Spectroscopic characterization of hydroxide and aqua complexes of Fe(II)-protoheme, structural models for the axial coordination of the atypical heme of membrane cytochrome b6f complexes

Article abstract of DOI:10.1021/ja052791g

Electronic absorption and resonance Raman (RR) spectra are reported for hydroxide and aqua complexes of iron(II)-protoporphyrin IX (Fe(II)PP) respectively formed in alkaline and neutral aqueous solutions. These compounds with weak axial ligand(s) represent a biomimetic approach of the unusual coordination of the atypical heme c_i of membrane cytochrome b ₆f complexes. Absorption spectra and spectrophotometric titrations show that Fe(II)PP in alkaline aqueous cetyltrimethylammonium bromide (CTABr) binds one hydroxide ion, forming a five-coordinated high-spin (HS) complex. In alkaline aqueous ethanol, we confirm the formation of a dihydroxy complex of Fe(II)PP. In the RR spectra of Fe(II)PP dissolved in neutral aqueous CTABr, a mixture of a four-coordinated intermediate spin form with an HS monoaqua complex (Fe(II)PP(H₂O)) was observed. The spectroscopic information obtained for Fe(II)PP(OH^-), Fe(II)PP(H₂O), and Fe(II)PP(OH ^-)₂ was compared with that previously reported for the 2-methylimidazole and 2-methylimidazolate complexes of Fe(II)PP, representative of the most common axial ligation in HS heme proteins. This investigation reveals a very remarkable analogy in the spectral properties of, in one hand, the Fe(II)PP(H₂O) and mono-2-methylimidazole complexes and, in the other hand, the Fe(II)PP(OH^-) and mono-2-methylimidazolate complexes. The comparisons of the absorption and RR spectra of Fe(II)PP(OH^-) and Fe(II)PP(OH^-)₂ clearly establish that both a redshift of the π-π electronic transitions and an upshift of the v₈ RR frequency are spectral parameters indicative of porphyrin doming in HS ferrous complexes. Based upon isotopic substitutions (¹⁶OH, ¹⁶OD^-, and ¹⁸OH^-), stretching modes of the Fe-OH bond(s) of a ferrous porphyrin were assigned for the first time, i.e., at 435 cm^-1 for Fe(II)PP(OH^-) (ν(Fe(II)-OH ^-)) and at 421 cm^-1 for Fe(II)PP(OH^-) ₂ (ν_s(Fe(II)-(OH^-)₂). The spectroscopic and redox properties of Fe(II)PP(H₂O), Fe(II)PP(OH _-), and heme c_i were discussed and favor a water coordination for the heme c_i iron.

Full text of DOI:10.1021/ja052791g

Published on Web 11/25/2005
Spectroscopic Characterization of Hydroxide and Aqua
Complexes of Fe(II)-Protoheme, Structural Models for the
Axial Coordination of the Atypical Heme of Membrane
Cytochrome b6f Complexes
Adrienne Gomez de Gracia, Luc Bordes, and Alain Desbois*
Contribution from the De´partement de Biologie Joliot-Curie, SerVice de Biophysique des
Fonctions Membranaires, CEA et URA CNRS 2096, CEA/Saclay,
F-91191 Gif-sur-YVette Cedex, France
Received April 29, 2005; Revised Manuscript Received October 14, 2005; E-mail: alain.desbois@cea.fr
Abstract: Electronic absorption and resonance Raman (RR) spectra are reported for hydroxide and aqua
complexes of iron(II)-protoporphyrin IX (Fe(II)PP) respectively formed in alkaline and neutral aqueous
solutions. These compounds with weak axial ligand(s) represent a biomimetic approach of the unusual
coordination of the atypical heme ci of membrane cytochrome b6f complexes. Absorption spectra and
spectrophotometric titrations show that Fe(II)PP in alkaline aqueous cetyltrimethylammonium bromide
(CTABr) binds one hydroxide ion, forming a five-coordinated high-spin (HS) complex. In alkaline aqueous
ethanol, we confirm the formation of a dihydroxy complex of Fe(II)PP. In the RR spectra of Fe(II)PP dissolved
in neutral aqueous CTABr, a mixture of a four-coordinated intermediate spin form with an HS monoaqua
complex (Fe(II)PP(H2O)) was observed. The spectroscopic information obtained for Fe(II)PP(OH-), Fe-
(II)PP(H2O), and Fe(II)PP(OH-)2 was compared with that previously reported for the 2-methylimidazole
and 2-methylimidazolate complexes of Fe(II)PP, representative of the most common axial ligation in HS
heme proteins. This investigation reveals a very remarkable analogy in the spectral properties of, in one
hand, the Fe(II)PP(H2O) and mono-2-methylimidazole complexes and, in the other hand, the Fe(II)PP(OH-)
and mono-2-methylimidazolate complexes. The comparisons of the absorption and RR spectra of
Fe(II)PP(OH-) and Fe(II)PP(OH-)2 clearly establish that both a redshift of the π-π electronic transitions
and an upshift of the v8 RR frequency are spectral parameters indicative of porphyrin doming in HS ferrous
complexes. Based upon isotopic substitutions (16OH-,16OD-, and 18OH-), stretching modes of the Fe-OH
bond(s) of a ferrous porphyrin were assigned for the first time, i.e., at 435 cm-1 for Fe(II)PP(OH-) (ν(Fe-
(II)-OH-)) and at 421 cm-1 for Fe(II)PP(OH-)2 (νs(Fe(II)-(OH-)2). The spectroscopic and redox properties
of Fe(II)PP(H2O), Fe(II)PP(OH-), and heme ci were discussed and favor a water coordination for the heme
ci iron.
form of heme ci.3 All these observations have renewed the
interest to study biomimetic heme model compounds to
Introduction
Recent crystallographic and spectroscopic investigations of
membrane plastoquinone:plastocyanin oxidoreductase or cyto-
chrome b6f complex (cyt b6f)1 have characterized an atypical
heme (heme ci or x) in a protein.2,3 The axial ligation of this
new heme is not clearly established but the X-ray diffraction
data suggest coordination by a water molecule or a hydroxide
ion.2 More recently, resonance Raman (RR) spectroscopy has
identified a high-spin (HS) species corresponding to the ferrous
understand the spectroscopic properties of heme ci in cyt b6f.
In the present work, we characterized two hydroxide complexes
and a monoaqua complex of iron(II)-protoporphyrin IX
(Fe(II)PP) using absorption and RR spectroscopies. To distin-
guish the electronic and steric properties of a water or hydroxide
ligand bound to a ferroheme from those of the widespread
histidylimidazole ligation found in ferrous myoglobins, hemo-
globins, c′-type cytochromes, or peroxidases, the spectroscopic
properties of Fe(II)PP complexed with these weak ligands were
compared with those of the high-spin (HS) 2-methylimidazole
(2MeImH) and 2-methylimidazolate (2MeIm-) complexes of
Fe(II)PP.
(1) Abbreviations used: cyt, cytochrome; CTABr, cetyltrimethylammonium
bromide; NaDS.; sodium dodecyl sulfate; TX-100, Triton X-100; EtOH,
ethanol; RR, resonance Raman; PP, dianion of protoporphyrin IX; TtbutPP,
dianion of R,R,R,R-5,10,15,20-tetrakis(o-(N-tert-butylcarbamoyl)phenyl)-
porphyrin; 4c, four-coordinated; 5c, five-coordinated; 6c, six-coordinated;
HS, high spin; LS, low spin; IS, intermediate spin; 2MeImH, 2-methylimi-
dazole; 2MeIm-, 2-methylimidazolate; N-MeIm, 1-methylimidazole; E1/2
,
Experimental Section
redox potential.
(2) (a) Stroebel, D.; Choquet, Y.; Popot, J.-L.; Picot, D. Nature 2003, 426,
413-418. (b) Kurisu, G.; Zhang, H.; Smith, J. L.; Cramer, W. A. Science
2003, 302, 1009-1014.
