Báo cáo khoa học: Reductive nitrosylation and peroxynitrite-mediated oxidation of heme–hemopexin doc

Reductive nitrosylation and peroxynitrite-mediated
oxidation of heme–hemopexin
Paolo Ascenzi
1,2
, Alessio Bocedi
2
, Giovanni Antonini
1
, Martino Bolognesi
3
and Mauro Fasano
4
1 Department of Biology and Interdepartmental Laboratory for Electron Microscopy, University ‘Roma Tre’, Rome, Italy
2 National Institute for Infectious Diseases IRCCS ‘Lazzaro Spallanzani’, Rome, Italy
3 Department of Biomolecular Sciences and Biotechnology and CNR-INFM, University of Milan, Italy
4 Department of Structural and Functional Biology and Center of Neuroscience, University of Insubria, Busto Arsizio, Italy
Heme scavenging by high- and low-density lipopro-
teins, serum albumin and hemopexin (HPX) provides
protection against heme and iron oxidative damage,
limits access by pathogens to heme, and contributes to
iron homeostasis by recycling the heme iron. During
the first seconds after the appearance of heme in the
plasma, > 80% of this powerful oxidizer binds to
high- and low-density lipoproteins, and only the
remaining 20% binds to serum albumin and HPX.
Serum albumin and HPX then remove the heme from
high- and low-density lipoproteins. Afterwards, heme
transits to HPX, which releases it into hepatic paren-
chymal cells only after internalization of HPX–heme
by specific receptor-mediated endocytosis. After deliv-
ering the heme intracellularly, HPX is released intact

into the bloodstream and the heme is degraded [1–15].
HPX–heme is formed by two four-bladed b-propel-
ler domains, resembling two thick disks that lock
Keywords
nitric oxide; peroxynitrite; peroxynitrite-
mediated oxidation; rabbit hemopexin;
reductive nitrosylation
Correspondence
P. Ascenzi, Department of Biology,
University ‘Roma Tre’, I-00146 Rome, Italy
Fax: +39 06 5517 6321
Tel: +39 06 5517 3200 ⁄ 2
E-mail:
(Received 14 September 2006, revised 14
November 2006, accepted 21 November
2006)
doi:10.1111/j.1742-4658.2006.05609.x
Hemopexin (HPX), which serves as a scavenger and transporter of toxic
plasma heme, has been postulated to play a key role in the homeostasis of
NO. In fact, HPX–heme(II) reversibly binds NO and facilitates NO scaven-
ging by O
2
. HPX–heme is formed by two four-bladed b-propeller domains.
The heme is bound between the two b-propeller domains, residues His213
and His266 coordinate the heme iron atom. HPX–heme displays structural
features of heme-proteins endowed with (pseudo-)enzymatic activities. In
this study, the kinetics of rabbit HPX–heme(III) reductive nitrosylation
and peroxynitrite-mediated oxidation of HPX–heme(II)–NO are reported.
In the presence of excess NO, HPX–heme(III) is converted to HPX–
heme(II)–NO by reductive nitrosylation. The second-order rate constant

for HPX–heme(III) reductive nitrosylation is (1.3 ± 0.1) · 10
1
1
m
)1
Æs
)1
,at
pH 7.0 and 10.0 °C. NO binding to HPX–heme(III) is rate limiting. In the
absence and presence of CO
2
(1.2 · 10
)3
m), excess peroxynitrite reacts
with HPX–heme(II)–NO (2.6 · 10
)6
m) leading to HPX–heme(III) and
NO, via the transient HPX–heme(III)–NO species. Values of the second-
order rate constant for HPX–heme(III)–NO formation are
(8.6 ± 0.8) · 10
4
and (1.2 ± 0.2) · 10
6
m
)1
Æs
)1
in the absence and pres-
ence of CO
2

, respectively, at pH 7.0 and 10.0 °C. The CO
2
-independent
value of the first-order rate constant for HPX–heme(III)–NO denitrosyla-
tion is (4.3 ± 0.4) · 10
)1
s
)1
, at pH 7.0 and 10.0 °C. HPX–heme(III)–NO
denitrosylation is rate limiting. HPX–heme(II)–NO appears to act as an effi-
cient scavenger of peroxynitrite and of strong oxidants and nitrating
species following the reaction of peroxynitrite with CO
2
(e.g. ONOO-
C(O)O
–
,CO
3
–
, and NO
2
).
Abbreviations
Hb, hemoglobin; HbI, haemoglobin I; HPX, hemopexin; Lb, leghemoglobin; Mb, myoglobin; Ngb, neuroglobin.
FEBS Journal 274 (2007) 551–562 ª 2006 The Authors Journal compilation ª 2006 FEBS 551
together at a 90° angle; the face of the N-terminal
b-propeller domain packs against one edge of the
C-terminal domain. Each propeller blade comprises a
four-stranded antiparallel b-sheet, with the first and
the fourth blades joined by disulfide bridges. The heme

is bound between the two four-bladed b-propeller
domains in a pocket formed by the interdomain linker
peptide. Residues His213 and His266 coordinate the
heme iron atom giving a stable bis-histidyl complex
(Fig. 1). Heme binding and release results from the
opening and closing of the heme-binding pocket, via
movement of the two b-propeller domains and ⁄ or the
interdomain linker peptide [16].
Evidence suggests that heme-bound plasma proteins
may display ligand binding (kinetic and thermody-
namic) capacity and pseudo-enzymatic properties. Fer-
rous high- and low-density heme–lipoproteins bind NO
[17]. Ferrous heme–serum albumin has been reported
to bind NO, O
2
and CO [14,18–21], and to exhibit
weak catalase and peroxidase activity [22]. HPX–
heme(II) binds CO and NO, however, O
2
induces
HPX–heme(II) oxidation [17,23–27]. Furthermore,
HPX–heme(III) binds cyanide [16]. NO appears to
modulate heme binding to HPX–heme and, in turn,
HPX–heme may play a key role in NO homeostasis
[17,25–27]. Indeed, O
2
has been reported to react with
HPX–heme(II)–NO yielding HPX–heme(III) and
NO
3

