From the
The low spin ferric and low and high spin ferrous forms of
myoglobin, bacterial cytochrome P-450-CAM, and chloroperoxidase have
been examined by Fe-K x-ray absorption edge spectroscopy. The positions
of the absorption edge and the shapes of preedge and edge regions of
imidazole adducts of ferric P-450-CAM and chloroperoxidase are
essentially the same when compared with thiolate-ligated ferric
myoglobin. As these three protein derivatives all have six-coordinate,
low spin, ferric hemes with axial imidazole and thiolate ligands, the
superposition of x-ray absorption edge spectral properties demonstrates
that the protein environment does not effect the spectra, provided one
compares heme iron centers with identical coordination numbers, spin
and oxidation states, and ligand sets. In contrast, a 0.96 eV
difference is observed in the energy of the absorption edge for
imidazole- and thiolate-ligated ferric myoglobin with the latter
shifted to lower energy as observed for ferrous myoglobin states.
Similarly, in the low spin ferric-imidazole and ferrous-CO states, the
energies of the absorption edge for chloroperoxidase and P-450-CAM are
shifted in the direction of the ferrous state (to lower energy) when
compared with those for analogous myoglobin derivatives. In the
deoxyferrous high spin state, comparison of the edge spectra of
chloroperoxidase with analogous data for cytochrome P-450-CAM suggests
that the electron density at the iron is similar for these two protein
states. The shifts observed in the energies of the x-ray absorption
edge for the thiolate-ligated states of these proteins relative to
derivatives lacking a thiolate ligand provide a direct measure of the
electron releasing character of a thiolate axial ligand. These results
therefore support the suggested role of the cysteinate proximal ligand
of P-450 as a strong internal electron donor to promote O-O bond
cleavage in the putative ferric-peroxide intermediate to generate the
proposed ferryl-oxo ``active oxygen'' state of the reaction
cycle.
Since molecular structure dictates the spectroscopic and
catalytic properties of metalloenzyme active sites, a thorough
understanding of the unique spectral features of cytochrome P-450 and
chloroperoxidase has long been sought to provide insight into the
mechanism of action of these two heme enzymes
(1, 2) .
P-450 and chloroperoxidase are unusual among heme enzymes in each
having a cysteine thiolate proximal ligand. With P-450, it has been
proposed that the cysteinate ligand plays a key mechanistic role as a
strong internal electron donor to facilitate cleavage of the O-O
bond of the putative iron-peroxide intermediate to generate the active
hydroxylation catalyst
(1, 2, 3, 4) .
Using Fe-K x-ray absorption edge spectroscopy, we have probed the
properties of the central metal of these two enzymes and of myoglobin,
which has a histidine proximal ligand. Shifts in the energy of the
x-ray absorption edge are observed that correlate with the presence of
the thiolate proximal ligand. The results described herein provide the
most direct evidence to date for the specific influence of the
cysteinate ligand on the properties and possibly on the reactivities of
the heme iron centers of cytochrome P-450 and chloroperoxidase.
Cytochrome P-450 is a ubiquitous heme iron monooxygenase found in
bacteria, mammals, and plants
(1, 2, 5, 6, 7) . Its ability to
activate one atom in dioxygen for insertion into unactivated C-H
bonds, with concomitant reduction of the other oxygen atom to water
(Reaction 1), has generated substantial interest in its mechanism of
action. The name P-450 was derived from the unusually red-shifted Soret
absorption band at 450 nm for the ferrous-CO derivative of the enzyme.
This property is clearly dependent on the presence of a cysteinate
thiolate proximal heme iron ligand (Fig. 1); the role that ligand
may play in the mechanism of the enzyme is less well established.
P-450-CAM, obtained from Pseudomonas putida grown on
(1 R)-camphor, catalyzes the stereo- and regio-specific
hydroxylation of camphor to the 5- exo alcohol
(6) .
On-line formulae not verified for accuracy REACTION 1
On-line formulae not verified for accuracy REACTION 2
On-line formulae not verified for accuracy REACTION 3
On-line formulae not verified for accuracy REACTION 4
The myoglobins are monomeric heme-containing oxygen carriers found in
the vertebrate muscle tissue. The proximal ligand to the heme iron of
the myoglobins is histidine
(10) (Fig. 1). The ability of
the myoglobins (and hemoglobins) to bind dioxygen reversibly has led to
intense scrutiny by x-ray crystallography as well as spectroscopy.