Preparations of the Fe(II)PP Complexes. Protohemin chloride
(Sigma) (Fe(III)PP(Cl-)) freshly dissolved in aqueous alkaline solution
(10 mM) has been used as a starting material. The neutral and alkaline
(3) De Vitry, C.; Desbois, A.; Redeker, V.; Zito, F.; Wollman, F.-A.
Biochemistry 2004, 3956-3968.
9
17634
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Hydroxide and Aqua Complexes of Fe(II)-Protoheme
A R T I C L E S
complexes of Fe(II)PP was prepared from Fe(III)PP(Cl-) by addition
of either phosphate buffer (pH 7.0) or hydroxide ion (NaOH or KOH),
either in aqueous cetyltrimethylammonium bromide (CTABr) (Sigma)
(0.5-2% (w/v)), aqueous sodium dodecyl sulfate (NaDS) (Bio Rad)
(0.5% (w/v)), aqueous Triton-X100 (TX-100) (SigmaUltra) (0.5% (w/
v)), or aqueous ethanol (EtOH) (Merck, spectroscopic grade) (50% (v/
v)). Heme reduction was achieved by anaerobic addition of a small
excess of freshly prepared sodium dithionite (Merck) to hemin solutions
either under vacuum or equilibrated with wet argon.4,5 The range of
heme concentrations was (6-10) × 10-6 M for the absorption
measurements and (2-10) × 10-4 M for the RR experiments.
D2O (isotopic enrichment: 99.8%), H218O (isotopic enrichment:
95.1%), and NaOD (isotopic enrichment: 98.5%, 9.8 N in D2O) were
purchased from Euriso-top S. A..
nm component in aqueous NaDS is significantly decreased when
compared to the 428 nm contribution observed in aqueous
CTABr (Figure 1A (spectrum (a)) and B (spectrum (a)). In the
visible region, a 4 nm difference in the peak position of the R
band is also detected. The absorption spectrum of hemin reduced
in TX-100 exhibits a single Soret band at 426 nm and visible
bands at 475, 545, and 564 nm. The observation of a hydroxide
form of Fe(II)PP in aqueous NaDS or in aqueous TX-100 was
not possible due to a detergent precipitation in concentrated
KOH.
The absorption spectrum of dithionite-reduced hemin in
neutral aqueous EtOH solution shows a Soret peak at 419 nm
and weak R/â bands at 567 and 543 nm (Figure 1C, spectrum
(a)). The titration of this complex by hydroxide produces a
compound with Soret, â, and R bands at 435, 557, and 591 nm,
respectively (Figure 1C, spectrum (b); Table 1). These band
maxima are essentially those observed previously for an
Fe(II)PP(OH-)2 complex (435, 558, and 590 nm, respectively).7
Spectroscopies. The electronic absorption spectra as well as the
spectrophotometric titrations were measured using a Cary 5E (Varian)
spectrometer. The equilibrium constants for hydroxide binding to Fe-
(II)PP were determined according to methods previously described.4a,6
As far as the monohydroxy complex of Fe(II)PP is concerned, some
microprecipitation due to the simultaneaous presence of detergent and
hydroxide was observed. To minimize the effect of light scattering,
the ratios (R) of the absorbances at 440 and 415 nm were determined
at a known hydroxide concentration. For the dihydroxy complex, the
ratio of the absorbances at 435 and 419 nm was similarly measured as
a function of the OH- concentration. The resonance Raman (RR) spectra
were obtained using a Jobin-Yvon U1000 Raman spectrometer equipped
with an N2-cooled charge-coupled device detector (Spectrum one, Jobin-
Yvon, France). The samples were excited in the Soret region with the
413.1 nm line of a Kr+ laser (Coherent Innova), the 441.6 nm line of
a He/Cd laser (Liconix), and the 457.9 and 476.5 lines of an Ar+ laser
(Coherent Innova). Improvement of the signal-to-noise ratios of RR
spectra was achieved by summations of scans of 30 s. For the
determination of the very low-frequency modes (< 200 cm-1), a
spectrometer (Jobin-Yvon HG2S) equipped with an EMI photomulti-
plier was used.4 The frequencies of the RR bands were determined
using a Grams/32 software (Galactic Industries) and methods previously
described.4d The frequencies of the weakest RR bands were character-
ized with 1-2 cm-1 uncertainties. For the strongest bands, the
Spectrophotometric Titrations. The equilibrium measure-
ments made from spectrophotometric titrations of Fe(II)PP by
hydroxide in aqueous CTABr solutions fit a binding of one
molecule of hydroxide to one molecule of Fe(II)PP (Figure 2
and data not shown). The equilibrium constant (KD) is 3.4 ×
10-1 M in 0.5% aqueous CTABr solution. The OH- affinity is
decreased to 4.5 × 10-1 M when the detergent concentration is
increased to 2%.
The titration of Fe(II)PP with OH- performed in aqueous
EtOH confirms the binding of two hydroxide anions to Fe(II)-
PP.7 No formation of an intermediate monohydroxy complex
was detected, and a KD of 5 × 10-2 M2 was measured
(Supporting Information S1).
RR Studies of Fe(II)PP in Aqueous Detergent Solution
at pH 7-8. When excited at 441.6 nm, the high-frequency
regions of RR spectra (1300-1700 cm-1) of the dithionite-
reduced hemin dissolved in neutral aqueous CTABr solutions
clearly show a mixture of two species (Figure 3). In particular,
the relative intensity of the two ν4 bands observed at 1371 and
1358 cm-1 is dependent on the concentration of CTABr (Figure
3, spectra (a) and (b)) but insensitive to the dithionite concentra-
tion (spectra not shown). Taking as a reference the RR spectra
of the bare Fe(II)PP complex excited at 514.5 or 488 nm,8 the
ν4 contribution arising from this four-coordinated (4c) inter-
mediate-spin (IS) form is observed at 1371 cm-1. Differences
in RR spectra more clearly confirm that hemin reduced by
dithionite produces the 4c IS Fe(II)PP form mixed with another
ferrous form (Figure 3, spectra (c) and (d)). The 1358 cm-1
band and minor bands detected at 1472 and 1525 cm-1 are
indicative of the formation of a ferrous HS species.9 This form
is RR-active using a blue excitation but RR-inactive when
excited in the green region.8e In the low-frequency RR spectrum,
the HS species displays two weak bands at 408 and 672 (ν7)
cm-1 (spectrum not shown).
uncertainty range is decreased to 0.5-1 cm-1
.
Results
Electronic Absorption Spectra. Hemin dissolved in aqueous
CTABr solution at pH 7 and then reduced by sodium dithionite
yields an absorption spectrum in which the R/â bands are
detected at 572 and 536 nm (Figure 1A, spectrum (a)).
Overlapping bands peaking at 392, 416, 428, and 441 nm are
observed in the Soret region. The Fe(II)PP complex formed in
aqueous CTABr but in a concentrated hydroxide solution (pH
g 14) results in a new spectral form with three separate
absorption bands at 440 (Soret), 561 (â), and 593 (R) nm (Figure
1A, spectrum (b); Table 1).
The absorption spectrum of hemin reduced by dithionite in
aqueous NaDS or in aqueous TX-100 is different from that
obtained in aqueous CTABr (Figure 1A (spectrum (a) and B
(spectra (a) and (b)). In the Soret region, a broad band with
four components (395, 405, 425, and 439 nm) is again observed
in aqueous NaDS. However, the relative intensity of the 425
(4) (a) Desbois, A.; Lutz, M. Biochim. Biophys. Acta 1981, 671, 168-176.
(b) Desbois, A.; Lutz, M. Eur. Biophys. J. 1992, 20, 321-335. (c) Othman,
S.; Le Lirzin, A.; Desbois, A. Biochemistry 1993, 32, 9781-9791. (d) Le
Moigne, C.; Schoepp, B.; Othman, S.; Verme´glio, A.; Desbois, A.
Biochemistry 1999, 38, 1066-1076.
(7) Keilin, J. Biochem. J. 1949, 45, 448-455.
(8) (a) Spiro, T. G.; Burke, J. M. J. Am. Chem. Soc. 1976, 98, 5482-5489.
