Mb) ∼ 1.4 Å /0.45 ) 3.1 Å . For FePPIX in glycerol, the ligand
3
accessible volume is expected to be smaller than the 1.4 Å distal pocket
3
of the protein, so the 1.3 Å bond length cube is probably an upper limit.
(
c) Recent temperature dependent ligand rebinding kinetics experiments
2, upper). This should be contrasted with the spectral evolution
performed in our lab suggest that this enthalpy barrier may be as high as
3 kJ/mol.
found for FePPIX in CTAB micelles (Figure 2, lower), which
5860 J. AM. CHEM. SOC.
9
VOL. 127, NO. 16, 2005

CO Rebinding to Protoheme
A R T I C L E S
0
∆
H ≈ 9 ( 2 kJ/mol
(10)
contribution, H D, can be taken to be approximately zero. Taking
D
0
HD ) ∆HD + H D, and using the ratio of the CO rebinding
rates as outlined in eqs 5-10, yields the distal pocket contribu-
tion to the barrier
Proximal and Distal Contributions to the Rebinding
Barrier. The advantage of systematic studies of a well-behaved
model system is that we can separately assess the proximal and
distal contributions to the enthalpic rebinding barrier. Dlott and
co-workers measured the temperature dependence of the CO
rebinding to FePPIX in glycerol (with no added ligand) and
H ≈ ∆H ≈ 9 ( 2 kJ/mol.
(12)
D
D
An early analysis32 of the low-temperature CO rebinding to Mb,
which also separated the proximal and distal contributions to
the rebinding barrier, found that a fit to the low-temperature
0
found that the barrier for rebinding, H BA, was quite small (∼1
7
32
data required a distal term of ∼7 kJ/mol and a (distributed)
proximal term of ∼5 kJ/mol. However, at room temperature
the photolyzed heme is no longer “quenched” by the frozen
protein (below the glass transition of the glycerol solvent) and
it can relax to a fully domed out-of-plane position. Using the
4
4,73c
kJ/mol).
This allows both the proximal contribution to the
barrier (due to the imidazole ligation) and the distal contribution
due to the protein matrix) to be directly assessed by using eqs
(
4
and 10.
The weak proximal ligand, observed in the absence of the
-MeIm, is consistent with a more in-plane position of the heme
32
simple model of Srajer et al., along with a difference between
the average iron out-of-plane position for temperatures below
2
iron and a much lower rebinding barrier. Thus, we denote the
(
∼0.2 Å) and above (∼0.4 Å) the solvent glass transition, the
barrier for FePPIX-CO in glycerol, associated with kBA(-Im),
32
room-temperature proximal barrier HP was predicted to
0
as H BA. Then the definition of ∆HP in eq 3 leads to ∆HP ) HP
increase from 5 to 12 kJ/mol. Thus, the overall barrier at room
0
-
H BA so that the absolute enthalpic barrier associated with
3
2
temperature was estimated to be HBA ≈ HP + HD ≈ 12kJ/
mol + 7kJ/mol ≈ 19 kJ/mol. These early predictions agree
surprisingly well with the MbCO rebinding barrier deter-
the proximal imidazole ligation can be found using HP ) ∆HP
0
44
0
+
H BA. Here, we have assigned the magnitude of H BA ≈ 1
kJ/mol to the weak proximal ligation of water, which is an
8
0,81
mined
near room temperature (HBA ) 18 ( 2 kJ/mol prior
approximation that falls within our margin of error. Given the
to distal relaxation) as well as with the room-temperature
measurements using the FePPIX model system presented here
0
44
uncertainty of H BA as measured by Miers et al., and its
assignment to the proximal part of the enthalpic barrier, we use
eq 4 with an expanded error margin to find
(
HP + HD ≈ 11 kJ/mol + 9 kJ/mol ) 20 kJ/mol). It is worth
32,64
emphasizing that this same simple model
also predicts a
-
1
-2
heme doming force constant of 22 ( 3 kcal mol Å ,
associated with the proximal rebinding barrier, which is
consistent with recently detected heme doming oscillations near
H ≈ 11 ( 2 kJ/mol
(11)
P
8
2-84
On the other hand, distal effects have also been implicated in
1 THz.
the rebinding process and extensive distal pocket mutation
In conclusion, we emphasize that a systematic analysis of
CO rebinding to a “well behaved” heme model compound has
been carried out so that the magnitude of the proximal and distal
contributions to the rebinding barrier can be separately deter-
mined. The results can be cross-checked by noting that the sum
of the two contributions HBA ) HP + HD ≈ 20 kJ/mol is very
studies5
,12,74-76
have led some workers to suggest that the distal
7
7-79
pocket is the main source of the ligand binding barrier.
We can estimate the distal contribution to the barrier using the
same approach as above because the 2-MeIm ligated FePPIX
(which has no distal pocket) can be compared to Mb, which
80,81
does have a distal pocket. Here, as above, we assume that the
close to independent measurements
of the MbCO rebinding
very small (∼1 kJ/mol) rebinding (enthalpy) barrier observed
barrier (HBA ) 18 ( 2 kJ/mol) and to the room temperature
barrier predicted by an early model.
44
32,80
for FePPIX in glycerol is purely proximal in origin (due to
the weakly bound proximal water ligand), and that the distal
Acknowledgment. This work is supported by NSF 0211816
and NIH DK035090 (PMC) and by NSF0240955 and NIH GM-
52002 (JTS).
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VOL. 127, NO. 16, 2005 5861