X-ray absorption spectroscopy using synchrotron radiation is a
powerful technique for high resolution studies of transition metal
active sites in biological molecules because it directly probes the
properties of the central metal
(11, 12, 13) .
The heme iron coordination structures of a variety of heme proteins
have been studied by means of extended x-ray absorption fine structure
(EXAFS)
Cytochrome P-450-CAM was purified from Pseudomonas putida grown on (1 R)-camphor by the method of Peterson et
al.(33) with minor modifications
(34) .
Chloroperoxidase was purified from C. fumago grown on fructose
(35) as reported previously
(36, 37) . Horse
heart myoglobin (Sigma) was purified as described by Dawson et al.(38) . All chemicals were obtained from Sigma or Aldrich
and were used as received. The derivatives of P-450, chloroperoxidase,
and myoglobin were prepared at
X-ray absorption spectra
were measured between 10 and 85 K using an Oxford Instruments
continuous flow liquid helium cryostat at the Stanford Synchrotron
Radiation Laboratory (SSRL) and the National Synchrotron Light Source
(NSLS). At SSRL, the data were collected as fluorescence excitation
spectra on unfocused wiggler beam lines 7-3 and 4-2 using a
Si
(220) double crystal monochromator detuned 50% at 7474 eV for
harmonic rejection. At NSLS, the data were collected on unfocused
bending magnet beam line X19A using a Si
(220) double crystal
monochromator. A Canberra 13-element solid state germanium array
detector was used in all experiments except one set at SSRL in which an
ionization chamber detector of the Stern-Lytle design was used. The
SSRL storage ring was operated under dedicated condition at 3.0 GeV and
35-95 mA. The NSLS storage ring was operated at 2.5 GeV and
90-200 mA.
Data reduction and analysis were performed with the
XFPAKG program package
(39, 40) . Energy calibration was
done using an internal iron foil standard by assigning the first
inflection point of the iron absorption edge at 7111.2 eV. The
calibration was fine tuned by individually comparing and recalibrating
each foil scan relative to a ``master'' scan until a
reproducibility of <0.06 eV was obtained. Calibrated scans were
further inspected individually for noise-caused inconsistencies, and
unacceptable scans were identified and rejected during further data
reduction. A weighted average of the remaining scans was calculated.
The first and the last scans were compared and showed that no
detectable photoreduction occurred for any of the samples. A preedge
subtraction was performed by fitting the postedge region with a smooth
polynomial, which was extrapolated into the preedge region and
subtracted. A one-segment spline was fit to the postedge region and
subtracted, at which point the data were normalized to an edge jump of
one.
The edge separation was quantified in three separate ways. The
edges were fit using the program EDG_FIT (written by Dr. G. N. George,
SSRL), which utilizes the double precision version of the public domain
MINPAK fitting library (Garbow, B. S., Hillstrom, K. E., and More, J.
J., Argonne National Laboratory). All spectra were fit over the range
7100-7190 eV. The preedge and all edge features including the
rising edge were all modeled by pseudo-Voigt line shapes (sums of
Gaussian and Lorenzian functions). A fixed ratio (1:1) of
Gaussian/Lorenzian were used for all functions. The number of functions
and their initial energy positions were determined from the second
derivative of the spectrum. An arctangent step function was applied to
model the total edge jump. The number of functions were minimized and
kept constant for samples of each group, such as
N-methylimidazole-ligated chloroperoxidase,
N-methylimidazole-ligated P-450, and 1-propanethiol-ligated
myoglobin. In the fits, the line-width, energy position, and height
were allowed to vary in a stepwise manner. The difference in edge
position was then calculated as the difference between the energy of
the maximum of the first highest transition for each edge. The
uncertainty in the determination is estimated to be
To test the rationale, the
edge spectra of the N-methylimidazole adducts of ferric
cytochrome P-450-CAM and chloroperoxidase were compared to that of a
1-propanethiolate adduct of ferric myoglobin (Fig. 2,
top). All three samples have imidazole and thiolate axial
ligands and a six-coordinate, low spin ferric iron. The shape and
energy of the absorption edge are essentially indistinguishable among
the three spectra. This conclusion is further supported in the
difference x-ray absorption spectra in the bottom of Fig. 2. In
separate experiments, it was found that the edge energies of ferric
myoglobin complexed with either N-methylimidazole,
4-methylimidazole, or unsubstituted imidazole were identical or very
similar (; data not shown); N-methylimidazole was
used for subsequent experiments. A similar experiment comparing the
edge energy for complexes of ferric myoglobin with aliphatic
(1-propanethiol) and aromatic ( p-chlorothiophenol) thiolate
ligands revealed a noticeable difference of 0.31 ± 0.03 eV in
the edge energy (; spectrum not shown); because cysteine is
an aliphatic thiol, 1-propanethiol was used in subsequent experiments.