(b) Kitagawa, T.; Ozaki, Y.; Teraoka, J.; Kyogoku, Y.; Yamanaka, T.
Biochim. Biophys. Acta 1977, 494, 100-114. (c) Kitagawa, T.; Teraoka,
J. Chem. Phys. Lett. 1979, 63, 443-446. (d) Nagai, K.; Kitagawa, T.;
Morimoto, H. J. Mol. Biol. 1980, 136, 271-289. (e) Bordes, L.; Desbois,
A., unpublished spectra.
(5) Leondiadis, L.; Momenteau, M.; Desbois, A. Inorg. Chem. 1992, 31, 4691-
4696.
(6) (a) Momenteau, M. Biochim. Biophys. Acta 1973, 304, 814-827. (b)
(9) Parthasarathi, N.; Hansen, C.; Yamaguchi, S.; Spiro, T. G. J. Am. Chem.
Soc. 1987, 109, 3865-3871.
Yoshimura, T.; Ozaki, T. Arch. Biochem. Biophys. 1984, 230, 466-482.
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A R T I C L E S
Gomez de Gracia et al.
Figure 1. Electronic absorption spectra of dithionite-reduced hemin. (A) in 0.5% aqueous CTABr solution at pH 7.0 (a) and in 3 M NaOH (b); (B) in 0.5%
aqueous NaDS at pH 8.0 (a) and in 0.5% aqueous TX-100 at pH 7.0 (b); (C) in 50% ethanol/water at pH 7.0 (a) and in 2.5 M NaOH (b). Heme concentration
) 10-5 M.
Table 1. Absorption Maxima (nm) of High-Spin Fe(II)PP
Complexes
complex
absorption (nm)
Fe(II)PP(H2O)a
Fe(II)PP(OH-)a
Fe(II)PP(OH-)2
428
440, 561, 593
435, 557, 591
433, 556
b
Fe(II)PP(2MeImH)a
Fe(II)PP(2MeIm-)a
441, 560, 592
a In 0.5 % aqueous CTABr solution. b In a 50 % EtOH/H2O mixture.
The spectra of hemin reduced in aqueous NaDS at pH 8.010
exhibits a Raman signature essentially corresponding to the 4c
IS Fe(II)PP, the contribution of the HS species being very minor
(Figure 4, spectrum (a)). In the ν3 region, the observation of a
band at 1495 cm-1 indicates the formation of a six-coordinated
(6c) low-spin (LS) form.
Figure 2. Plot of 1/(R - R0) versus 1/[OH-]. R represents the ratio of the
absorbances at 440 and 415 nm, R0 being the R value obtained at pH 7.0.
The line corresponds to an affinity constant (KD) of 3.4 × 10-1 M and fits
with a correlation coefficient of 0.995.
(10) The addition of phosphate buffer to aqueous NaDS produces a precipitation
of the detergent.
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Hydroxide and Aqua Complexes of Fe(II)-Protoheme
A R T I C L E S
Figure 3. High-frequency regions (1260-1660 cm-1) of RR spectra of
dithionite-reduced hemin (a) in 0.5% CTABr aqueous solution, pH 7; (b)
in 2% CTABr aqueous solution, pH 7. (c) spectrum (a) minus spectrum
(b). (d) spectrum (b) minus 0.6 × spectrum (a). Excitation: 441.6 nm.
Figure 5. High-frequency regions (1250-1650 cm-1) of RR spectra of
dithionite-reduced hemin (a) in 0.5% CTABr aqueous solution and 3 M
NaOH; (b) in 50% EtOH/H2O and 2.5 M NaOH. Excitation: 441.6 nm.
The intensity of the ν4 bands (1357 cm-1) has been divided by a factor of
4. Above spectrum (b), the bands marked by an asterisk are due to free
ethanol.
Figure 4. High-frequency regions (1300-1670 cm-1) of RR spectra of
dithionite-reduced hemin (a) in 0.5% NaDS aqueous solution, pH 8; (b) in
0.5% TX-100 aqueous solution, pH 7. Excitation: 441.6 nm. The band
marked by an asterisk is due to a plasma line.
Figure 6. Low-frequency regions (200-850 cm-1) of RR spectra of
dithionite-reduced hemin (a) in 0.5% CTABr aqueous solution, 3 M NaOH;
(b) in 50% EtOH/H2O solution, 2.5 M NaOH. Excitation: 441.6 nm. The
bands marked by an asterisk are due to the Raman cell.
As far as the RR spectra of hemin reduced in aqueous TX-
100 are concerned, the bands observed at 1358, 1472, and 1563
cm-1 in the ν4, ν3, and ν2 region, respectively, are clearly
associated with the major formation of an HS complex (Figure
4, spectrum (b)). Small contributions of the 4c IS Fe(II)PP form
and of a 6c LS complex are however seen in the ν3 region (1502
and 1496 cm-1, respectively). In the low-frequency regions,
major bands corresponding to this HS form are observed at 256,
287, 301, 349 (ν8), 367, 376, 411, 548, 586, 675 (ν7), and 716
cm-1 (Supporting Information S2). The substitution of H216O
by H218O produces a clear upshift of two bands. On one hand,
a 287 cm-1 band is shifted to 295 cm-1. On the other hand, the
ν8 mode is shifted by 2 cm-1 from 349 to 351 cm-1. A shoulder
on the high-frequency side of the 411 cm-1 band is also
upshifted upon H216O f H218O substitution (Supporting Infor-
mation S2)
marker ν4 appears at 1357 cm-1 (Figure 5). The skeletal ν2,
ν38, and ν3 modes are identified at 1560, 1522, and 1472 cm-1
,
respectively. Strong contributions of vinyl modes are detected
at 1623, 1616 (ν(CadCb), 1424 (δ(dCbH2)), and 1306 (δ(CaHd
)) cm-1 11
.
The low-frequency RR spectrum of the monohydroxy com-
plex formed in aqueous CTABr is presented in Figure 6
(spectrum (a)). The ν7 and ν8 modes are detected at 673 and
347 cm-1, respectively. On the nonresonant Raman contribution
of the cuvette, a very broad feature is observed in the 400-
450 cm-1 region. Considering its overall width at half-height
(54 ( 1 cm-1), this feature appears to be a cluster of overlapping
bands. To distinguish its different components, we have
measured the RR spectra of the monohydroxy complex with
RR Studies of Fe(II)PP in Alkaline Aqueous CTABr
Solution. The RR spectrum of the Fe(II)PP complex formed in
alkaline CTABr solutions exhibits marked differences with
respect to that observed for the neutral form of Fe(II)PP (Figures
3 and 5). In the high-frequency regions, a single electron density
(11) (a) Choi, S.; Spiro, T. G.; Langry, K. C.; Smith, K. M. J. Am. Chem. Soc.
1982, 104, 4337-4344. (b) Hu, S.; Smith, K. M.; Spiro, T. G. J. Am. Chem.
Soc. 1996, 118, 12638-12646.
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A R T I C L E S
Gomez de Gracia et al.
Figure 9. Low-frequency regions (200-730 cm-1) of RR spectra of the
dihydroxy complex of Fe(II)PP excited at 413.1 (a), 441.6 (b), 457.9 (c),
and 476.5 nm (d). Spectrum (e): nonresonant Raman spectrum of the cell
containing a 50% EtOH/H2O solution, excited at 441.6 nm.
Figure 7. Low-frequency regions (200-730 cm-1) of RR spectra of the
monohydroxy complex of Fe(II)PP excited at 413.1 (a), 441.6 (b), 457.9
(c), and 476.5 nm (d).
clearly shifted to 414 (( 1) cm-1. This shift is confirmed by
the difference spectrum 16OH- minus 18OH- (spectrum (d) in
Figure 8).
RR Spectra of Fe(II)PP in Alkaline EtOH/H2O Solution.
The high-frequency regions of RR spectra of the dihydroxy
complex of Fe(II)PP formed in 50% EtOH/H2O show a strong
resemblance in frequency as well as in relative intensity with
the spectra of the monohydroxy complex (Figures 5 and 6).
