Heme/copper assembly mediated nitrite and nitric oxide interconversion

Article abstract of DOI:10.1021/ja3083818

The heme_a3/Cu_B active site of cytochrome c oxidase is responsible for cellular nitrite reduction to nitric oxide; the same center can return NO to the nitrite pool via oxidative chemistry. Here, we show that a partially reduced heme/Cu assembly reduces NO₂^- ion, producing nitric oxide. The heme serves as the reductant, but the Cu ^II ion is also required. In turn, a μ-oxo heme-Fe ^III-O-Cu^II complex facilitates NO oxidation to nitrite; the final products are the reduced heme and Cu^II-nitrito complexes.

Full text of DOI:10.1021/ja3083818

Communication
pubs.acs.org/JACS
Heme/Copper Assembly Mediated Nitrite and Nitric Oxide
Interconversion
Shabnam Hematian, Maxime A. Siegler, and Kenneth D. Karlin*
Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218, United States
S
* Supporting Information
ABSTRACT: The hemea3/CuB active site of cytochrome
c oxidase is responsible for cellular nitrite reduction to
nitric oxide; the same center can return NO to the nitrite
pool via oxidative chemistry. Here, we show that a partially
−
reduced heme/Cu assembly reduces NO2 ion, producing
nitric oxide. The heme serves as the reductant, but the CuII
ion is also required. In turn, a μ-oxo heme-FeIII−O−CuII
complex facilitates NO oxidation to nitrite; the final
products are the reduced heme and CuII−nitrito
complexes.
itrogen oxides (NOx) are components of great interest in
−
Nboth biological and environmental sciences. Nitric oxide
Figure 1. Cytochrome c oxidase (CcO) functioning in nitrite (NO2 )/
nitric oxide (NO) interconversion as part of its role in regulation of O2
balance. The availability of O2 influences the redox state of the CcO
containing the hemea3/CuB binuclear center.
(NO) is an important cellular signaling molecule and a
powerful vasodilator involved in many physiological and
1
−
pathological processes. Nitrite (NO2 ) is the one-electron-
oxidized product of endogenous NO metabolism. Recent
studies indicate that nitrite plays a critical biological role by
serving as a biochemical circulating reservoir for NO, in
particular under conditions of physiologic hypoxia (low O2
tensions; see also below) and ischemia. The nitrite-to-NO
conversion represents an important alternative source of NO to
the classical oxygen-dependent L-arginine-derived generation of
NO catalyzed by nitric oxide synthase (NOS).2 Subsequently,
In turn, in normoxia, high local O2 concentrations do not
allow nitrite to compete as an oxidant at the CcO binuclear
center; thus, NO/O2 binding is noncompetitive,7 and hemea3/
CuB oxidizes NO back to nitrite (Figure 1) to rejoin the storage
pool. NO is thought to first attack oxidized CuB, formally giving
CuI−NO+; the latter hydrolyzes to nitrite.8,9
In this report, we describe a chemical system involving a
heme/Cu assembly-mediated interconversion of these impor-
tant nitrogen oxides. A partially reduced/oxidized state, with
reduced heme and oxidized copper ion (i.e., FeII···CuII)
efficiently converts nitrite to NO. When a fully oxidized
FeIII···CuII heme/Cu complex is employed, NO is readily
oxidized to nitrite. The overall reactions are represented by eqs
1 and 2:
−
suggested conserved roles for the NO2 /NO pool in cellular
processes include oxygen sensing and oxygen-dependent
modulation of intermediary metabolism.3 It is now considered
that in order to stimulate NO signaling, nitrite reductase
activity occurs widely in differing cellular environments and is
effected by a variety of proteins/enzymes, including hemes,
those with molybdenum,4 and what draws our current interest,
cytochrome c oxidases (CcO’s).3,5
−
(P)FeIICuII + NO2 → (P)FeIII−O−CuII + NO
(1)
The link between nitrite/NO redox interconversion and O2
sensing is thought to occur in mitochondria at the CcO
binuclear hemea3/CuB center; CcO is the terminal enzyme of
the mitochondrial respiratory chain. Here, molecular oxygen
consumption (i.e., O2 reduction to water) is down-regulated in
hypoxia by increased NO generation via CcO nitrite reductase
activity, as reduced heme/Cu centers dominate when the O2
concentration is low.3a,4,6 The NO thus generated inhibits CcO
activity by reversibly binding to hemea3 in place of O2, resulting