Figure 1:
Schematic representation of the active
site heme iron coordination structures of cytochrome P-450
( P-450) and chloroperoxidase ( CPO) ( left)
and of myoglobin ( right) showing the endogenous proximal
ligands and exchangeable distal sites.
Chloroperoxidase is an unusual heme enzyme isolated from the fungus
Caldariomyces fumago(1, 2, 8, 9) that functions as a peroxidase (Reaction 2), catalase
(Reaction 3), or halogenation catalyst (Reaction 4). Well documented
spectroscopic similarities to P-450 support the conclusion that
chloroperoxidase also has a cysteinate proximal ligand (Fig. 1).
That chloroperoxidase, in contrast to P-450, is a peroxide-dependent
halogenation catalyst and is incapable of dioxygen activation has
focused attention on what must be significantly different
structure-function relationships for the two thiolate-ligated heme
enzymes.
(
)
spectroscopy
(14, 15, 16, 17, 18, 19, 20) .
Using this technique, it is possible to determine
Fe-N
and Fe- X
bond distances to an accuracy of about ± 0.02 Å. We
have previously characterized four P-450 states and two
chloroperoxidase derivatives with this technique
(21, 22, 23, 24) . X-ray crystal
structures have been reported for three of the four P-450 states to
have been examined by EXAFS
(25, 26, 27) . The
metal-ligand bond distances obtained with these two methods are in
close agreement
(13) . As usually applied in the analysis of
first coordination shells for solution samples using empirical phase
and amplitude functions, EXAFS is insensitive to the three-dimensional
arrangement of atoms. The x-ray absorption edge spectrum, on the other
hand, contains information about the oxidation state, structure, and
coordination geometry of the heme iron
(28, 29, 30, 31, 32) . We have
systematically examined parallel derivatives of the three proteins,
making careful comparison of the energy and shape of the absorption
edge under conditions where the oxidation state, coordination number,
and spin state are kept constant within a particular group being
compared. In this way, the specific effect of the thiolate ligand that
is present in P-450 and chloroperoxidase on the properties of the
central heme iron has been quantified.
4 °C in 75 mM
potassium phosphate buffer (pH 7.0, 6.0, and 7.0, respectively)
containing 25% (v/v) ethylene glycol to final protein concentrations of
2.5 mM. The ligand complexes of the ferric proteins were
generated by adding the neat ligand ( N-methylimidazole) or
concentrated ligand stock solutions in ethanol (1-propanethiol,
p-chlorothiophenol, 4-phenylimidazole) or water (imidazole
with pH adjustment) to the proteins. The formation and the homogeneity
of the complexes were monitored by electronic absorption spectral
changes of the peak at 630-640 nm of the native ferric proteins
as well as by electron paramagnetic resonance spectroscopy at 77 K for
the low spin ligand complexes. The deoxy-ferrous derivatives of P-450,
chloroperoxidase, and myoglobin were prepared under N
in a
2-mm cuvette by first reducing the ferric proteins (without ethylene
glycol) with a few grains of solid sodium dithionite and then adding
anaerobic ethylene glycol ( volume). Ferrous-CO derivatives were then
prepared by gently bubbling with CO. Reduction and CO complex formation
were again monitored by optical absorption spectroscopy between 600 and
700 nm. The samples were transferred to lucite cells with Kapton
windows using an gas-tight syringe under air (ferric samples) or
N
(ferrous samples) and were stored at 77 K. The
homogeneity and integrity of the protein before and after the x-ray
absorption measurements were verified by electron paramagnetic
resonance (for the ferric samples directly in the lucite cell) or by
optical absorption spectroscopy (for anaerobically diluted ferrous
samples in the presence or absence of CO, and for the diluted,
ferrous-CO form of the ferric samples of P-450 and chloroperoxidase).