However, the peripheral modes at 1618 (vinyl CadCb), 1426
(ν42), and 1302 (vinyl δ(CaHd)) cm-1 are shifted by 2-4 cm-1
(Figure 5). Among the skeletal porphyrin modes, only ν2 is
clearly downshifted by 2 cm-1 in changing CTABr by EtOH
(Figure 5).
In the low-frequency regions, the frequency of ν7 remains
unaffected (673 cm-1) (Figure 6). On the contrary, the ν8 mode
is downshifted from 347 for the monohydroxy complex to 343
cm-1 for the dihydroxy complex. Similarly, a broad band
observed at 155 cm-1 for the monohydroxy complex is
downshifted at 149 cm-1 in the RR spectrum of the dihydroxy
complex (spectra not shown). Another major spectral difference
between the two hydroxide species concerns the 400-450 cm-1
regions. Like in the case of the spectra of the Fe(II)PP(OH-)
complex, the broad feature covering this region cannot be
considered as a single band. However, its width at half-height
is significantly smaller than that observed for the monohydroxy
complex (40 ( 1 versus 54 ( 1 cm-1). In fact, the hydroxide
complex formed in EtOH/H2O exhibits two overlapping bands
at 408 (( 1) and 421 (( 1) cm-1 (Figure 6). These bands
exhibits slight changes in relative intensity when the excitation
is changed at 413.1 or 457.9 nm (Figure 9, spectra (a)-(c)).
The low-frequency bands of Fe(II)PP(OH-)2 become weak or
inactive with the 476.5 nm excitation, a band observed at 439
cm-1 being a solvent contribution (Figure 9, spectra (d) and
(e)). The ligand deuteration (16OH- f 16OD-) provokes a small
downshift of the 421 cm-1 shoulder to 417 (( 1) cm-1 (Figure
10, spectra (a) and (b)). This small shift is also identified in the
difference spectrum (not shown). The substitution of 16OH- by
18OH- induces a strong shift of the 421 cm-1 band under the
vinyl deformation mode, resulting in a single broad band peaking
at 404 cm-1 (Figure 10, spectrum (c)). All the other RR bands
are unaffected by the change in mass of the hydroxide ion
Figure 8. Low-frequency regions (200-730 cm-1) of RR spectra of Fe-
(II)PP in alkaline CTABr aqueous solution (0.5%). (a) 3 M Na16OH in
H216O (100% 16OH-); (b) 3 M NaOD in D2O (99% OD-); (c) 3 M Na16-
OH in H218O (92.3% 18OH-, 7.7% 16OH-) (d) Difference spectrum (a)-
(c). Spectra (a)-(c) were normalized on the intensity of the ν7 band. The
bands marked by an asterisk are due to the cell.
different Soret excitations. In this way, three main components
are thus observed between 400 and 450 cm-1, i.e., at 409, 421,
and 435 cm-1 (Figure 7). The 409 cm-1 band is likely a vinyl
deformation mode in analogy with the 409-410 cm-1 band
observed in the RR spectra of the 2MeImH and 2MeIm-
complexes of Fe(II)PP excited at 441.6 nm.4b Thus, at least one
of the two remaining bands (421 and 435 cm-1) could be a
candidate for a vibration involving the axial Fe-OH bond since
they are absent in the RR spectra of Fe(II)PP(2MeImH) and
Fe(II)PP(2MeIm-).4b
The ligand deuteration in Fe(II)PP(OH-) (16OD- in D216O
versus 16OH- in H216O) produces a small downshift of the high-
frequency component of the cluster from 435 (( 1) cm-1 to
431 (( 1) cm-1 (Figure 8, spectra (a) and (b)). In the difference
spectrum (16OH- minus 16OD-), a small S-shaped signal
confirms the isotopic sensitivity of the 435 cm-1 band (spectrum
not shown). When the solvent contains a major concentration
of 18OH- and H218O, we observed a more important change in
RR spectrum (Figure 8, spectrum (c)). The 435 cm-1 band is
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Hydroxide and Aqua Complexes of Fe(II)-Protoheme
A R T I C L E S
skeletal ν2 and ν4 modes were, respectively, observed at 1542
and 1346 cm-1 for the monoaqua complex and at 1540 and
1344 cm-1 for the mono-N-MeIm complex. It is thus clear that
the monoaqua and mono-N-MeIm complexes of Fe(II)[TtbutPP]
adopt very similar electronic structures. In the Fe(II)[TtbutPP]-
(H2O) complex, the coordinated water molecule was proposed
to be H-bonded by at least one well-oriented carbonyl group of
a peripheral picket.5
The present RR observation of an Fe(II)PP(H2O) complex
formed in neutral aqueous CTABr solution confirms a very
similar electronic structure for monoaqua and monoimidazole
complexes of a “biological” Fe(II)-porphyrin (Tables 2 and 3).
The RR spectra of the Fe(II)PP(H2O) complex show an HS
signature with ν4, ν3, and ν2 bands at 1357, 1472, and 1557
cm-1 (Table 2). In the interaction between Fe(II)PP(H2O) and
the CTABr micelle, the positively charged headgroups of the
detergent certainly play a role in a relative stabilization of the
bound water molecule. In this line, the anionic NaDS detergent
has a poor stabilizing effect for the formation of the Fe(II)PP-
(H2O) complex. This marked difference between CTABr and
NaDS illustrates the influence of the electrostatic interactions
on the binding of water to Fe(II)PP.
As far as the hemin reduced in TX-100 is concerned, its
absorption and RR spectra exhibit an HS signature very similar
to that observed for the Fe(II)PP(H2O) complex formed in
aqueous CTABr. Thus, a water binding to Fe(II)PP could be
favored in the neutral TX-100 detergent when compared to the
cationic CTABr detergent. However, the hydrophilic part of TX-
100 is constituted by a poly(ethylene glycol) chain in which
the terminal hydroxyl group has coordinating properties likely
very similar to that of H2O. Two possibilities can therefore be
considered. A first one is a direct binding of a water molecule
to Fe(II)PP. Alternatively, Fe(II)PP can bind the terminal ethanol
group of TX-100, this O-donor ligand interacting with a water
molecule via a H-bond. Considering the H216O fH218O change
in aqueous TX-100 that induces clear upshifts of two identified
low-frequency bands, the isotopic sensitivity of these bands is
likely due to a strong coupling of the axial Fe-ROH stretch
(with R ) H from water or -CH2-CH2 from TX-100) with a
pyrrole deformation mode (at 287 cm-1 in H216O or 295 cm-1
in H218O) and the pyrrole stretching mode (ν8 at 349 or 351
cm-1).
Figure 10. Low-frequency regions (320-480 cm-1) of RR spectra of Fe-
(II)PP in EtOH/H2O solution. (a) 2.5 M Na16OH in EtOH/H216O (100%
16OH-); (b) 2.5 M NaOD in EtOH/D2O (99% OD-, 1% OH-); (c) 2.5 M
Na16OH in EtOH/H218O (90.6% 18OH-, 9.4% 16OH-; (d) difference
spectrum (a) - (c)). Spectra (a)-(c) were normalized on the intensity of
the ν7 band. The bands marked by an asterisk are due to the cell.
(Figure 10, spectrum (d)). It is important to note that both the
mono- and dihydroxy complexes exhibit a 421 cm-1 band.
However, the origin of the two bands is clearly different. For
the dihydroxy complex, this band is sensitive to 16OH-/16OD-
and 16OH-/18OH- substitutions. On the contrary, the 421 cm-1
band of the monohydroxy complex exhibits no sensitivity to
the isotopic changes.