–
, by way of the ferric heme-bound peroxynitrite
intermediate HPX–heme(III)–N(O)OO. Afterwards,
peroxynitrite dissociates from HPX–heme(III)–
N(O)OO and isomerizes to nitrate. HPX–heme(III)
may reduce back to HPX–heme(II) and bind heme lig-
ands [27]. The recommended IUPAC nomenclature for
peroxynitrite is oxoperoxonitrate; for peroxynitrous
acid, it is hydrogen oxoperoxonitrate. The term per-
oxynitrite is used in the text to refer generically to
both ONOO
)
and its conjugate acid HOONO [28].
Here, the kinetics of reductive nitrosylation of
HPX–heme(III) and peroxynitrite-mediated oxidation
of HPX–heme(II)–NO are reported. HPX–heme(II)–
NO appears to act as an efficient scavenger of peroxy-
nitrite and of strong oxidants and nitrating species
following the reaction of peroxynitrite with CO
2
[e.g.
ONOOC(O)O
–
,CO
3
and NO
2
). Our results have been
analyzed in parallel with those of related heme–protein
systems.

Results and Discussion
Reductive nitrosylation of HPX–heme(III)
Addition of NO (either gaseous or dissolved in the
buffer solution) to the HPX–heme(III) solution causes
a shift in the maximum of the optical absorption spec-
trum in the Soret band from 414 nm, i.e. HPX–
heme(III), to 419 nm, i.e. HPX–heme(II)–NO, and a
corresponding change in the extinction coefficient from
e
414
¼ 1.16 · 10
5
m
)1
Æcm
)1
to e
419
¼ 1.45 · 10
5
m
)1
Æcm
)1
(Fig. 2A). The optical absorption spectrum of the reac-
tion product HPX–heme(II)–NO (Fig. 2A) is identical
to that obtained by adding NO to HPX–heme(II)
[26,27]. Accordingly, optical absorption spectroscopic
changes for the reaction of HPX–heme(III) with NO
were not reversible. Pumping off gaseous NO causes a

shift in the maximum of the optical absorption spec-
trum in the Soret band from 419 nm, i.e. HPX–
heme(II)–NO, to 428 nm, i.e. HPX–heme(II), and a
corresponding change in the extinction coefficient from
e
419
¼ 1.45 · 10
5
m
)1
Æcm
)1
to e
428
¼ 1.47 · 10
5
m
)1
Æcm
)1
.
The optical absorption spectra of HPX–heme(III),
HPX–heme(II)–NO and HPX–heme(II) here deter-
mined correspond to those reported in the literature
Fig. 1. Rabbit ferric HPX–heme structure, including the coordinating
heme-iron residues His213 and His266 (PDB entry: 1QJS) [16]. The
N-terminal domain (residues 1–208) is shown at the top. The C-ter-
minal domain (residues 228–435) is shown at the bottom. The
arrow indicates the interdomain linker peptide (residues 209–227).
Heme group and His213 and His266 residues are shown in black.

The figure was drawn with
SWISS-PDB-VIEWER [75]. For details, see
text.
Pseudo-enzymatic properties of heme–hemopexin P. Ascenzi et al.
552 FEBS Journal 274 (2007) 551–562 ª 2006 The Authors Journal compilation ª 2006 FEBS
[24,26,27,29]. As already reported for Glycine max leg-
hemoglobin (Lb) [30], sperm whale myoglobin (Mb)
[31], horse cytochrome c [31], human neuroglobin
(Ngb) [32], Scapharca inaequivalvis hemoglobin I (HbI)
[33] and human hemoglobin (Hb) [31], our data indi-
cate that the reaction of HPX–heme(III) with an excess
NO leads to reduction of the heme–Fe(III) atom and
generation of the HPX–heme(II)–NO species (Table 1).
Over the whole NO concentration range explored,
the time course of reductive nitrosylation of HPX–
heme(III) (2.6 · 10
)6
m) conforms to a single-exponen-
tial decay for > 90% of its course between 360 and
460 nm (Fig. 2B). The pseudo-first-order rate constant
for HPX–heme(III) reductive nitrosylation (i.e. k)is
wavelength independent. The plot of k versus [NO] is
linear (Eqn 2) with a y-intercept at 0, indicating that
the reverse reaction rate is negligible (k
off
<1·
10
)4
s
)1

); the slope of the plot of k versus [NO] corres-
ponds to k
on
¼ (1.3 ± 0.1) · 10
1
m
)1
Æs
)1
(Fig. 2C,
Table 1). The first-order rate constant for HPX–
heme(II)–NO
+
conversion to HPX–heme(II)* (i.e. h)
must exceed by at least one order of magnitude
the value of k (6.5 · 10
)3
s
)1
) obtained at the highest
NO concentration investigated (5.0 · 10
)4
m), i.e.
h >7· 10
)2
s
)1
(Scheme 1, Fig. 2C, Table 1), other-
wise a hyperbolic plot of k versus [NO] would be
observed [31,34,35].