These examinations revealed that no autoxidation of the ferrous samples
had occurred and that less than 5% protein denaturation took place
during sample manipulation or irradiation.
0.1 eV. The
edge separation was also estimated from the difference in the second
derivative at the inflection point of the highest rising edge
transition and by calculating an average energy difference for a number
of points at the same height for the highest rising edge transition for
samples under comparison, giving results consistent with those from the
fits above.
Rationale
The rationale of the present study is
that the energy and shape of the x-ray absorption edge for two heme
iron protein derivatives should be the same if the two iron centers
have identical oxidation and spin states and matching axial ligands.
Once that premise is established, it should then be possible to change
one of the ligands while keeping the oxidation and spin states constant
in order to specifically probe the electron donor properties of that
ligand. This approach has been used in the present study to directly
examine the effect a thiolate sulfur donor axial ligand has on the
energy and shape of the x-ray absorption edge relative to the influence
of a histidine nitrogen donor ligand.
Figure 2:Top, comparison of the Fe-K x-ray
absorption edge spectra of the N-methylimidazole adducts of
ferric cytochrome P-450-CAM ( N- P- 450,
dottedline) and chloroperoxidase
( N- CPO, solidline) and the
1-propanethiolate complex of ferric myoglobin ( S-myoglobin,
dashedline). Bottom, difference x-ray
absorption spectra between S-myoglobin
( S- Mb) and N-CPO ( solidline) and between S-Mb and N-P-450 ( dottedline).
Having established that heme iron complexes with identical oxidation
and spin states and axial ligand sets have nearly indistinguishable
x-ray absorption edge spectra, the effect of changing one of the axial
ligands was then examined. The edge spectra of the
N-methylimidazole and 1-propanethiolate adducts of ferric
myoglobin are shown in the top of Fig. 3. A clear shift
of 0.96 eV to lower energy is observed in the energy of the rising edge
for the thiolate-bound case relative to the imidazole adduct. The shift
is especially evident in the difference spectrum displayed in the
bottom of Fig. 3. A shift to lower energy is typically
seen in the edge upon reduction of ferric heme iron complexes. This
experiment thus demonstrates that the energy of the x-ray absorption
edge is sensitive to the presence of a thiolate axial ligand.
Furthermore, since the shift in the edge energy is to lower energy upon
thiolate binding ( i.e. the same as for reduction of the ferric
heme iron), this suggests that the thiolate ligand serves as a stronger
electron donor to the iron than does an imidazole ligand.
Figure 3:Top, comparison of the Fe-K x-ray
absorption edge spectra of 1-propanethiolate ferric myoglobin
( S-myoglobin) ( dottedline) and
N-methylimidazole ferric myoglobin ( N-myoglobin)
( solidline). Bottom, difference x-ray
absorption spectrum between N-myoglobin
( N- Mb) and S-myoglobin
( S- Mb).
Low Spin Ferric State
The x-ray absorption edge
spectra of the N-methylimidazole adducts of ferric cytochrome
P-450-CAM, chloroperoxidase, and myoglobin are displayed in the top of Fig. 4. The edge energies for the P-450-CAM and
chloroperoxidase samples occur at lower energy than that of the
myoglobin complex. Since the only difference among imidazole-ligated
ferric P-450, chloroperoxidase, and myoglobin is the presence of a
cysteine thiolate axial ligand in P-450 and chloroperoxidase compared
with the histidine imidazole ligand for myoglobin, it is concluded that
the edge shift seen in Fig. 4is indicative that the thiolate
sulfur of cysteinate is a stronger electron donor than the imidazole
nitrogen of histidine. The edge differences, as displayed in the
bottom of the figure, are very similar in magnitude (1
eV) to that seen when comparing imidazole- and thiolate-ligated
myoglobin (0.96 eV, Fig. 3), indicating strong similarity in the
electronic and geometric environment among imidazole-ligated ferric
P-450 and chloroperoxidase, and thiolate-ligated ferric myoglobin.
Figure 4:Top, comparison of the Fe-K x-ray
absorption edge spectra of the N-methylimidazole adducts of
ferric cytochrome P-450-CAM ( N- P- 450,
dottedline), chloroperoxidase
( N- CPO, dashedline), and myoglobin
( N- Mb, solidline).