Discussion
Monoaqua Complex of Fe(II)PP. In the 441.6 nm excited
RR spectra of ferroprotoheme dissolved in neutral aqueous
CTABr, we observed the formation of an HS Fe(II)PP form
mixed with the well-identified 4s IS Fe(II)PP complex.8 An HS/
IS spin equilibrium is hardly compatible for a 4c complex. Given
the very low concentration of OH- at pH 7 and the low affinity
of Fe(II)PP for OH-, this HS species cannot be attributed to a
residual Fe(II)PP(OH-) complex. We can also exclude the
binding of either Br- or degradation products of dithionite.12
Although water is a weak ligand and thus has a very low affinity
for Fe(II)-hemes, one can finally consider a water binding when
Fe(II)PP is dissolved in neutral aqueous CTABr. The dissocia-
tion constant of water to Fe(II)-deuteroheme in wet toluene was
estimated to be 10 M.13 Stabilizing factors however can
significantly increase the affinity of H2O for Fe(II)-porphy-
rins.5,14 In this vein, a monoaqua complex of a “picket-fence”
Fe(II)-porphyrin (Fe(II)[TtbutPP]) was fully characterized by
absorption and RR spectroscopies.5 The spectral properties of
this monoaqua complex strongly resemble those of monoimi-
dazole complexes of the homologue ferrous porphyrin. In the
absorption spectra, the Soret and â bands of Fe(II)[TtbutPP]-
(H2O) were, respectively, observed at 435 and 546 nm, while
those of the N-methylimidazole (N-MeIm) complex peaked at
438 and 544 nm.5 The RR spectra of the H2O and N-MeIm
complexes of Fe(II)[TtbutPP] also show great analogies. The
Finally, the RR observation of an HS aqua complex mixed
with the IS 4c Fe(II)PP form is consistent with the multicom-
ponent Soret band observed in the spectra of hemin reduced in
CTABr or NaDS. The Soret band of Fe(II)PP(OH-) in aqueous
detergent peaks at 440 nm (vide infra). Considering that the
deprotonation of an axial ligand of Fe(II)PP produces a marked
redshift of the visible absorption bands,15a the Soret maximum
of the Fe(II)PP(H2O) complex is expected to be in the 430 nm
region. Comparing the absorption spectra of hemin reduced in
aqueous CTABr with those of hemin reduced in aqueous NaDS
or TX-100, the 428 nm component can be assigned to the Soret
contribution of Fe(II)PP(H2O) (Table 1).
Monohydroxy Complex of Fe(II)PP. The formation of
monohydroxy complexes of Fe(II)-porphyrins has been observed
(12) The addition of KBr up to 1 M does not significantly change the RR
spectrum. The presence of a large excess of sodium dithionite (103 equiv/
heme) has no influence on the RR spectra.
(13) Brault, D.; Rougee, M. Biochemistry 1974, 13, 4591-4597.
(14) Maillard, P.; Schaeffer, C.; Te´treau, C.; Lavalette, D.; Lhoste, J.-M.;
Momenteau, M. J. Chem. Soc., Perkin Trans. 2 1989, 1437-1442.
(15) (a) Mincey, T.; Traylor, T. G. J. Am. Chem. Soc. 1979, 101, 765-766. (b)
Silver, J.; Lukas, B. Inorg. Chim. Acta 1983, 80, 107-113. (c) Lexa, D.;
Momenteau, M.; Save´ant, J.-M.; Xu, F. Inorg. Chem. 1985, 24, 122-127.
(d) Kadish, K. M.; Larson, G.; Lexa, D.; Momenteau, M. J. Am. Chem.
Soc. 1975, 97, 282-288.
9
J. AM. CHEM. SOC. VOL. 127, NO. 50, 2005 17639

A R T I C L E S
Gomez de Gracia et al.
Fe(II)PP
Table 2. Frequencies (cm-1) of the High-Frequency RR Modes of High-Spin Fe(II)PP Complexes
Fe(II)PP
Fe(II)PP
(H2O)
(ROH) (R
)
H
Fe(II)PP
(OH-
Fe(II)PP
(OH-
Fe(II)PP
(2MeImH)
(CTABr)
or sCH2sCH2)
)
)
(2MeIm-
)
2
mode
(CTABr)
(TX-100)
(CTABr)
(EtOH)
(CTABr)
ν(CadCb)
1626
1616
1603
1626
1620
1605
1623
1616
1605
1583
1560
1549
1522
1472
1440
1424
1395
1357
1305
1624
1618
1605
1585
1562
1550
1522
1472
1624
1618
1604
1584
1560
1549
1525
1471
1445
1428
1393
1355
1307
1624
1619
1605
1586
1559
1548
1525
1473
1443
1426
1393
1357
1307
ν(CadCb)
ν10
ν37
ν2
1557
1563
ν11
ν38
1525
1472
1524
1472
1447
1425
1392
1358
1305
ν3
δ(dCbH2)s
ν28
1426
1396
1357
1302
ν12
ν4
1358
δ(CaHd)
Table 3. Frequencies (cm-1) of the Low-Frequency RR Modes of High-Spin Fe(II)PP Complexes
Fe(II)PP
Fe(II)PP
(H2O)
(ROH) (R
or CH2
)
CH2)
H
Fe(II)PP
(OH-
Fe(II)PP
(OH-
Fe(II)PP
(2MeImH)
(CTABr)
Fe(II)PP
−
−
)
)
(2MeIm-
)
2
mode
(CTABr)
(TX-100)
(CTABr)
(EtOH)
(CTABr)
ν15
γ11
752
672
752
716
752
719
697
673
585
545
435
421
409
377
752
720
758
718
700
675
586
541
755
716
699
675
587
542
ν7
675
586
548
673
585
ν48
γ21
ν(Fe-OH-)
421
δ(CâCaCb)
δ(CâCcCd)
408
411
376
408
379
366
343
304
257
410
380
409
377
367
349
299
255
227
132
ν8
349
301
256
347
302
257
349
298
258
206
139
γ7
ν9
ν(Fe-Nax)
155
149
but their spectroscopic properties were not fully determined.15
Our spectrophotometric titrations of Fe(II)PP by hydroxide in
aqueous CTABr clearly show the binding of one hydroxide ion
to the iron atom. The low affinity of OH- for Fe(II)PP (ca. 4
× 10-1 M) originates from the null charge of the Fe(II)-
porphyrinate system. However, environment effects can strongly
increase the affinity of OH- for the ferrous ion.15c The 5c
Fe(II)PP(OH-) complex and the 6c Fe(II)PP(OH-)(H2O) com-
plex are thus two plausible monohydroxy complexes formed
in aqueous CTABr. This alkaline form shows λmax at 440, 561,
and 593 nm. These spectral features are very similar to those
of the 2MeIm- complex of Fe(II)PP in aqueous CTABr (441,
560, and 592 nm) (Table 1). A parallel situation occurs in the
family of the Fe(II)-tetraphenylporphyrins with a close similarity
in absorption spectra for the OH- and 2MeIm- complexes.15c,16
As far as the RR spectra are concerned, the frequencies of the
skeletal ν2 (1560 cm-1), ν3 (1472 cm-1), ν4 (1357 cm-1), ν10
(1605 cm-1), and ν38 (1522 cm-1) modes confirm the HS state
of the monohydroxy complex of Fe(II)PP (Table 2). It is of
importance to note that all these frequencies are also very close
to those determined for the 2MeImH and 2MeIm- complexes
of Fe(II)PP (Table 2). In the low-frequency regions of RR
spectra, the ν8 mode is observed at 347 cm-1 (Table 3). This
RR frequency is again close to that determined for the ν8 mode
of various 5c imidazole and imidazolate complexes of Fe(II)-
PP (345-349 cm-1).4a,b Analysis of the absorption and RR
spectra and the fact that the aqueous detergents stabilize the 5c
ligation states via an interfacial effect4a,b are thus most consistent
with the formation of a 5c complex in aqueous CTABr solution,
i.e., the formation of Fe(II)PP(OH-).17
To our knowledge, no axial Fe(II)-OH- mode has been
characterized for an Fe(II)-porphyrin complex. On the contrary,
a number of ν(Fe(III)-OH) modes was assigned in the 450-
575 cm-1 region of RR spectra of ferric heme proteins and
model compounds.18 Bands recognizable at 257, 300, 347, 366,
and 377 cm-1 in the RR spectra of Fe(II)PP(OH-) was already
seen in the Soret-excited RR spectra of Fe(II)PP(2MeImH) and
(17) (a) A Fe(II)-tetraphorphyrin(OH-)(H2O) complex has been studied by X-ray
crystallography.18b The Fe(II) ion was found to sit above the porphyrin
plane towards the OH- ligand with an Fe-OH distance of 2.18 Å.