in cellular O2 accumulation (Figure 1). Some of the NO
produced also participates in hypoxic signaling, the up-
regulation of nuclear genes needed in response to the inherent
dangers of low cellular O2 concentrations.3
−
(P)FeIII−O−CuII + NO → (P)FeIICuII + NO2
(2)
The nitrite reductase chemistry,10 here however in a heme/
Cu chemical system, consisted of the iron(II) complex (F8)FeII
[F8 ≡ tetrakis(2,6-difluorophenyl)porphyrinate(2−)]11 and a
preformed copper(II)−nitrito complex [(tmpa)CuII(NO2)][B-
(C6F5)4] [tmpa ≡ tris(2-pyridylmethyl)amine]; the latter was
synthesized by adding AgNO2 to a chloride precursor
[(tmpa)CuII(Cl)][B(C6F5)4], and its X-ray structure revealed
an O-bound nitrito ligated Cu(II) ion (Scheme 1).12
Received: August 23, 2012
© XXXX American Chemical Society
A
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Communication
Scheme 1. Heme/Copper Assembly-Mediated Reduction of
Nitrite to Nitric Oxide
Scheme 2. Heme/Copper Assembly-Mediated Oxidation of
Nitric Oxide to Nitrite
When 2 equiv of (F8)FeII were mixed with 1 equiv of
[(tmpa)CuII(NO2)]+ under a N2 atmosphere in acetone at
room temperature (RT), a reaction ensued, and on the basis of
UV−vis, electron paramagnetic resonance (EPR), and IR
spectroscopies, a 1:1 mixture of the heme−nitrosyl species
(F8)FeII(NO) and the μ-oxo complex [(F8)FeIII−O−
CuII(tmpa)]+ were produced.12 These products were readily
identified, having been previously thoroughly characterized.13
To determine whether the heme or the copper ion is the
reductant in this one-electron process (NO2− → NO), we also
carried out the reaction in which nitrite was added to the
oxidized heme complex [(F8)FeIII]SbF613b (binding of nitrite to
the ferric heme14 was indicated by a large UV−vis change)12
and then the reduced complex [(tmpa)CuI(MeCN)]+ 12,15 was
added. In this case there was no reaction (Scheme 1), even over
a period of days.12 Control experiments showed that nitrite
reacts only very slowly with (F8)FeII and not at all with
[(tmpa)CuI(MeCN)]+. Moreover, no nitrite reductase activity
was observed for the fully reduced metal combination, nitrite
plus (F8)FeII and [(tmpa)CuI(MeCN)]+.12
Figure 2. (a) UV−vis spectra of (F8)FeIII−O−CuII(tmpa)][B(C6F5)4]
(1) (blue) and (F8)FeII(NO) generated from 1 + NO(g) (12 μM in
acetone at RT) (red). (b, c) EPR spectra of (b) the products of the
reaction of (F8)FeIII−O−CuII(tmpa)][B(C6F5)4] and NO (red) and
(c) an authentic sample of a 1:1 mixture of F8FeII(NO) and
[(tmpa)Cu(NO2)][B(C6F5)4] (green). The EPR spectra were
recorded at 20 K (1 mM in MeTHF).
heterobinuclear complex led to rapid reaction (Scheme 2)
and formation of nitrite, which bound to Cu(II); the (F8)FeII
formed17 in this redox reaction was trapped by a second
equivalent of NO to give (F8)FeII(NO). UV−vis (Figure 2) and
IR (νNO = 1688 cm−1)12 spectroscopies directly indicated
nitrosyl complex formation. Nitrite analysis employing capillary
electrophoresis revealed that this ion was produced in 95%
yield.12 EPR spectroscopy confirmed that a copper(II)−nitrito
complex was produced (Figure 2); a sample taken from the
reaction mixture was identical in all regards to that of an
authentic sample of a 1:1 mixture of (F8)FeII(NO) and
[(tmpa)CuII(NO2)]+.
It is important to explain why the reaction required 2 molar
equiv of NO (Scheme 2). The second equivalent was not
involved in the redox chemistry but was needed to trap the free
(F8)FeII produced by the NO oxidase chemistry. When (F8)FeII
is present, it effects the reverse reaction, namely, reduction of
Cu(II)-bound nitrite to give NO (Scheme 1). This was
These observations indicate that the heme is the reductant in
this heme/Cu nitrite reductase chemistry. The need for 2 equiv
of (F8)FeII is due to the well-known high affinity of NO to bind
ferrous hemes.16 The initially formed NO reacts very rapidly
with (F8)FeII; thus, if the reaction were carried out with
equimolar quantities of (F8)FeII and [(tmpa)CuII‑(NO2)]+,
only half of the iron would be available to reduce nitrite, and
the rest would trap the NO as (F8)FeII(NO). The role of the
CuII ion appears to be to provide a Lewis acid interaction with
−
nitrite, facilitating NO2 (N−O) bond cleavage and stabiliza-
tion of the resulting oxo anion via eventual formation of
[(F8)FeIII−O−CuII(tmpa)]+.