Values of k
on
for reductive nitrosylation of HPX–
heme(III), horse cytochrome c(III) [31,36] and human
Ngb(III) [32] are lower than those reported for G. max
2
Lb(III) [30], sperm whale Mb(III) [31,36], S. inaequi-
valvis HbI(III) [33] and human Hb(III) [31], possibly
reflecting heme–Fe(III) atom hexa-coordination [16,37,
38] (Table 1). Values of k
off
for NO dissociation from
the heme(III)–NO proteins considered range between
<1 · 10
)4
and 1.4 · 10
1
s
)1
(Table 1), reflecting the
different stability of the heme–Fe(III)–NO complexes
[30–33,36]. Values of h for NO
+
dissociation from
HPX–heme(II)–NO
+
, human Ngb(II)–NO
+
[32] and
S. inaequivalvis HbI(III) [33] are larger than those

reported for reductive nitrosylation of G. max Lb(III)
[30], sperm whale Mb(III) [31,36], horse cytochrome c
[31,36] and human Hb(III) [31] (Table 1).
Values of k for reductive nitrosylation of HPX–
heme(III) (Fig. 2C), human Ngb(III) [32], G. max
Lb(III) [30] and S. inaequivalvis HbI [33] depend line-
arly on NO concentration over the whole range
explored (i.e. between 5.0 · 10
)5
and 1.2 · 10
)3
m). In
contrast, values of k for reductive nitrosylation of
sperm whale Mb(III) [31], horse cytochrome c [31] and
human Hb(III) [31] do not increase linearly with
the NO concentration but tend to level off at
[NO] > 2 · 10
)5
m. The transient heme(III)–NO spe-
cies was observed during reductive nitrosylation
of G. max Lb(III) [30], sperm whale Mb(III) [31],
horse cytochrome c(III) [31] and human Hb(III) [31]
Fig. 2. Kinetics of NO-mediated reductive nitrosylation of HPX–
heme(III), at pH 7.0 and 10.0 °C. (A) Steady-state and kinetic differ-
ence absorption spectra (line and circles, respectively) in the Soret
region of HPX–heme(III) minus HPX–heme(II)–NO. (B) Time course
of reductive nitrosylation of HPX–heme(III), k ¼ 420 nm. The NO
concentration was 1.0 · 10
)4
M (trace a), 2.0 · 10

)4
M (trace b),
and 5.0 · 10
)4
M (trace c). The time course analysis according to
Eqn (1) allowed us to determine the following values of k:
1.2 · 10
)3
s
)1
(trace a), 2.8 · 10
)3
s
)1
(trace b), and 6.7 · 10
)3
s
)1
(trace c). (C) Dependence of the pseudo-first-order rate constant for
reductive nitrosylation of HPX–heme(III) (i.e. k) on the NO concen-
tration. The continuous line was generated from Eqn (2) with
k
on
¼ (1.3 ± 0.1) · 10
1
M
)1
Æs
)1
. The HPX–heme(III) concentration

was 2.6 · 10
)6
M.
P. Ascenzi et al. Pseudo-enzymatic properties of heme–hemopexin
FEBS Journal 274 (2007) 551–562 ª 2006 The Authors Journal compilation ª 2006 FEBS 553
only. Furthermore, the intermediate species HPX–
heme(III)*, HPX–heme(III)–NO, HPX–heme(II)–
NO
+
, HPX–heme(II)* and HPX–heme(II) (Scheme 1)
were not detected. This suggests the following consid-
erations. (a) Formation of HPX–heme(III)* does not
appear to be rate limiting, as observed for reductive
nitrosylation of Ngb(III) [32] and horse cytochrome c
[31]. (b) HPX–heme(III)–NO hydrolyzes very rapidly
under neutral conditions, similarly to human Ngb(III)–
NO [32] and S. inaequivalvis HbI [33] and human
Hb(III)–NO [31]. By contrast, G. max Lb(III)–NO
[30], sperm whale Mb(III)–NO [31] and horse cyto-
chrome c(III)–NO [31] are rather stable under neutral
conditions and are hydrolyzed at a significant extent
under alkaline conditions. (c) HPX–heme(II) nitrosyla-
tion [27] is faster than the NO-induced reduction of
the heme–Fe(III) atom, as observed for G. max Lb(II)
[30,39], sperm whale Mb(II) [31,40], horse cyto-
chrome c(II) [31,36], mouse Ngb(II) [32,41] (highly
homologous to human Ngb [38,42,43]), S. inaequivalvis
HbI(II) [33,44] and human Hb(II) [31,39,40,45]. How-
ever, nitrosylation of hexa-coordinate HPX–heme(II),
horse cytochrome c(II) [36] and mouse Ngb(II) [41]

(highly homologous to human Ngb [38,42,43]) is
slower than NO binding to penta-coordinate G. max
Lb(II) [39], sperm whale Mb(II) [40], S. inaequivalvis
HbI(II) [44] and human Hb(II) [39,40,45,46] (Table 1).
As a whole, NO binding to HPX–heme(III), human
Ngb(III) [32] and S. inaequivalvis HbI(II) [33] appears
to be rate limiting (i.e. k < h; Scheme 1), whereas the
conversion of heme(II)–NO
+
to heme(II) is rate limit-
ing for reductive nitrosylation of G. max Lb(III) [30],
sperm whale Mb(III) [31], horse cytochrome c [31] and
human Hb(III) [31] (i.e. k > h, Scheme 1).
Effect of CO
2
on peroxynitrite-mediated oxidation
of HPX–heme(II)–NO
In the absence and presence of CO
2
, mixing of the
HPX–heme(II)–NO and peroxynitrite solutions causes
a shift in the optical absorption maximum of the Soret
band from 419 nm, i.e. HPX–heme(II)–NO, to 420 nm,
i.e. HPX–heme(III)–NO, and a corresponding change
in the extinction coefficient from e
419
¼ 1.45 · 10
5
to e
420

¼ 1.59 · 10
5
m
)1
Æcm
)1
(Fig. 3A). The HPX–
heme(III)–NO solution undergoes a shift in the optical
absorption maximum of the Soret band from 420 nm,
i.e. HPX–heme(III)–NO, to 414 nm, i.e. HPX–
heme(III), and a corresponding change in the extinction
coefficient from e
420
¼ 1.59 · 10
5
m
)1
Æcm
)1
to e
414
¼
1.16 · 10
5
m
)1
Æcm
)1
(Fig. 3A). The optical absorption
spectrum of HPX–heme(III) obtained by mixing the