Considering the Fe-OH2 distance (2.95 Å), there is no evidence for a water
coordination to the Fe(II) ion. The coordination properties of water are
expected to be similar to those of alcohol. The coordinating Fe-O distances
are in the 2.15-2.35 Å range.18c Finally, the negative OH- ligand is
expected to destabilize the binding of a weaker ligand (i.e., water) in the
trans position favouring the formation of a 5c complex.11a (b) Fleischer, E.
B.; Miller, C. K.; Webb, L. E. J. Am. Chem. Soc. 1964, 86, 2342-2347.
(c) Barkigia, K. M.; Palacio, M.; Sun, Y.; Nogues, M.; Renner, M. W.;
Varret, F.; Battioni, P.; Mansuy, D.; Fajer, J. Inorg. Chem. 2002, 41, 5647-
5649.
(16) (a) Brault, D.; Rougee, M. Biochem. Biophys. Res. Commun. 1974, 57,
654-659. (b) Landrum, J. T.; Hatano, K.; Scheidt, W. R.; Reed, C. A. J.
Am. Chem. Soc. 1980, 102, 6729-6735. (c) Mandon, D.; Ott-Woelfel, F.;
Fischer, J.; Weiss, R.; Bill, E.; Trautwein, A. X. Inorg. Chem. 1990, 29,
2442-2447.
9
17640 J. AM. CHEM. SOC. VOL. 127, NO. 50, 2005

Hydroxide and Aqua Complexes of Fe(II)-Protoheme
A R T I C L E S
Fe(II)PP(2MeIm-).4a,b The ligand deuteration in Fe(II)PP(OH-)
porphyrin is nearly planar with an expanded core corresponding
to Fe-N(pyrrole) distances of 2.06-2.09 Å.21
produces a downshift of 4 cm-1 for a specific band at 435 cm-1
.
The wavelengths of the absorption maxima of Fe(II)PP(OH-)2
(435, 557, and 591 nm) are close to those of bis-alkoxide and
bis-phenoxide complexes of ester derivatives of Fe(II)PP (427-
436, 558-565, and 593-595 nm).15a,22 These features are in
agreement with an HS state for the ferrous ion. The positions
of the absorption bands of the HS Fe(II)-porphyrins are sensitive
to the nature of the axial ligand(s) and the porphyrin substituents
as well as the structure and the environment of the Fe(II)-
porphyrin complex. In particular, the negatively charged ligands
produce red-shifted absorption spectra.15a,16 The data obtained
for Fe(II)PP(OH-) and Fe(II)PP(OH-)2 confirm this trend (Table
1). Nevertheless, the redshifts (2-5 nm) of the R, â, and Soret
bands of Fe(II)PP(OH-) relative to those of Fe(II)PP(OH-)2
(Table 1) can be interpreted in the frame of an out-of-plane
distortion of the porphyrin macrocycle for the monohydroxy
complex. This distortion is likely a moderate doming similar
to that observed for crystallized 5c HS ferrous complexes.16c,20
Redshifts of the absorption bands were recently established for
ruffled and saddled 6c LS Fe(II)-porphyrin complexes.23 This
study shows that a redshift of the absorption bands is also
operative when a planar HS Fe(II)-porphyrin becomes domed.
When the mass effect is localized on the oxygen atom of the
ligand (16OH- versus 18OH-), we observe a downshift of this
band by ca. 21 cm-1. Assuming an isolated Fe-OH harmonic
oscillator with Fe and OH as oscillating units and masses of
56, 17, 18, and 19 amu for Fe, 16OH, 16OD, and 18OH,
respectively, the theoretical shifts are -9.4 cm-1 for the 16OH
f
16OD substitution and -17.9 cm-1 for the 16OH f 18OH
substitution.19 The 435 cm-1 band is thus assigned to a mode
involving a stretching of the axial bond (ν(Fe(II)-OH-)) likely
coupled with a stretching of the Fe(II)-N(pyrrole) bonds. It is
interesting to note that the experimental sensitivity to ligand
deuteration of the 435 cm-1 band is weak (4 cm-1) when
compared to the theoretical shift (9.4 cm-1). This effect is
consistent with a bent geometry for the Fe(II)-O-H grouping.
Such a conformation in fact minors the mass effect of the
terminal proton or deuteron.19 The frequency of the ν(Fe(III)-
OH) mode of a 5c HS ferriporphyrin was determined at 541
cm-1 18d The 435 cm-1 frequency reflects a weaker Fe-OH
.
bond in Fe(II)PP(OH-) than that in the ferric complex. The
strong Fe(III)-OH bond observed for the ferric derivative is
likely related to its ionic character.
Dihydroxy Complex of Fe(II)PP. In aqueous ethanol
solution, the spectrophotometric titration of Fe(II)PP with
OH- confirms the formation of a dihydroxy complex, i.e.,
Fe(II)PP(OH-)2.7 The characterization of the Fe(II)PP(OH-) and
Fe(II)PP(OH-)2 complexes thus offers the possibility to compare
the spectroscopic properties of two ferrous HS complexes with
identical steric and electronic effects provided by the ligand
but differing in coordination geometry.
We have no X-ray data on mono- and bis-(OH-) complexes
of Fe(II)-porphyrins. However, the structures of several 5c and
6c HS Fe(II)-porphyrin complexes have been determined and
can be used as a reference for each compound. On one hand,
the single OH- binding to Fe(II)PP is expected to produce a
porphyrin with an out-of-plane displacement of the iron atom.
The most typical examples of this coordination geometry
concern the Fe(II)- porphyrin complexes axially coordinated by
an imidazole ring or an electronegative oxygen donor.16c,20 In
these 5c HS complexes, the Fe-N(pyrrole) bond lengths are in
the 2.09-2.11 Å range and the distance between the porphyrin
center and the N(pyrrole) atoms is 2.03-2.05 Å.16c,20 On the
other hand, the porphyrin structure of the Fe(II)PP(OH-)2
complex is expected to be similar to that of bis-alcohol or bis-
tetrahydrofuran complexes.17c,21 In these 6c HS complexes, the
The high-frequency region of RR spectra are essentially
identical for Fe(II)PP(OH-) and Fe(II)PP(OH-)2. In passing
from the mono- to the dihydroxy complex, small shifts (2-4
cm-1) are however detected for the skeletal ν2 mode as well as
for several vinyl modes. The frequency shifts of the vinyl modes
may have two origins. In the one hand, the different environ-
ments for the complex formation could induce different polar
effects and/or steric effets on the peripheral groups when the
porphyrin interacts with either the water and ethanol molecules
or the detergent micelle. On the other hand, the different
conformations of the porphyrin macrocycle (domed versus
planar) could change the electronic and kinematic coupling of
the vinyl groups with the adjacent pyrrole rings.
As far as the frequency shift of ν2 is concerned, it could be
assigned to the different conformations adopted by the macro-
cycle in Fe(II)PP(OH-) and Fe(II)PP(OH-)2. For 6c LS Fe-
(II)-porphyrins complexes, the ν2 frequency was found to be
sensitive to porphyrin ruffling but insensitive to porphyrin
saddling.23 The downshifted ν2 frequency observed for
Fe(II)PP(OH-) could be associated with the expected por-
phyrin doming induced by the binding of a single hydroxide
ligand. Considering that the same type of pyrrole movement
occurs in the saddled and domed conformations,24 a conclusion
(18) (a) Desbois, A.; Lutz, M.; Banerjee, R. Biochemistry 1979, 18, 1510-
1518. (b) Asher, S. A.; Schuster, T. M. Biochemistry 1979, 18, 5377-
5387. (c) Han, S.; Ching, Y. C.; Rousseau, D. L. Nature 1990, 348, 89-
90. (d) Reed, R. A.; Rodgers, K. R.; Kushmeider, K.; Spiro, T. G.; Su Y.