To demonstrate that heme/copper assemblies can mediate
NO oxidation to nitrite, as occurs biologically in order to
remove excess NO when it is not needed and return it to the
nitrite pool (see above), we employed [(F8)FeIII−O−
CuII(tmpa)]+. Addition of NO to this fully oxidized
B
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Journal of the American Chemical Society
Communication
demonstrated as follows: When 25 mL of a 10 μM solution
containing a mixture of (F8)FeII(NO) and [(tmpa)-
CuII(NO2)]+ produced by the reaction of [(F8)FeIII−O−
CuII(tmpa)]+ with excess NO (green spectrum in Figure 3) was
by oxo transfer (see above)19 with reduction of (F8)FeIII. The
closely related species [(F8)FeIII−(OH)−CuII(tmpa)]+ and/or
heme-only complexes do not enable this reaction. However, as
already mentioned, it is the CuB in CcO that is thought to
oxidize NO,9 and it is well-known that NO can react with CuII
complexes,25 affording nitrite. This may imply that in CcO it is
a particular coordination environment, a specific structural and/
or redox state, that is required for mediation of the NO oxidase
chemistry.
Further investigations will include our probing of the
mechanisms of the reactions described in this report. For
nitrite reduction, critical mechanistic components will certainly
include the heme reductive capability and the nitrite−Cu
binding mode (e.g., O- vs O,O′- vs N-bound). As our heme and
Cu centers have switched redox capabilities compared with
CcO, we also wish to change the heme or the Cu ligand to
make the Cu center a better oxidant than the heme. For our
heme/Cu NO oxidation chemistry, we are uncertain about
which metal is the real oxidant,18 so further investigations are
required; as is often the case, “the devil is in the details”.
Figure 3. UV−vis spectra of a mixture of (F8)FeII(NO) (λmax = 399
nm) and [(tmpa)CuII(NO2)]+ derived from the NO oxidase chemistry
before (green) and after (red) the addition of 2 equiv of (F8)FeII. The
red spectrum shows the presence of a 2:1 mixture of (F8)FeII(NO)
and [(F8)FeIII−O−CuII(tmpa)]+ (435 nm, sh). The new second
equivalent of (F8)FeII(NO) was derived from the nitrite reductase
chemistry described by Scheme 1. See the text for further explanation.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic and analytical details; UV−vis, IR, and EPR spectra;
cyclic voltammetry and capillary electrophoresis results; and X-
ray structural details and CIF files. This material is available free
of charge via the Internet at http://pubs.acs.org.
titrated with 25 mL of a 20 μM solution (i.e., 2 equiv) of
(F8)FeII, the product solution (red spectrum in Figure 3)
contained ∼5 μM [(F8)FeIII−O−CuII(tmpa)]+ along with 10
μM (F8)FeII(NO); the spectral intensity was equivalent to that
observed in the starting mixture because of dilution. This
proves that the backward reaction can and does occur, that is,
that [(tmpa)CuII(NO2)]+ reacts first with (F8)FeII in a 1:1
stoichiometry to give NO, which is then trapped by the second
equivalent of (F8)FeII.
AUTHOR INFORMATION
■
Corresponding Author
karlin@jhu.edu
Notes
The authors declare no competing financial interest.
We also tested the μ-hydroxo complex [(F8)FeIII−(OH)−
CuII(tmpa)]2+ for “NO oxidase” chemistry, but upon addition
of NO, there was no nitrite production (Scheme 2).12 Instead,
very slow (hours) reductive nitrosylation18 occurred, and all of
the heme present was converted to (F8)FeII(NO). It is thus
clear that the μ-oxo complex ([(F8)FeIII−O−CuII(tmpa)]+ is
efficient or at least special in its ability to effect a redox reaction
(formally FeIII → FeII) that includes oxo transfer.19
ACKNOWLEDGMENTS
■
This research was supported by the National Institutes of
Health (GM060353 to K.D.K.). We also thank Dr. Richard C.
Himes for help in synthesizing [(tmpa)CuII(Cl)][B(C6F5)4].
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