HPX–heme(II)–NO and peroxynitrite solutions (e
414
¼
1.16 · 10
5
m
)1
Æcm
)1
) (Fig. 3A) corresponds to that
reported in the literature [24,26,27]. Values for the opti-
cal absorption maximum and extinction coefficient of
HPX–heme(II)–NO, HPX–heme(III)–NO and HPX–
heme(III) are unaffected by CO
2
. Analogous to G. max
Lb [30], horse Mb [47], human Ngb [32] and human
Hb [28,47], our data indicate that the reaction of HPX–
heme(II)–NO with an excess peroxynitrite leads to oxi-
dation of the heme–Fe(II) atom and generation of the
HPX–heme(III) species.
Over the whole peroxynitrite concentration range
explored (1.5 · 10
)5
)2.5 · 10
)4
m), the time course
for the peroxynitrite-mediated oxidation of HPX–
heme(II)–NO (2.6 · 10
)6

m) corresponds to a bipha-
sic process, in the absence and presence of CO
2
(1.2 · 10
)3
m) (Fig. 3B). Values of the pseudo-first-order
Table 1. Kinetic parameters for reductive nitrosylation of ferric heme-proteins (for details, see Scheme 1). ND, not determined.
5
Heme-protein k
on
(M
)1
Æs
)1
) k
off
(s
)1
) h (s
)1
) l
on
(M
)1
Æs
)1
) l
off
(s
)1

)
Rabbit HPX–heme(III) 1.3 · 10
1a
<1· 10
)4a
>7· 10
)2a
6.3 · 10
3b
9.1 · 10
)4b
Human Ngb(III)
Fast reacting form
c
2.1 · 10
1
2.5 · 10
)3
>2· 10
)1
ND ND
Slow reacting form
c
2.9 2.5 · 10
)3
>5· 10
)2
ND ND
Mouse Ngb(III)
d

ND ND ND 2 · 10
5
2.0 · 10
)4
S. inaequivalvis HbI 3.2 · 10
1e
<1· 10
)3e
>6· 10
)1e
1.6 · 10
7f
ND
Horse cytochrome c 7.2 · 10
2g
4.4 · 10
)2g
< 4.0 · 10
)3h
8.3
g
2.9 · 10
)5g
Human Hb(III)
a subunits 1.7 · 10
3i
6.5 · 10
)1i
1.3 · 10
)3j

2.6 · 10
7k
4.6 · 10
)5l
b subunits 6.4 · 10
3i
1.5
i
1.3 · 10
)3j
2.6 · 10
7k
2.2 · 10
)5l
G. max Lb(III) 1.4 · 10
5m
3.0
m
4.8 · 10
)4m
1.2 · 10
8l
2.4 · 10
)5l
Sperm whale Mb(III) 1.9 · 10
5g
1.4 · 10
1g
< 8.8 · 10
)4h

1.7 · 10
7n
1.2 · 10
)4n
a
pH 7.0 and 10.0 °C; this study.
b
pH 7.0 and 10.0 °C [27].
c
pH 7.0 and room temperature [32].
d
pH 7.0 and 25.0 °C [41].
e
pH 7.5 and
20.0 °C [33].
f
pH 7.0 and 20.0 °C [44].
g
pH 6.5 and 20.0 °C [36].
h
pH < 8.3 and 20.0 °C [31].
i
pH 7.0 and 20.0 °C [74].
j
pH 7.0 and 20.0 °C
[31].
k
pH 7.0 and 20.0 °C [45].
l
pH 7.0 and 20.0 °C [39].

m
pH 7.0 and 20.0 °C [30].
n
pH 7.0 and 20.0 °C [40].
Pseudo-enzymatic properties of heme–hemopexin P. Ascenzi et al.
554 FEBS Journal 274 (2007) 551–562 ª 2006 The Authors Journal compilation ª 2006 FEBS
rate constant for the formation of and the first-order
decay of the transient HPX–heme(III)–NO species (i.e. b
and d, respectively) are wavelength independent.
As shown in Fig. 3C, the first step of kinetics for per-
oxynitrite-mediated oxidation of HPX–heme(II)–NO
(b
on
in Scheme 2) is a bimolecular process as observed
under pseudo-first-order conditions. The plot of b
versus [peroxynitrite] is linear (Eqn 6) with a y-intercept
at 0, indicating that the reverse reaction rate is negli-
gible; the slope of the plot of b versus [peroxynitrite]
corresponds to b
on
¼ (8.6 ± 0.8) · 10
4
m
)1
Æs
)1
and
(1.2 ± 0.2) · 10
6
m

)1
Æs
)1
in the absence and presence
of CO
2
, respectively (Table 2). By contrast, the second
step (d in Scheme 2) follows a peroxynitrite-independ-
ent monomolecular behavior (Fig. 3D), the average
value of d is (4.3 ± 0.4) · 10
)1
s
)1
(Table 2).
Fig. 3. Kinetics of peroxynitrite-mediated oxidation of HPX–heme(II)–NO in the absence and presence of CO
2
, at pH 7.0 and 10.0 °C. (A)
Steady-state and kinetic difference absorption spectra (line and symbols, respectively) in the Soret region of HPX–heme(II)–NO minus HPX–
heme(III) (line and triangles) and fully populated HPX–heme(III)–NO minus HPX–heme(III) (squares). Steady-state and kinetic difference
absorption spectra were independent of CO
2
. (B) Time course of the peroxynitrite-induced conversion of HPX–heme(II)–NO to HPX–heme(III)
by way transient HPX–heme(III)–NO formation, in the absence (trace a) and presence (trace b) of CO
2
, k ¼ 425 nm. The time course
analysis according to Eqns (3–5) allowed us to determine the following parameters: b ¼ 4.5 s
)1
and d ¼ 4.3 · 10
)1
s