O. Inorg. Chem. 1990, 29, 2881-2883. (e) Rodgers, K. R.; Reed, R. A.;
Spiro, T. G. New J. Chem. 1992, 16, 533-536. (f) Boffi, A.; Das, T. K.;
della Longa, S.; Spagnuolo, C.; Rousseau, D. L. Biophys. J. 1999, 77,
1143-1149.
(22) Ainscough, E. W.; Addison, A. W.; Dolphin, D.; James, B. R. J. Am. Chem.
Soc. 1978, 100, 7585-7591.
(23) (a) Picaud, T.; Le Moigne, C.; Loock, B.; Momenteau, M.; Desbois, A. J.
Am. Chem. Soc. 2003, 125, 11616-11625. (b) Le Moigne, C.; Picaud, T.;
Boussac, A.; Loock, B.; Momenteau, M.; Desbois, A. Inorg. Chem. 2003,
42, 6081-6087.
(24) (a) The saddled and domed conformations produce pyrrole tilts about the
CR-CR axes. For these two types of porphyrin conformation, only the
direction of the tilts differs. The pyrrole rings of the saddled porphyrins
are displaced, alternatively, above and below the mean plane of the core.
Upon doming, the direction of the tilt is the same for the four pyrroles.
The sensitivities of the ν2, ν3, ν10 frequencies upon porphyrin ruffling were
related to the twisting of the methine bridges.21b,c Doming and saddling
change the Fe-N(pyrrole) bond lengths without any twisting of the CR-
Cm-CR bonds. (b) Shelnutt, J. A.; Medforth, C. J.; Berber, M. D.; Barkigia,
K.; Smith, K. M. J. Am. Chem. Soc. 1991, 113, 4077-4087. (c) Jentzen,
W.; Simpson, M. C.; Hobbs, J. D.; Song, X.; Ema, T.; Nelson, N. Y.;
Medforth, C. J.; Smith, K. M.; Veyrat, M.; Mazzanti, M.; Ramasseul, R.;
Marchon, J.-C.; Takeuchi, T.; Goddard, W. A., III; Shelnutt, J. A. J. Am.
Chem. Soc. 1995, 117, 11085-11097.
(19) (a) Lechner, F. Monatsh. Chem. 1932, 61, 385-396. (b) Cross, P. C.; Van
Vleck, J. H. J. Chem. Phys. 1933, 1, 350-356. (c) Wilson, E. B., Jr. J.
Chem. Phys. 1939, 7, 1047-1052.
(20) (a) Jameson, G. B.; Molinaro, F. S.; Ibers, J. A.; Collman, J. P.; Brauman,
J. I.; Rose, E.; Suslick, K. S. J. Am. Chem. Soc. 1980, 102, 3224-3237.
(b) Momenteau, M.; Scheidt, W. R.; Eigenbrot, C. W.; Reed, C. A. J. Am.
Chem. Soc. 1988, 110, 1207-1215. (c) Ellison, M. K.; Schulz, C. E.;
Scheidt, W. R. Inorg. Chem. 2002, 41, 2173-2181. (d) Nasri, H.; Fischer,
J.; Weiss, R.; Bill, E.; Trautwein, A. J. Am. Chem. Soc. 1987, 109, 2549-
2550.
(21) (a) Reed, C. A.; Mashiko, T.; Scheidt, W. R.; Spartalian, K.; Lang, G. J.
Am. Chem. Soc. 1980, 102, 2302-2306. (b) Lecomte, C.; Blessing, R. H.;
Coppens, P.; Tabard, A. J. Am. Chem. Soc. 1986, 108, 6942-6950.
9
J. AM. CHEM. SOC. VOL. 127, NO. 50, 2005 17641

A R T I C L E S
Gomez de Gracia et al.
that the frequency of ν2 could be sensitive to porphyrin doming
seems however unlikely. In this line, it is important to consider
that the frequency of ν2 is strongly influenced by the peripheral
vinyl groups.25 Therefore, the downshifted ν2 frequency of
Fe(II)PP(OH-) can be alternatively attributed to either a steric
effect or an electronic perturbation on the vinyl groups and not
to a direct effect of the porphyrin doming. We favor this latter
interpretation taking into account the behavior of other skeletal
modes. In particular, the ν3 mode that is vinyl-insensitive
exhibits no frequency shift in the RR spectra of the two
hydroxide complexes (Table 2). In the RR spectra of Fe-heme
complexes, this skeletal mode is affected to different extents
by the porphyrin distortions.26
In the low-frequency regions of RR spectra, the ν8 mode is
downshifted from 347 to 343 cm-1 when the coordination is
changed from 6 to 5 (Table 3). This mode involves ν(Fe-
N(pyrrole)) and ν(CR-Cm).11b,27 For 6c LS complexes of
ferroporphyrins, the ν8 frequency was found to be upshifted by
the ruffling as well as by the saddling of the porphyrin
macrocycle.23 For the HS complexes presently investigated, the
small vinyl influence on ν8 strongly supports that its frequency
is upshifted upon change in porphyrin conformation (Table 3).
In other terms, the increased frequency of the in-plane ν8 mode
does not follow the increase in length of the Fe-N(pyrrole)
bonds following the porphyrin doming but is related to the
accompanying decrease in core size.16c,20 In conclusion, both a
redshift of the absorption bands and an upshift of the ν8 RR
mode are spectral parameters corresponding to a porphyrin
doming in HS complexes. A similar behavior was observed upon
porphyrin saddling in 6c LS complexes.23
recently revealed the presence of an extra-heme in cyt b6f.2 The
detection of this additional heme ci had no precedence since it
was not predicted using conventional biochemical and spectro-
scopic methods. In particular, heme ci is not directly observable
in the absorption spectra of cyt b6f due to the superimpositions
of the strong electronic transitions of the LS hemes (f, bL and
bH), chlorophyll-a, and â-carotene.
Heme ci is located in the Qi site, close to heme bH. This
location blocks any contact between the substrate and heme bH.
Heme ci is therefore an obligate intermediate in the linear
electron transfers between photosystem II and photosystem I
as well as in the cyclic electron transfers around photosystem
I. This role implies a redox potential for heme ci close to, or
slightly higher than, that of heme bH (-70 ( 25 mV).28 The
peripheral composition of heme ci is not fully determined, but
it was classified as a c-type on the basis of its thioether link
with a conserved cysteine residue.2a,3 Its most unusual property
however concerns its axial ligation since no amino acid is able
to axially bind the iron atom.2a In the fifth ligand position, an
electronic density was assigned to either a water molecule or a
hydroxide ion but does not establish a true coordination of this
exogenous molecule. The proximity of one propionate group
of heme bH appears to stabilize through H-bonding this trapped
or bound OH- or H2O molecule.
An RR investigation of a monomeric form of Chlamydomonas
reinhardtii cyt b6f has recently identified weak contributions
of heme ci.3 Among strong RR bands arising from hemes f, bH,
and bL, the ν4, ν3, and ν2 frequencies of reduced heme ci were
determined at 1354, 1471, and 1576 cm-1, respectively. This
set of frequencies was found close to those previously deter-
mined for various HS cyt c′ and is thus consistent with an HS
state for the ferrous heme ci.3,30 The absence of water or
hydroxide coordination to reduced heme ci would produce a
spectral signature corresponding to a 4c IS complex.
To our knowledge, no axial heme mode involving the Fe-
(II)-(OH-)2 bonds was assigned. The experimental shifts
observed for a ligand specific band at 421 cm-1 (4 cm-1 for
the 16OH-
f
f
16OD- substitution and 17 cm-1 for the 16OH-
18OH- substitution) allow its assignment as a mode of the
In vivo, a carrier G with a redox potential (E1/2) of about
-30 mV has been identified by absorption spectroscopy as a
c′-type cytochrome exchanging electrons with heme bH.30a,b
Given their similarities in functional and spectroscopic proper-
ties, heme ci can be tentatively associated with carrier G.3,30
The classical c′-type cytochromes constitute a unique class of
cytochromes that exhibit an HS 5c state for its ferrous state.