Bottom, difference x-ray absorption spectra between N-Mb and
N-CPO ( solidline) and between N-Mb and N-P-450
( dottedline).
Furthermore the preedge and edge shapes, and energy positions of
N-methyl-imidazole-chloroperoxidase and
N-methylimidazole-P-450-CAM are very similar to those of
1-propanethiolate-myoglobin. Since myoglobin has a distal histidine
imidazole, as established by x-ray crystallography and optical methods,
the x-ray absorption edge spectra reflect the similarities of the
electron donating properties of the thiolate ligand in the
N-methylimidazole adducts of chloroperoxidase and P-450-CAM,
and 1-propanethiolate-ligated myoglobin.
Low Spin Ferrous State
The edge spectra of low
spin ferrous-CO myoglobin, chloroperoxidase, and P-450-CAM are shown in
Fig. 5
. The energies of the edges for ferrous-CO chloroperoxidase
and P-450-CAM occur at lower energy than for the corresponding
myoglobin analog. This is consistent with the energy differences seen
for the low spin ferric state and again demonstrates the greater
electron donating effect of the thiolate ligand relative to an
imidazole ligand on the electronic properties of the iron in low spin
ferrous-CO chloroperoxidase and P-450-CAM.
Figure 5:
Comparison of the Fe-K x-ray absorption
edge spectra of the ferrous-CO derivatives of cytochrome P-450-CAM
( P-450, dash-dot line), chloroperoxidase ( CPO, dotted
line) and myoglobin ( Mb, solid line). Inset,
expanded plot of the 7110-7120 eV region of the
spectra.
The preedge 1 s 3 d transitions of these three heme protein CO
adducts are split into two peaks, A and B (Fig. 5, inset). This transition is generally
electric dipole forbidden but quadrupole allowed and can gain intensity
from 3 d-4 p mixing. The iron 3 d orbitals
split into two degenerate e
orbitals and three
degenerate t
orbitals in an perfect octahedral
ligand field. For the low spin ferrous state, the three
t
orbitals are occupied by all six d electrons, leaving the e
orbitals,
d
High Spin Ferrous
The Fe-K edge spectra of
deoxyferrous high spin chloroperoxidase and P-450-CAM are shown in
Fig. 6
. It would be expected for the ferrous oxidation state that
the edge position is less sensitive to the electron donating character
of the axial ligand, as electron density at the iron is inherently
higher. Ferrous-CO samples, on the other hand, contain an electron
withdrawing CO ligand that decreases the electron density at the iron,
leaving it more sensitive to the presence of the electron donating
thiolate ligand. Although no crystal structure has been published for
high spin deoxyferrous P-450-CAM, our previous EXAFS results for this
state have clearly demonstrated that it has a sulfur donor axial ligand
(23) . The shape and the edge position in the spectrum of
deoxyferrous chloroperoxidase is very similar to that of deoxyferrous
P-450-CAM (Fig. 6), suggesting that the electron density at the
iron is similar for these two proteins. The only significant difference
is the somewhat higher preedge intensity in the spectrum of
deoxyferrous chloroperoxidase.
Figure 6:
Comparison of the Fe-K x-ray absorption
edge spectra of the deoxyferrous derivatives of cytochrome P-450-CAM
( P-450, dotted line) and chloroperoxidase ( CPO, solid
line).
In summary, direct evidence has been
obtained through the use of Fe-K x-ray absorption edge spectroscopy to
show that a thiolate axial ligand is more electron releasing to the
central heme iron than an imidazole axial ligand. In both the low spin
six-coordinate ferric and ferrous states of cytochrome P-450,
chloroperoxidase, or myoglobin, the absorption edge is shifted by about
1.0 eV to lower energy when comparing thiolate-ligated adducts with
complexes of the same oxidation and spin state that lack a thiolate
ligand. Because the edge also shifts to lower energy upon reduction of
ferric iron to the ferrous state, the shift in the edge energy for
thiolate-bound complexes provides a direct measure of the
electron-releasing character of a thiolate axial ligand. These results
therefore support the suggested role of the cysteinate proximal ligand
of cytochrome P-450 as a strong internal electron donor to promote
O-O bond cleavage in the putative ferric-peroxide intermediate to
generate the proposed ferryl-oxo ``active oxygen'' state of
the reaction cycle
(1, 2, 3) .
Table:
Relative edge position shift
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.