)1
(trace a), and
b ¼ 5.9 · 10
1
s
)1
and d ¼ 4.5 · 10
)1
s
)1
(trace b). The peroxynitrite concentration was 5.0 · 10
)5
M. (C) Dependence of the pseudo-first
order rate constant for the peroxynitrite-induced conversion of HPX–heme(II)–NO to HPX–heme(III)–NO (i.e. b) on the peroxynitrite concen-
tration, in the absence (diamonds) and presence (squares) of CO
2
. The continuous line was calculated according to Eqn (6) with
b
on
¼ (8.6 ± 0.9) · 10
4
M
)1
Æs
)1
in the absence of CO
2
(diamonds), and b
on
¼ (1.2 ± 0.2) · 10

6
M
)1
Æs
)1
in the presence of CO
2
(squares). (D)
Dependence of the first order rate constant for NO dissociation from HPX–heme(III)–NO (i.e. d) on the peroxynitrite concentration, in the
absence (diamonds) and presence (squares) of CO
2
. The average value of d is (4.3 ± 0.4) · 10
)1
s
)1
. The HPX–heme(II)–NO concentration
was 2.6 · 10
)6
M. The CO
2
concentration was 1.2 · 10
)3
M.
Table 2. Kinetic parameters for peroxynitrite-mediated oxidation of
ferrous nitrosylated heme-proteins (for details, see Scheme 2).
Heme–protein [CO
2
](M) b
on
(M

)1
Æs
)1
) d (s
)1
)
Rabbit
HPX–heme(II)–NO
a
– 8.6 · 10
4
4.3 · 10
)1
1.2 · 10
)3
1.2 · 10
6
4.3 · 10
)1
Human Hb(II)–NO
b
– 6.1 · 10
3
 1
1.2 · 10
)3
5.3 · 10
4
 1
G. max

Lb(II)–NO
c
– 8.8 · 10
3
2.5
1.0 · 10
)3
1.2 · 10
5
2.5
Horse Mb(II)–NO –
d
3.1 · 10
4d
 1.2 · 10
1d
1.2 · 10
)3e
1.7 · 10
5e
1.1 · 10
1e
Human Ngb(II)–NO
f
– 1.3 · 10
5
1.2 · 10
)1
a
pH 7.0 and 10.0 °C; this study.

b
pH 7.2 and 20.0 °C [48].
c
pH 7.3
and 20.0 °C [30].
d
pH 7.5 and 20.0 °C [47].
e
pH 7.0 and 20.0 °C
[47].
f
pH 7.2 and 20.0 °C [32].
P. Ascenzi et al. Pseudo-enzymatic properties of heme–hemopexin
FEBS Journal 274 (2007) 551–562 ª 2006 The Authors Journal compilation ª 2006 FEBS 555
As observed for HPX–heme(II)–NO (Fig. 3C), val-
ues of the pseudo-first-order rate constant b (Scheme 2)
for peroxynitrite-mediated oxidation of G. max Lb(II)–
NO [30], horse Mb [47], human Ngb(II)–NO [32] and
human Hb(II)–NO [28,47] depend linearly over the
whole peroxynitrite concentration range explored, in
the absence and presence of CO
2
. Values of b
on
for per-
oxynitrite-mediated oxidation of HPX–heme(II)–NO
and human Ngb(II)–NO [32] exceed those reported for
G. max Lb(II)–NO [30], horse Mb [47] and human
Hb(II)–NO [28,47] (Table 2). CO
2

facilitates peroxy-
nitrite-mediated oxidation of HPX–heme(II)–NO
(Fig. 3C), G. max Lb(II)–NO [30], horse Mb [47] and
human Hb(II)–NO [28,47] increasing values of b
on
(Table 2). A similar observation was made for the reac-
tion of G. max Lb(II)–O
2
[48], sperm whale Mb(II)–O
2
[49] and human Hb(II)–O
2
[50] with peroxynitrite in
the absence and presence of CO
2
.
In the presence of CO
2
, peroxynitrite changes from
a two- to a one-electron oxidant. In fact, CO
2
reacts
rapidly with peroxynitrite leading to ONOOC(O)O
–
(second-order rate constant is  3 · 10
4
m
)1
Æs
)1

),
which in turn decays very rapidly to CO
3
–
and NO
2
(first-order rate constant is  5 · 10
5
s
)1
). CO
3
–
and
NO
2
are stronger oxidant and nitrating agents than
peroxynitrite; NO
2
nitrates with preference Tyr and
Trp residues [51,52]. Although CO
2
facilitates the
nitration of heme–protein aromatic residues by peroxy-
nitrite [53], optical absorbance spectroscopy for HPX
between 230 and 500 nm indicates that no appreciable
aromatic nitration takes place (data not shown). This
suggests that the CO
2
-induced increase in k

on
for per-
oxynitrite-mediated oxidation of HPX (
3
Table 2),
reflects oxidation of the heme–Fe atom by CO
3
–
rather
than conformational transition(s) depending on the
nitration of Tyr and Trp residues by NO
2
.
As observed for HPX–heme(III)–NO (Fig. 3D), val-
ues of the first-order rate constant d (Scheme 2) for
NO dissociation from G. max Lb(III)–NO [30], horse
Mb [47], human Ngb(III)–NO [32] and human
Hb(III)–NO [28,47] are unaffected by CO
2
, ranging
between 1 · 10
)1
and 1.2 · 10
1
s
)1
(Table 2). The dis-
sociation of heme(III)–NO adducts is facilitated by the
consumption of NO via its reaction with peroxynitrite
excess and⁄ or with the reactive species generated