They have redox potentials varying from -10 to +100 mV.31
Its heme is axially ligated by a H-bonded imidazole ring.29,3e´
The comparison of the RR frequencies observed for reduced
heme ci with those presently determined for H2O and OH-
complexes of Fe(II)PP unambiguously establishes that heme ci
is coordinated by a water or hydroxide molecule. Unfortunately,
the RR frequency of the skeletal porphyrin modes cannot
distinguish the H2O and OH- ligations. Only, the characteriza-
tion of the absorption spectrum of reduced heme ci and/or the
axial bonds. Assuming a linear three-body oscillator for the
HO-Fe-OH grouping, the expected shifts are -11.9 cm-1
upon ligand deuteration and -22.8 cm-1 upon 16OH- f 18OH-
substitution. Upon 16OH- f 16OD- substitution, we observe a
small experimental shift of the 421 cm-1 band. Like in the case
of the monohydroxy complex, this effect is again associated
with the bent structure of the Fe-O-H and Fe-O-D bonds.
All these observations are thus in agreement with the assignment
of the 421 cm-1 band to a symmetric stretching mode of the
axial Fe-OH bonds (νs(Fe(II)-(OH-)2). In the ferric series, a
νs(Fe(III)-(OH-)2) mode was characterized at 447 cm-1 for
an HS dihydroxy complex of a water-soluble Fe(III)-porphyrin.18e
According to a mechanism similar to that already seen for the
monohydroxy complexes, the strongest Fe-OH- bonds ob-
served for the dihydroxy ferric compound likely originate from
an electrostatic interaction between the OH- anion and the
charged iron atom.
Heme ci of Cytochrome b6f. The comparisons of the
crystallographic structures of cyt bc1 and cyt b6f complexes have
(28) (a) Pierre, Y.; Breyton, C.; Kramer, D.; Popot, J.-L. J. Biol. Chem. 1995,
270, 29342-29349. (b) Kramer, D. M.; Croft, A. R. Biochim. Biophys.
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(29) Othman, S.; Richaud, P., Verme´glio, A.; Desbois, A. Biochemistry 1996,
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4351. (c) Choi, S.; Spiro, T. G. J. Am. Chem. Soc. 1983, 105, 3683-3692.
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Kroneck, P. M. H.; Shelnutt, J. A. Biochemistry 1998, 37, 12431-12442.
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Joliot, A. Biochim. Biophys. Acta 1988, 933, 319-333. (c) Zhang, H.;
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17642 J. AM. CHEM. SOC. VOL. 127, NO. 50, 2005

Hydroxide and Aqua Complexes of Fe(II)-Protoheme
A R T I C L E S
frequency of the Fe-ligand stretching mode would be clearly
informative. From this study on model compounds, one can
however conclude that a coordination change from imidazole
(or imidazolate) to H2O (or OH-) does not drastically modify
the electronic structure of the Fe(II)-heme. A parallel situation
occurs for homologue ferric complexes.18f Consequently, an
axial substitution of a H-bonded imidazole ring (i.e., the case
of a typical cyt c′)29 by a H-bonded H2O molecule (as one of
the two possibilities of axial ligation for heme ci) is expected
to produce small effects on the oxidation potential of heme.
For a given Fe-porphyrin complex in a given solvent, the redox
potential (E1/2) of the metal atom is influenced by the axial
coordination and particularly by the electronegativity of the
ligand(s).15c,d,33,34 For example, deprotonation at a heme-bound
imidazole is known to produce a decrease in E1/2 of 700 mV.33a
The monohydroxy complexes of Fe-“basket handle” porphyrins
also exhibit very negative potentials.15c This large effect is
directly related to the preferential stabilization of the oxidized
heme through a charge compensation between the Fe(III)-heme
which carries one formal positive charge and the anionic ligand.
As the basicity of the axial ligand is increased, the ferric state
is stabilized relative to the ferrous state and consequently a more
negative E1/2 is observed. A similar charge effect on the redox
potential can be operative when water and hydroxide complexes
of Fe(II)- and Fe(III)-porphyrins are considered.15c,34,35 The axial
deprotonation of the Fe(III)- and Fe(II)-porphyrin(H2O) com-
plexes obviously increases the ligand basicity. The OH- ligand
is a stronger σ-donor species than H2O, and consequently, the
ferric state is stabilized relative to the ferrous state. A largely
more negative E1/2 is thus expected for the Fe(III)/Fe(II)PP-
(OH-) redox couple when compared to the Fe(III)/Fe(II)PP-
(H2O) couple in the same environment. Moreover, the cyt b6f
complex uses a so-called Q-cycle to couple electron transfer
with proton translocation. In this transmembrane complex, heme
ci is on the stromal face of the Qi site where proton uptake and
reduction of plastoquinone occur. The coupling of ligand
deprotonation with a decreased redox potential in an Fe-
porphyrin(H2O)/(OH-) complex like heme ci could therefore
originate a redox-dependent proton pump very analogous to that
proposed for the Fe-porphyrin(imidazole)/(imidazolate) systems.4b
In addition to the similar properties of the Fe-porphyrin(H2O)/
(OH-) and Fe-porphyrin(imidazole)/(imidazolate) couples, the
low affinities of the H2O and OH- ligand for the Fe(II)-
porphyrins offer an interesting flexibility in terms of ligand
exchange. In this line, heme ci is a likely binding site for a
physiological ligand like plastoquinone. To conclude about the
two possibilities of axial ligation for heme ci, a coordination of
the iron atom of heme ci by a water molecule appears to be the
most plausible on the basis of a redox potential close to that of
heme bH. The E1/2 value for the aqua complex of a water soluble
Fe(III)-/Fe(II)-porphyrin was estimated to be -230 mV.34a This
value can be positively shifted by 200-300 mV taking into
account the degree of hydrophobicity of the heme cavity in
proteins.36 A OH- binding to heme ci would produce a too
negative E1/2 value.
Supporting Information Available: Plot of 1/(R - R0) vs
1/[OH-]2 and low frequency regions (220-730 cm-1) of RR
spectra of Fe(II)PP in 0.5% TX-100 aqueous solution at pH
7.0. This material is available free of charge via the Internet at
http://pubs.acs.org.
JA052791G
(35) (a) Following a mechanism similar to that observed for the histidylimidazole
ligand,4c,36b hydrogen-bonding at the coordinated H2O or OH- molecule
can modulate the electronegativity of the O(ligand) atom and thus the redox
potential. For heme ci, this effect would in great part depend on the
ionization state of one of the propionic groups of heme bH. An increased
electronegativity of the O(H2O) atom is conditioned by an interaction with
an ionized proponiate group (FePP-OH2- - --O2C(propionate)). On the
contrary, a decreased electronegativity of the OH- ligand would be operative
with a protonated propionate group (FePP-HO-- - -HO2C(propionic acid)).
(b) Valentine, J. S.; Sheridan, R. P.; Allen, L. C.; Kahan, P. C. Proc. Natl.
Acad. Sci. U.S.A. 1979, 76, 1009-1013.
(32) Finzel, B. C.; Weber, P. C.; Hardman, K. D.; Salemme, F. R. J. Mol. Biol.
1985, 186, 627-643.
(33) (a) Quinn, R.; Strouse, C. E.; Valentine, J. S. Inorg. Chem. 1983, 22, 3934-
3940. (b) Bottomley, L. A.; Kadish, K. M. Inorg. Chem. 1981, 20, 1348-
1357. (c) Nesset, M. J. M.; Shokhirev, N. V.; Enemark, P. D.; Jacobson,
S. E.; Walker, F. A. Inorg. Chem. 1996, 35, 5188-5200.
(34) (a) Laverman, L. E.; Ford, P. J. Am. Chem. Soc. 2001, 123, 11614-11622.
(b) Jones, S. E.; Srivatsa, G. S.; Sawyer, D. T.; Traylor, T. G.; Mincey, T.
C. Inorg. Chem. 1983, 22, 3903-3910.
(36) (a) Kassner, R. J. Proc. Natl. Acad. Sci. U.S.A 1972, 69, 2263-2267. (b)
Kassner, R. J. J. Am. Chem. Soc. 1973, 95, 2674-2677. (c) Stellwagen, E.
Nature 1978, 275, 73-74.
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