during peroxynitrite decomposition (e.g. NO
2
). Under
anaerobic conditions, the reaction of NO with peroxy-
nitrite leads to N
2
O
3
and H
2
O, at pH < 7. At
pH > 7, NO reacts with NO
2
leading to N
2
O
3
,in
turn, N
2
O
3
reacts with peroxynitrite leading to NO
2
–
and NO
2
[28].
The transient species heme(III)–NO was observed
during peroxynitrite-mediated oxidation of HPX–

heme(II)–NO (Fig. 3B). This intermediate is also seen
in G. max Lb(II)–NO [30], horse Mb [47], human
Ngb(II)–NO [32] and human Hb(II)–NO [28,47]. By
contrast, the transient penta-coordinate derivative of
HPX–heme(III), i.e. HPX–heme(III)*, (Scheme 2) was
not observed. This transient was also never observed in
ligand-binding reaction(s) to human Ngb(III) [32]. This
suggests the following: (a) NO dissociation from
heme(III)–NO represents the rate-limiting step for per-
oxynitrite-mediated oxidation of the heme(II)–NO
proteins considered [28,30,32,47]; and (b) the HPX–
heme(III)* fi HPX–heme(III) reaction (Scheme 2) does
not appear to be rate limiting, as reported for peroxy-
nitrite-mediated oxidation of human Ngb(II)–NO [32].
Under the experimental conditions, kinetic and
spectroscopic properties of HPX–heme(II)–NO were
unaffected by decomposed peroxynitrite.
Conclusions
Our data represent the first evidence for reductive
nitrosylation of HPX–heme(III) and for peroxyni-
trite-mediated oxidation of HPX–heme(II)–NO. As a
general remark, the few data available from litera-
ture concerning the reductive nitrosylation of ferric
heme–proteins and the peroxynitrite-mediated oxida-
tion of ferrous nitrosylated heme-proteins (in the
absence and presence of CO
2
) are reported for the
purpose of a comparison with those of HPX–heme
(Tables 1,2).

Although HPX–heme(III) reduction and nitrosyla-
tion occur physiologically and modulate HPX–heme
complex (de-)stabilization [27,54–56], reductive nitrosy-
lation of HPX–heme(III) appears too slow to occur
in vivo despite NO concentrations > 10
)5
m under
pathological conditions [57–61]. The same considera-
tions hold also for reductive nitrosylation of human
Ngb(III), sperm whale Mb(III), G. max Lb(III) and
human Hb(III) [30–32]. However, as shown in Table 1,
heme reduction kinetics are facilitated in bis-histidyl
hexa-coordinate heme–proteins (i.e. HPX–heme and
human Ngb) [62].
The reactivity of peroxynitrite with HPX–heme(II)–
NO (Table 2) is high enough to protect against
peroxynitrite-mediated damage, and to impair the for-
mation of strong oxidants and nitrating agents (e.g.
ONOOC(O)O
–
,CO
3
–
and NO
2
), in the absence and
presence of CO
2
[63]. In fact, values of the second-
order rate constant b

on
for peroxynitrite-mediated oxi-
dation of HPX–heme(II)–NO are larger than those for
the reaction of peroxynitrite with (macro)molecular
targets (e.g. cysteine residues;  4 · 10
3
m
)1
Æs
)1
) and
with CO
2
( 3 · 10
4
m
)1
Æs
)1
) [51,52] (Table 2).
Pseudo-enzymatic properties of heme–hemopexin P. Ascenzi et al.
556 FEBS Journal 274 (2007) 551–562 ª 2006 The Authors Journal compilation ª 2006 FEBS
The high value of the reaction rate of HPX–
heme(II)–NO with peroxynitrite (i.e. b
on
; Table 2) may
reflect structural features reminiscent those of heme–
proteins endowed with (pseudo-)enzymatic activities
[64]. Indeed, the imidazole ring of the proximal His266
residue of HPX–heme is eclipsed with respect to the

heme N–Fe–N coordination bonds [16], as observed
for the proximal His residue in horseradish peroxidase
[65], and in Alcaligenes eutrophus and Escherichia coli
flavohemoglobins [66,67], although His266 is rotated
by 90° with respect to heme propionates in HPX–
heme. Furthermore, the negatively charged residue
Glu226 occurs in the neighborhood of the proximal
His266 residue of HPX–heme [16]. Similarly, horserad-
ish peroxidase [65], and A. eutrophus and E. coli flavo-
hemoglobins [66,67] all display Asp or Glu residues in
the neighborhood of the proximal His. In horseradish
peroxidase [65], and A. eutrophus and E. coli flavoh-
emoglobins [66,67], the proximal Asp⁄ Glu residue is
hydrogen-bonded to the proximal His ND1 atom,
partly setting the orientation of the proximal imidazole
[66,67]. In HPX–heme the orientation of the proximal
imidazole is defined by a hydrogen bond connecting
the carbonyl O atom of Ser267 to the His266 ND1
atom, whereas the carboxylate of Glu226 falls at
 0.45 nm from the proximal imidazole ring [16].
Although the local stereochemistry makes unlikely the
achievement of a hydrogen bond between His266 and
Glu226 in HPX–heme(III), Glu226 may modulate
heme binding to HPX and HPX–heme ligand binding
capacity by affecting the protonation state of the prox-
imal His266 residue via electrostatic control of the resi-
due pK
a
[26].
In conclusion, our results describe a curious situ-

ation in which heme binding to a nonheme-protein
(i.e. HPX) confers (although transiently) functional
properties (e.g. peroxynitrite scavenging) and may be
predictive of (pseudo-enzymatic) function(s) of heme-
carriers (e.g. heme–albumin as well as high and low
density heme–lipoproteins). The system studied here
may suggest that the effects arising from heme binding
to HPX might have some role in the regulation of bio-
logical functions. Because these effects involve tran-
sient reactive functions, dependent on the interaction
with specific molecules (i.e. the heme), they have been
called ‘chronosteric’ effects [68].
Experimental procedures
Chemicals
Hemin [iron(III)–protoporphyrin(IX)] was obtained from
Sigma Chemical Co. (St Louis, MO). Gaseous NO was
purchased from Aldrich Chemical Co. (Milwaukee, WI).
NO was purified by flowing through a NaOH column in
order to remove acidic nitrogen oxides. The NO stock
solution was prepared by keeping in a closed vessel the
1.0 · 10
)1
m phosphate buffer solution (pH 7.0) under
purified NO, at 760.0 mmHg and 20.0 °C, anaerobically.
The solubility of NO in the aqueous buffered solution is
2.05 · 10
)3
m, at 760.0 mmHg and 20.0 °C. The NO
stock solution was diluted with degassed 1.0 · 10
)1

m
phosphate buffer to reach the desired concentration
[26,27,69]. The 1.0 · 10
)1
m phosphate buffer solution
was kept under helium. Peroxynitrite was prepared from
KO
2
and NO and from HNO
2
and H
2
O
2
, under anaer-
obic conditions. Peroxynitrite was purified by freeze frac-
tionation. The peroxynitrite concentration was determined
by measuring the optical absorbance at 302 nm (e
302
¼
1.67 · 10
3
m
)1
Æcm
)1
). The peroxynitrite stock solution was
diluted with degassed 1.0 · 10
)2
m NaOH to reach the

desired concentration. The 1.0 · 10
)2
m NaOH solution
was kept under helium. Decomposed peroxynitrite was
prepared by acidification of the peroxynitrite solution
with HCl, then the solution was neutralized with
1.0 · 10
)1
m NaOH [53,70,71]. For the experiments car-
ried out in the absence of CO
2
, the 1.0 · 10
)1
m phos-
phate buffer and the 1.0 · 10
)2
m NaOH solutions were
prepared fresh daily, thoroughly degassed, and kept
under helium. Experiments in the presence of CO
2
(1.2 · 10
)3
m) were carried out by adding to the protein
solution the required amount from a freshly prepared
5.0 · 10
)1
m sodium bicarbonate solution. The CO
2
con-
centration is always expressed as the true concentration

in equilibrium with HCO
3
–
. The value of the constant
of the hydration–dehydration equilibrium
4
CO
2
þ H
2
O $
H
þ
þ HCO

3
at pH 7.0 and 10.0 ° C is 6.38 · 10
)7
m.
The bicarbonate concentration present during the reac-
tions was 9.5 · 10
)3
m [28,30,32,47,53]. All the other
chemicals were obtained from Merck AG (Darmstadt,
Germany). All products were of analytical or reagent
grade and used without purification unless stated.
HPX
Rabbit serum HPX was prepared as reported previously
[25,72]. Protein contaminants were < 3% of the HPX sam-
ple as judged by gel electrophoresis and N-terminal amino

acid sequence determination. The HPX–heme(III) solution
(2.0 · 10
)6
)1.5 · 10
)4
m) was prepared by adding 1.2-
molar excess of the HPX solution to the heme(III) solution
(1.0 · 10
)1
m phosphate buffer, pH 7.0), at 10.0 °C [26,27].
Under these conditions, no free heme is present in solution
[17,25,26,72]. In fact, the value of the dissociation equilib-
rium constant for heme binding to HPX is < 10
)9
m
[25,72]. The HPX–heme(II)–NO solution (2.6 · 10
)6
m)
was prepared by reductive nitrosylation of HPX–
heme(III) under anaerobic conditions, i.e. by adding to
P. Ascenzi et al. Pseudo-enzymatic properties of heme–hemopexin
FEBS Journal 274 (2007) 551–562 ª 2006 The Authors Journal compilation ª 2006 FEBS 557
HPX–heme(III) either gaseous NO or the buffered NO
solution (see below).
Reductive nitrosylation of HPX–heme(III)
The value of the second-order rate constant for reductive
nitrosylation of HPX–heme(III) (k
on
) was determined by
mixing the HPX–heme(III) solution (final concentration

2.6 · 10
)6
m) with the NO solution (final concentration,
1.0 · 10
)4
)5.0 · 10
)4
m) under anaerobic conditions, at
pH 7.0 (1.0 · 10
)1
m phosphate buffer) and 10.0 °C [30–33].
No gaseous phase was present. Kinetics was monitored
between 360 and 460 nm. Under all experimental condi-
tions, final pH measured after mixing ranged always
between 6.9 and 7.1. For an homogeneous comparison
with the available functional data [26,27], kinetics was
obtained at 10.0 °C.
Time courses were fitted to the minimum reaction mech-
anism represented by Scheme 1 [30–33], where HPX–heme*
indicates the transient penta-coordinate HPX–heme species.
Values of the NO-dependent pseudo-first-order rate con-
stant for reductive nitrosylation of HPX–heme(III) (i.e. k)
have been determined from data analysis, according to
Eqn (1) [30–33]:
½HPX–hemeðIIIÞ
t
¼½HPX–hemeðIIIÞ
i
 e
kt

ð1Þ
The value of the second-order rate constant for reductive
nitrosylation of HPX–heme(III) (i.e. k
on
) was obtained
from the linear dependence of k on the NO concentration
(i.e. [NO]) according to Eqn (2) [30–33]:
k ¼ k
on
½NOð2Þ
The difference optical absorption spectrum in the Soret
region of HPX–heme(III) minus HPX–heme(II)–NO was
obtained under steady-state conditions by subtracting the
absorbance change in HPX–heme(II)–NO from that of
HPX–heme(III).
The kinetic difference optical absorption spectrum in the
Soret region of HPX–heme(III) minus HPX–heme(II)–NO
was reconstructed from the difference optical absorption
spectrum of HPX–heme(II)–NO minus HPX–heme(II)–
NO (De ¼ 0.0 m
)1
Æcm
)1
) obtained under steady-state
conditions plus the total absorbance changes of the HPX–
heme(III) reductive nitrosylation process.
Peroxynitrite-mediated oxidation of
HPX–heme(II)–NO
Values of the second-order rate constant for peroxynitrite-
mediated conversion of HPX–heme(II)–NO to HPX–

heme(III)–NO (i.e. b
on
) and of the first-order rate
constant for NO dissociation from the HPX–heme(III)–NO
complex (i.e. for the formation of HPX–heme(III); d)
were determined by rapid mixing the HPX–heme(II)–NO
solution (final concentration 2.6 · 10
)6
m) with the peroxy-
nitrite solution (final concentration, 1.5 · 10
)5
m to
2.5 · 10
)4
m) under anaerobic conditions, at pH 7.0
(1.0 · 10
)1
m phosphate buffer) and 10.0 °C, in the absence
and presence of CO
2
(1.2 · 10
)3
m) [28,30,32,47]. The dead
time of the SX18MV-R rapid-mixing stopped-flow appar-
atus (Applied Photophysiscs Ltd, Leatherhead, UK) was
1.6 ms. No gaseous phase was present. Kinetics was monit-
ored between 360 and 460 nm. Under all the experimental
conditions, the final pH value measured after mixing ranged
always between 6.9 and 7.1. Kinetics was obtained at
10.0 °C in order to avoid loss of the initial part of the

HPX–heme(II)–NO fi HPX–heme(III)–NO reaction especi-
ally in the presence of CO
2
, and for an homogeneous com-
parison with the available functional data [26,27].
The time courses were fitted to two consecutive mono-
exponential processes according to the minimum reaction
mechanism depicted in Scheme 2 [28,30,33,47].
Values of the (pseudo-)first-order rate constants for the
formation of the HPX–heme(III)–NO complex (i.e. b) and
for NO dissociation from the transient HPX–heme(III)–NO
complex (i.e. for the formation of HPX–heme(III); d) have
been determined from data analysis, according to Eqns (3–
5) [73]:
½HPX–heme(II)–NO
t
¼½HPX–heme(II)–NO
i
 e
bt
ð3Þ
Scheme 1.
Pseudo-enzymatic properties of heme–hemopexin P. Ascenzi et al.
558 FEBS Journal 274 (2007) 551–562 ª 2006 The Authors Journal compilation ª 2006 FEBS
½HPX–heme(III)–NO
t
¼½HPX-heme(II)–NO
i
ðb ððe
bt=ðdbÞ

Þ
þðe
dt=ðbdÞ
ÞÞÞ ð4Þ
½HPX–heme(III)
t
¼½HPX–heme(II)–NO
i
ð½HPX–heme(II)–NO
t
þ½HPX–heme(III)–NO
t
Þð5Þ
The value of b
on
was obtained from the linear dependence
of b on the peroxynitrite concentration (i.e. [peroxynitrite])
according to Eqn (6) [28,30,32,47]:
b ¼ b
on
½peroxynitriteð6Þ
The difference optical absorption spectrum in the Soret
region of HPX–heme(II)–NO minus HPX–heme(III) was
obtained under steady-state conditions by subtracting the
absorbance change of HPX–heme(III) from that of HPX–
heme(II)–NO.
The kinetic difference optical absorption spectra in the
Soret region of HPX–heme(II)–NO minus HPX–heme(III)
and of HPX–heme(III)–NO minus HPX–heme(III) were
reconstructed from the difference optical absorption spec-

trum of HPX–heme(III) minus HPX–heme(III) (De ¼
0.0 m
)1
Æcm
)1
) obtained under steady-state conditions plus
the absorbance changes of the overall process HPX–
heme(II)–NO + HOONO fi HPX–heme(III) + NO and
of the partial reaction HPX–heme(III)–NO fi HPX–
heme(III) + NO (Scheme 2).
The absolute optical absorption spectrum of HPX–
heme(III)–NO in the Soret region was reconstructed from
the optical absorption spectrum of HPX–heme(III)
obtained under steady-state conditions plus the absorbance
changes of the partial reaction HPX–heme(III)–
NO fi HPX–heme(III) + NO (Scheme 2).
Nitration of HPX Tyr and Trp residues was investigated
by optical absorbance spectroscopy [53]. Briefly, the heme
was removed from HPX–heme(III), obtained by reacting
HPX–heme(II)–NO (1.5 · 10
)4
m) with peroxynitrite
(1.0 · 10
)3
m), by mixing the HPX–heme(III) solution
(1.5 · 10
)4
m) with the cold ()20.0 °C) acid acetone solu-
tion (2–3 mL of 2.0 m HCl per liter of acetone). One vol-
ume of HPX–heme(III) in water was added slowly with

vigorous stirring to about 30 vol of the cold acid acetone
solution. The precipitated HPX was collected by centrifuga-
tion and then dissolved in a minimum amount of water.
The dissolved HPX was dialyzed in the cold against a dilute
bicarbonate solution (1.0 · 10
)3
m) and subsequently
against 1.0 · 10
)2
m phosphate buffer at pH 7.0. The pro-
tein precipitate was removed by centrifugation [69]. Then,
the optical absorbance spectrum of HPX ( 1 · 10
)4
m)
was recorded between 230 and 500 nm.
Data analysis
All experiments were carried out at least in quadruplicate.
The results are given as mean values plus or minus the
standard deviation. All data were analyzed using matlab
(The Math Works Inc., Natick, MA).
Acknowledgements
This work was partially supported by grants from the
Ministry for Education, University, and Research of
Italy (University ‘Roma Tre’, Rome, Italy, ‘CLAR
2005’ to PA) and from the Ministry for Health of Italy
(National Institute for Infectious Diseases IRCCS
‘‘Lazzaro Spallanzani’’, Rome, Italy, ‘Ricerca Corrente
2005’ to PA).
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